SCIENCE LABORATORY TECHNOLOGY

A Comprehensive Manual for Laboratory Technicians

SIR DOMINIC MAGUT

SCIENCE LABORATORY TECHNOLOGY
 SCIENCE LABORATORY TECHNOLOGY
DEDICATION

To my dear wife Sussy, children; Mike, Faith and Denies, My parents;

Benson and Rabecca,My brothers; Leonard ,Walter,Godfrey,Duncan ,
Vincent, My sisters ;Jailyne and Lillian not forgeting to mention the
entire Kapchemaget family .
 SCIENCE LABORATORY TECHNOLOGY

ACKNOWLEDGEMENT.
I would most sincerely wish to give credit to the adminstration , the
teaching and non teaching staff and students of Kaimosi College of
Research and technology for having given me the opportunity to work
with them . It is indeed through their blessed hands that I have written
this book .

Specifically I thank to Mr. Nixon Zavani,- The HOD , Mr. Samson

shavanga ,Mr. Abel Bateri, Mr .Jared Kalerwa ,Mrs. Mabel Atsiaya,
Mrs. Lydia wanyonyi, Mrs. Jane Lumadede, Mrs. Mercy
Muraran,Mrs. Florence Boiyo, Mr. Albert mackenzie, Mr. Jonathan
Mugoya , Mr. Felix Khalwale and Mrs. Judith Ingaitsa who are all
wonderful members of the Applied science department in Kaimosi
College of Research and Technology.

I also wish to express my thanks to all my beloved church members at

Morongiot Baptist church for their prayers and moral support .

Lastly , my sincere appreciation goes to all who contributed in one

way or another in typesetting , editing and publishing this book .

May God bless you in all your endeavours.

 SCIENCE LABORATORY TECHNOLOGY
CHAPTER ONE

PROSPECTS FOR LAB

TECHNICIANS
Science Laboratory technology is a wide field that deals with the
technical management of any scientific laboratory. Modern trends in
laboratory technology require technician and technologists with
knowledge in scientific concepts, who are able to gather, analyze and
synthesis scientific information ,using laboratory instruments and
techniques , and at the same time maintaining high organizational
and safety standards .
Science Laboratory technologists and technicians study and analyze
the chemical, biological or physical composition and behavior of matter
through experiments in the laboratories . they are the personnel
responsible for all laboratory-based tasks, which include sampling,
testing, measuring, recording and analyzing results in biological,
chemical, physical and life sciences. They also provide all the required
technical support to enable the laboratory to function effectively whilst
adhering to correct procedures and health and safety guidelines.
Science laboratory technicians carry out fundamental tests as part of a
scientific team. These tests assist in the advancement and development
of modern medicine and science .
The main function of a scientific laboratory technician is to perform the
specific scientific procedures that allow scientists to perform the more
complex analytical processes of the laboratory

Their typical tasks involve ;

 Performing laboratory tests in order to produce reliable and precise
data to support scientific investigations;
 Carrying out routine tasks accurately and following strict
methodologies to carry out analyses;
 Preparing specimens and samples;
 Maintaining and operating standard laboratory equipment,
 Ensuring the laboratory is well-stocked and resource;
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 Recording and sometimes interpreting results to present to senior
colleagues;
 Using computers and performing mathematical calculations for the
preparation of graphs;
 Keeping up to date with technical developments, especially those
which can save time and improve reliability;
 Demonstrating practical procedures if working with students ;
 Conducting searches on identified topics relevant to the research;
 Following and ensuring strict safety procedures and safety checks.

Skills needed include ;

 Enthusiasm for exploring
 Curiosity and creativity
 Analytical & quantitative
 abilities
 Desire for precision
 Diligence
 Innovative talents
 Information handling & organization
 Organize and maintain accurate records
 Statistical awareness
 Oral & written communication
 Numerical computation
 Ability to concentrate for long periods of time
 Problem solving
 Teamwork
 Operate scientific equipment
Science laboratory technology is a multimillionaire field that
comprises other hundreds of technical laboratory specialties . Some
of these fields have been in existence for the past many years while
other new ones have emerged that are tailored to meet the demands of
specific scientific niches or industries . In this book we have listed just
a few of them , But you will notice that they all require the application
of almost the same skills and expertise .

Biochemists
Biochemistry is the study of the structure, composition, and chemical
reactions of substances in living systems. Biochemistry includes the
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sciences of molecular biology; immunochemistry; neurochemistry; and
bioinorganic, bioorganic, and biophysical chemistry.
Biochemistry is applied to medicine, dentistry, and veterinary
medicine. In food science, biochemists research ways to develop
abundant and inexpensive sources of nutritious foods, determine the
chemical composition of foods, and develop methods to extract
nutrients from waste products, or invent ways to prolong the shelf life
food products. In agriculture, biochemists study the interaction of
herbicides with plants. They examine the structure-activity
relationships of compounds, determine their ability to inhibit growth,
and evaluate the toxicological effects on surrounding life.
Biochemistry spills over into pharmacology, toxicology, physiology,
microbiology, and clinical chemistry. In these areas, a biochemist may
investigate the mechanism of a drug action; engage in viral research;
conduct research pertaining to organ function; or use chemical
concepts, procedures, and techniques to study the diagnosis and
therapy of disease and the assessment of health.

Biotechnologist
Biotechnology is the application of biological organisms, systems, or
processes by various industries to learning about the science of life and
the improvement of the value of materials and organisms such as
pharmaceuticals, crops, and livestock. Biotechnology depends on the
ability to manipulate chemical structure .It is a relatively new and fast-
developing field that integrates knowledge from several traditional
sciences: biochemistry, chemistry, microbiology, and chemical
engineering.
Biotechnology is a source of great promise for innovations ranging
from improving the diagnosis and treatment of hereditary diseases, to
safer drugs, to more environmentally friendly herbicides and
pesticides, to microbial processes to clean up the environment. Making
these promises a reality requires rethinking some fundamental
assumptions.
Because biotechnology requires a grasp of many different scientific
disciplines, chemists suggest starting in high school with courses in
biology, chemistry, and genetics. In college, you can gain a sense of
whether this area is for you by taking a good molecular biology course.
Analytical chemists
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Analytical chemistry is the science of obtaining, processing, and
communicating information about the composition and structure of
matter. In other words, it is the art and science of determining what
matter is and how much of it exists.
Analytical chemists perform qualitative and quantitative analysis; use
the science of sampling, defining, isolating, concentrating, and
preserving samples; set error limits; validate and verify results through
calibration and standardization; perform separations based on
differential chemical properties; create new ways to make
measurements; interpret data in proper context; and communicate
results. They use their knowledge of chemistry, instrumentation,
computers, and statistics to solve problems in almost all areas of
chemistry. Analytical chemists are employed in all aspects of chemical
research in industry, academia, and government. They do basic
laboratory research, develop processes and products, design
instruments used in analytical analysis, teach, and work in marketing
and law. Analytical chemistry is a challenging profession that makes
significant contributions to many fields of science.

Chemical Engineers

Chemical engineers apply the principles of chemistry, math, and

physics to the design and operation of large-scale chemical
manufacturing processes. They translate processes developed in the lab
into practical applications for the production of products such as
plastics, medicines, detergents, and fuels; design plants to maximize
productivity and minimize costs; and evaluate plant operations for
performance and product quality.
Chemical engineers design and operate plants and processes for large-
scale production of chemical products. They use chemistry, physics,
and mathematical equations to solve real problems and design ways to
produce products safely and economically.
Chemical engineers typically work in manufacturing plants, research
laboratories, or pilot plant facilities. They work around large-scale
production equipment that is housed both indoors and outdoors. Often
they are required to wear safety protective equipment, such as hard
hats, goggles, and steel-toe shoes. Workdays may involve of moving
from place to place within a facility. Chemical engineers also work in
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business and management offices; these positions, however, often
require visiting research and production facilities. Interaction with
other people who are part of a team is critical to the success of projects.

Agricultural chemists
Agricultural chemistry focuses on chemical compositions and changes
involved in the production, protection, and use of crops and livestock.
It seeks to control and understand the processes by which humans
obtain food and fiber for them-selves and feed for their animals.
Agricultural chemists work with food producers to increase yields,
improve quality, and reduce costs. They also study the causes and
effects of bio-chemical reactions related to plant and animal growth,
seek ways to control these reactions, and develop chemical products
that provide help in controlling these reactions. Chemical products
developed to assist in the production of food, feed, and fiber include
herbicides, fungicides, insecticides, plant growth regulators, fertilizers,
and animal feed supplements.
Research projects for agricultural chemists cover many fields of
inquiry, including the development of a molecule or chemical
compound that controls a weed or other pest; the development of that
molecule for full-scale manufacturing; modifications to the molecule,
so that it works for longer periods of time or at lower dosages; and
testing for the impact and fate of the chemical in food and the
environment.

Environmental chemists

Environmental chemists focus on collecting and analyzing samples,

developing remediation programs, changing production processes to
yield a more environmentally friendly product, providing expert advice
on safety and emergency response, or dealing with government
regulations and compliance issues.

Forensic scientists
A forensic scientist is a professional who analyzes evidence that is
brought in from crime scenes and reaches a conclusion based on tests
run on that piece of evidence. A forensic chemist's job is to identify and
characterize the evidence as part of the larger process of solving a
crime. Forensic scientist rarely conducts any investigative work; they
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handle the evidence collected from the crime scene. Evidence may
include hair samples, paint chips, glass fragments, or blood stains.
Understanding the evidence requires tools from many disciplines,
including chemistry, biology, materials science, and genetics.
Forensic scientist applies knowledge from diverse disciplines such as
chemistry, biology, materials science, and genetics to the analysis of
evidence found at crime scenes or on/in the bodies of crime suspects.
The field is a combination of criminalistics and analytical toxicology.
Criminalistics is the qualitative examination of evidence using methods
such as microscopy and spot testing, whereas analytical toxicology
looks for evidence in body fluids through a range of instrumental
techniques from optical methods (UV, infrared, X-ray) to separations
analyses (gas chromatography, HPLC, and thin-layer
chromatography). Mass spectrometry is also frequently used since it
provides the strongest evidence in court.

Geochemists
Geochemists study the occurrence and distribution of chemical
elements in rocks and minerals. They also study the movement of these
elements into soil and water systems. Their work contributes to natural
resource use and environmental management policies. For example,
analytical work done by geochemists guides’ oil exploration, helps
improve water quality, and is used to develop remediation plans to
clean up toxic waste sites.

Inorganic chemists
Inorganic chemistry is the study of the synthesis and behavior of
inorganic and organometallic compounds. It has applications in every
aspect of the chemical industry–including catalysis, materials science,
pigments, surfactants, coatings, medicine, fuel, and agriculture. Their
work is based on understanding the behavior and the analogues for
inorganic elements, and how these materials can be modified,
separated or used–often in product applications. Their jobs often
involve understanding the chemical properties of these materials and
manipulating them, sometimes via reaction with other materials, to
achieve particular desired properties.
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Pharmaceutical chemistry

pharmaceutical chemistry is the application of chemical research

techniques to the synthesis of pharmaceuticals. During the early stages
of medicinal chemistry

development, scientists were primarily concerned with the isolation of

medicinal agents found in plants. Today, scientists in this field are also
equally concerned with the creation of new synthetic drug compounds.
Pharmaceutical chemistry is almost always geared toward drug
discovery and development.
They also work on improving the process by which other
pharmaceuticals are made. Most chemists work with a team of
scientists from different disciplines, including biologists, toxicologists,
pharmacologists, theoretical chemists, microbiologists, and
biopharmacists. Together this team uses sophisticated analytical
techniques to synthesize and test new drug products and to develop the
most cost-effective and environmentally friendly means of production.
Organic chemists

Organic chemistry is that branch of chemistry that deals with the

structure, properties, and reactions of compounds that contain carbon.
It is the science of designing, synthesizing, characterizing, and
developing applications for molecules that contain carbon. Organic
chemists create and study organic compounds, the reactions that
produce them, and their chemical and physical properties. They create
and explore new uses for new or existing organic materials. They carry
out synthesis reactions and isolations in a laboratory environment
using sophisticated instruments such as nuclear magnetic resonance;
gas and liquid chromatography; and infrared, ultraviolet, and visible
spectroscopy. Most of the instruments are computer driven and
controlled, so computer literacy is required.

Physical chemists
Physical chemistry applies physics and math to problems that interest
chemists, biologists, and engineers.
Physical chemists work in a variety of different industries, but their
common goal is to discover, test, and understand the fundamental
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physical characteristics of a material—be it solid, liquid, or gas.
Precision and attention to detail make their work similar to analytical
chemistry, although physical chemists also stress the importance of
applying knowledge of math and physics to develop an understanding
of the material. They work with both their minds and hands,
manipulating complex sets of data and operating sophisticated lab
instrumentation.

Hazardous waste management chemists

Hazardous waste management chemists generally use analytical

chemistry skills to determine the composition of materials deemed
hazardous, working either in a lab or in the field. Teamwork is a key
element. Biologists, toxicologists, and water and soil chemists work
together to evaluate hazardous wastes and develop strategies for
disposal or cleanup.

Science Writers and Journalists

Science writers describe discoveries and commercial developments in
all branches of science, engineering, medicine, and environmental
science. They explain the impact these discoveries have on the lives of
average individuals.
Science writers usually work in one of four career areas: science
journalism, public communications, technical writing in industry, and
editing. Science journalists write articles for general circulation
magazines, science magazines geared to the general public, magazines
for scientists and engineers, and newspapers. Some work for television
and radio networks. Science writers specializing in public
communications prepare press releases and reports for government
agencies, research universities, research institutes, and professional
societies. Those working at universities and research institutes often
assist researchers in preparing grant proposals. Technical writers in
industry prepare technical bulletins, technical advertising, and press
releases, and they assist in writing technical papers. Science editors
edit articles for science and technology journals, magazines, and books,
as well as government reports.
 SCIENCE LABORATORY TECHNOLOGY

Textile chemists
Textile chemistry is primarily an applied form of chemistry. It is a
highly specialized field that applies the principles of the basic fields of
chemistry to the understanding of textile materials and to their
functional and esthetic modification into useful and desirable items.
Textile materials are used in clothing, carpet, tire yarn, sewing thread,
upholstery, and air bags, to name a few examples.
The study of textile chemistry begins with the knowledge of fibers
themselves-both natural and synthetic. Because synthetic fibers are
such an important part of today's textile business, the field includes
many who are trained as polymer chemists. The interaction between
textile chemistry and materials science is also increasing. Textile
chemistry includes the application of the principles of surface
chemistry to cleaning processes and modifications such as dyeing and
finishing. It encompasses organic chemistry in the synthesis and
formulation of the products used in these processes
.
Water chemists
Water chemists study the impact of water on other elements in the
systems and how other elements in these systems affect the quality of
water. Water chemists also contribute to the design and
implementation of processes and policies to manage these effects.
Water chemists generally work on interdisciplinary teams that may
include scientists with expertise in soil culture, geology, aquatic
biology, statistics, forestry, hydrogeology, chemistry, mathematical
modeling, and database management. The

teams study and monitor a given ecosystem or industrial process; they

discover the impact of water on other elements of the system and,
conversely, how these other elements affect the quality of the water.

Food technologist
The fundamental work of food technologist is to offer techniques for
preservation, conservation and processing of food items to be
packaged. Additionally, they check the compliance of procedure during
the food processing and ensure that there is no contamination and
adulteration. Ensuring top quality nutritional value in the food
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products by putting quality raw materials is an additional job for a food
technologist.

Pulp and paper chemists

Pulp and paper chemists focus their work on the industrial paper-
making process. Much of their job is geared towards improving
efficiency, making the process more cost-effective and environmentally
friendly.

Polymer chemists

Polymer chemists develop polymers so they can be used to make

ingredients for products with unique physical and chemical properties.
They manipulate large, complex molecules and capitalize on the
connections between their molecular structure and the properties that
make them useful.
Polymer chemists work in many industries, creating a variety of
synthetic polymers such as polythene, plastics agricultural chemicals;
paints and adhesives; and biomedical applications such as artificial
skin and developing new polymers that are less expensive or that
outperform traditional materials and replace those that are scarce.

Science consultant

Consultants play a combined role of journalist, lawyer, and teacher;

they gather information, shape it for a particular situation, and educate
their clients. In the chemical industry, consultants study products,
markets, manufacturing processes, environmental regulations, and
patents. With this information they assist executives in making
business decisions concerning new products, acquiring other
companies, or reorganizing internally.

Applied biologists
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Applied biology is the study of the nature, composition, and utilization

of biological processes and systems. Applied biology deal with the
application of several areas of biology in medicine, industry,
agriculture and environment.
Applied biologists study the processes of living systems plants, insects,
viruses, microorganisms, and mammals and apply the knowledge in
developing new products e.g. drugs ,pesticides , fertilizers or in sewage
treatment .
 SCIENCE LABORATORY TECHNOLOGY
CHAPTER TWO

LABORATORY DESIGN
AND LAYOUT

When designing even a single laboratory, the aim is to provide

attractive, well-equipped facilities that support pupils as they engage
with and learn about science.
Exciting, high-quality practical work is a vital part of pupils’ science
experience and provision for this is what makes science
accommodation unique. However, ambitions for high standards of
science accommodation are often thwarted, to a greater or lesser
extent, by constraints such as a lack of:
• Knowledge about what the facilities might look like.
• Flexibility to create the optimum size and shape of rooms within
an existing building.
• Budget – planned or unplanned – with costs overrunning.
• Knowledge about the essentials required for health and safety.
Laboratory designing and layout process is a very tasking exercise
since every precaution must be taken to ensure that all features
intended for that laboratory are factored in so as to avoid or minimize
inconveniences that my be costly to correctin future .One way to avoid
these inconveniences is by letting teachers, technicians, support staff
and pupils to be involved throughout all the stages of design and build.
This requires teamwork ,time, financial resources and coordination

Planning site for a Science Laboratory

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The chemicals, materials, equipment and services involved in science
laboratories make them a ‘danger area’ under health and safety
regulations. Science laboratories should be sited
(a) Away from cafeterias , health units and other residential places
(b) Away from traffic e.g. roads , railway lines ,etc
(c) In proximity to workshops ,botanical gardens , animal houses and
green house
(d) It should not be far away from delivery points
(e) It should be oriented in east to west direction so as to avoid stray
light from interfering with chemicals which can explode due to
exposure to sunlight.
It is also essential that they are supervised by suitably qualified staff ,
are properly equipped for health and safety, and are not used for
inappropriate activities. The same considerations apply to the Prep
Room, the chemicals store, the radioactives store and some equipment
stores.
Only equipment in frequent use should be stored in a laboratory; all
other equipment and materials should have dedicated storage.
Teachers’ marking and personal preparation are best provided for away
from the laboratory. Most management activities are best
accommodated in planned spaces, while social activities, eating and
drinking should certainly be accommodated separately from the
‘danger areas’. A laboratory is not the most suitable place for a tutor-
base.
Prep Room(s)
The purpose of these areas is to support the practical work taking place
in laboratories. They are for the preparation, maintenance and repair
of equipment and materials and for the disposal of materials when they
are finished with. As such, they are for the use of science technicians
(and science teachers who are preparing or practicing practical
activities) with administration facilities focused on the support of
practical work.
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Prep room

Stores

• Chemicals: A separate, secure storeroom for hazardous chemicals is

essential. It should open into the Prep Room and be separately
ventilated.
• Radioactives: Radioactive sources must be stored in a metal
cupboard within a secure storeroom. This storeroom is usually one
used mainly for general equipment or paper resources.
• Other storerooms: These will be needed for gas cylinders, general
equipment, paper, textbooks, ICT equipment, etc. Most of these will
need to be secure as a protection from theft or damage. Some
equipment (e.g. vacuum glassware, high voltage equipment) will also
need additional security for health and safety reasons. All stores should
be easily accessible from the Prep Room(s) and the laboratories.

Management, administration and individual staff work space

Proper provision for these functions will promote higher standards of
leadership and morale. It will also ensure that the laboratories and
Prep Room(s) are able to function safely as practical areas.
Staff are entitled to toilet provision within easy reach of their teaching
areas. Distributed provision for pupils is an increasing trend and can
mean that pupils take better care of the facilities.
Toilets for the disabled should also be easily accessible from science
areas.
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Cleaning equipments storage of cleaning equipment should be
entirely separate to science accommodation, including science stores.

The Laboratory – Good standards

Space
A minimum floor area of 90m2. Its shape should be rectangular, or
close to it, with a
length to width ratio of between 1:0.8 to 1:1.1.Circulation spaces are
vital.
the space should provide the following zones :
• Two (teacher) presentation areas, each on different walls – one for
demonstration, one with a teacher base (the base and demonstration
area could be on the same wall).
• Fume cupboard zone.
• Wall zone for health and safety equipment .
• Display areas.
• Presentation areas to have good sight lines for all pupils.
• Emergency or escape doors. All doors should be fire doors, with view
panels and automatic closers . These should be wide enough and tall
enough to permit the passage of large equipment and wheel chairs .
Lifts
Where science accommodation is on more than one floor, or is not on
the ground floor, a passenger lift is required to meet the requirements
for those with Disability.
Any lift should be wide enough/tall enough to accommodate
wheelchairs and support staff , mobile fume cupboards, etc.
Security
Labs should be locked when unoccupied, and supervised by suitably
qualified science staff when open.

Furniture and Equipment

There are a number of important issues to consider when planning a
good working, ergonomic school science laboratory:

• Pupil benches: These should have a work surface area ≥ 0.36m2

per pupil, with linear frontage ≥ 600mm per pupil. The height of pupil
benches should be 900mm, with good clearance for legs when the pupil
is seated at the bench (270mm top of stool to underside of bench) . All
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services, except water, should be within 600mm of the pupil position in
at least one layout.

• Perimeter benching: This should be on two walls maximum, with

the height to match pupil benching, and having adequate structure to
support it, especially on long stretches.
• Demonstration bench/service bollard: This should have all
services (e.g., water, gas, electricity), and be the same height as pupil
benches, with no teacher dais.
• Teacher base: This is a secure storage area for the teacher’s
personal items while conducting a science lesson.
• Presentation areas: Writing whiteboards, as well as one/both
with projection screen.
• Display areas: With display boards.
• Adjustable height bench: to cater for those with disability.
• Equipment trolleys: Again, heights should match benches, with
planned spaces for parking and access.
• Cupboards , shelves and drawer for storage of lab items
• Door hinges: These door hinges should be open self-closing.
• Stools: Supply one per pupil, plus four spare stools for teacher and
support staff . Stool design should include large area leg ends or skids.
Allow enough leg space for people to sit at benches .
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Laboratory stools

Ergonomic considerations should include a back rest if possible.

Storage
Pupils usually bring a significant number of items such as books,
stationery materials and items of clothing into the school science lab.
Simply leaving such personal items on workbenches or floors, or
draping clothing over the backs of stools can pose serious health and
safety risks, and coming into contact with chemicals may well cause
irreparable damage. For these reasons, proper storage of pupils’
belongings are essential.

• Bags and coats: Place these near to the main door (but not too
near to cause an obstruction). Allow space for each pupil’s bag and one
coat/bag hook per pupil.
• Ready-use equipment: Items such as bunsen burners, mats,
tripods, etc., may need tray units or cupboards in the lab, generally
placed under perimeter benching. All other equipment and materials
storage should be in Prep Room(s) shelving or adjacent stores.

Services
It is imperative that provision of services be a main consideration in
any school science lab. Recommendations include the following:
• Pipes and cabling: These should be installed in or behind
benching, walls, or floors. None should be hanging from a ceiling
(except for ceiling-mounted data projectors). Pipes should be colour-
coded according to contents and show the direction of flow . Pupil
services (gas and electricity) outlets should be within 600mm of pupil’s
work/seating places. Services should be arranged so that pupils do not
all face outwards around the walls when undertaking practical work.
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• Gas taps: It is recommended that one gas tap per pupil, spaced
around the lab . Other taps should be fitted on demonstration benches,
perimeter benching and serving the fume cupboard. Do not place gas
taps under cupboards, curtains or blinds, or in front of windows.
Ensure that all taps are of robust design, with definite on/off positions
and anti-rotate fixing to bench, with non-return valves and restrictors
in nozzles
• Gas pipes: All gas pipes should concealed if made of copper and or
exposed if made of steel .
• Electrical Sockets: one electrical socket per pupil is
recommended. As with gas taps, other sockets should be provided for
demonstrations benches, perimeter benching and the fume cupboard.
Electrical sockets should be of robust design, facing away, with shield,
if placed near to water.
• Electrical circuits: School science labs usually require at least two
‘master’ 30 amp ring-main circuits to feed the standard mains sockets.
Other, separate circuits not under the ‘master’ control may be needed
for computers, data projectors, fish
tanks, fume cupboards.
• Water supply: This should be of high enough pressure to operate
science equipment.
• Sink: One sink is recommended per 6 pupils spaced around the lab,
and each should be large enough for experimental equipment
(recommended: ≥ 300mm x 200mm x depth150mm)with one cold tap
per sink. One larger experimental sink on should be fitted on the
demonstration bench with two cold taps. A further sink should be
included in the fume cupboard. One large sink with drainer on a
perimeter bench should be installed for wash up and hand wash, with
both hot and cold taps being provided. .
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• Water Taps: These should be of robust design, the top being ≥
300mm above benches, and with the spout ≥ 225mm above the sink.
Tap controls should be located on the front (see Appendix: Robust
tap). Fixing or locating plates should be used to prevent anti-rotating
of the taps. Domestic taps are unsuitable for science labs.
• Eyewash station: An eye wash station must be readily accessible
within the Science Laboratory. A constant supply of clean, cold water
should be available.
• Drains: Easily accessible drainage system should be placed under
each sink. Chemically-resistant high-density polythene or
polypropylene should be specified for pipe work
• Controls: Gas, electricity and water should all have master controls
(emergency shut-off s) in the science lab, easily accessible by teacher,
but not by pupils.
• Demonstration area (bench or service bollard): Should have
all services, i.e., at least two gas taps and four mains electricity sockets,
large experimental sink and all ICT services. Fire and health and safety
equipment should be nearby.
• ICT: if possible, Internet/intranet access for pupils and staff should
be provided, along with data projection and accompanying screen.
Telephones should be available as part of the school
telecommunications system.
• Fume cupboards: Should be installed, commissioned and
maintained . A-level chemistry labs should be supplied with at least
two fume cupboards. For demonstrations, supply one for every two
laboratories.
Fume cupboards should be sited away from doors and not in a corners
Adequate air supply is required. Extraction should be quiet in
operation .
• Fire and Health & Safety
There are number of key fire, health and safety measures to be
considered within any school science lab. They include:
• The need for noise, fire, smoke and fumes to be contained. This can
be a problem when movable walls are fitted.
• Emergency alarm points should be installed in each science lab or
close to them.
• An emergency eyewash station should be provided in each science lab
that is readily accessible.
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• Fire extinguishers and fire blanket in each lab; one extinguisher and
the blanket to be readily accessible, located by the demonstration
bench. Extinguishers should not be water based due to substantial
electrical circuits and items within the science lab, and preferably not
powder-based, which causes severe damage to equipment.
• First Aid kit in each lab, readily accessible.

Environment
Poorly-designed heating, lighting, ventilation, etc., can create
significant problems within a school science lab. Apart from
considerable health and safety issues resulting from poor
environments, teachers and pupils alike may suffer during lessons,
resulting in poor learning outcomes.
Major environmental issues include:
• Floors to be level, with no ramps. They should also be impervious to
water, resistant to chemicals, and non-slip; the manufacturers cleaning
instructions/arrangements should be made part of the school’s overall
cleaning procedures.
• Windows are good for natural light,
Windows should open for ventilation, and be easily accessible. Other
than on the ground floor, windows should have restricted openings,
which can nevertheless be opened fully in the event of an emergency.
• Lighting should be ≥ 300lux on work surfaces, plus the provision of
task lighting.

The Prep Room – good standards

Most of the items that apply to the school science lab also apply to the
school science Prep Room.
 SCIENCE LABORATORY TECHNOLOGY

A prep room

(i) Position and pace

• The Prep Room should be central to the laboratories that it serves.
Ideally, it should consist of one large area with stores leading off (the
chemicals store in particular). If science accommodation is on more
than one floor, then plans should accommodate one Prep Room per
floor.
• The recommended floor area for a Prep Room and stores is ≥ 0.5m2
per pupil work space, with other spaces in addition. For example, six
labs with 30 pupil places = 180 x 0.5 = 90m2 (or the size of one science
lab). Typical percentages of spaces within this are: practical work
areas: 30%, storage: 30%, mobile storage:
10%, office: 10%, circulation: 20%.

(ii) Access and Security

• A large Prep Room need two doors, with one having an access/egress
door direct to the corridor (fire escape) – not through the science lab.
Doors should be locked at all times when unoccupied, and supervised
by science technicians or science teachers when open.
• For delivery of hazardous materials, an outside door close to, or
directly in, the preparation area of the science accommodation should
be fitted. This can be shared with the Design Technology (DT)
department if science and DT are located next to each other.
• If the Prep Room is not on the ground floor, a lift will be required for
manual handling. A specialised hoist between the Prep Rooms (NOT in
 SCIENCE LABORATORY TECHNOLOGY
labs) is more expensive and less versatile than an appropriate
passenger lift. Such a passenger lift should be within easy reach of the
Prep Room(s).
Zones
Prep Room work areas should be considered in terms of zones. If prep
rooms are on different floors, some zones may be prioritized on a floor
(e.g., main chemical preparation on one floor along with the chemical
store). Standard work zones include:
• Wet preparation/washing up: Two large sinks (chemical
resistant) plus double drainers, hot and cold tall taps, all with good
drainage. An emergency eyewash should be positioned to this
preparation area.
• Chemicals preparation/dispensing: Sited by the chemicals
store (may be one of Prep Room ‘wet’ areas), plus ducted fume
cupboard and good ventilation.
• Dry preparation/repair and maintenance: This should
include a ≥ 2000mm linear bench, ≥ six mains electrical sockets and a
metal working vice. Local exhaust ventilation should be fitted if large
amounts of soldering will be carried out.
• Collation/return of equipment: Floor plans should allocate
specific areas for trolleys and free bench areas.
• Administration: This is a dry area for the administration of Prep
Room activities, away from other preparation zones. This zone should
have computer and ICT access, cabinets for filing documents and
appropriate shelving.
Storage of hazardous materials
There are three groups of hazardous materials that should be stored
separated from each other. Essential details are:
 SCIENCE LABORATORY TECHNOLOGY

A chemical store
• Chemicals: The floor area should be ≥ 10m2, larger for schools with
1000 pupils or more and larger for A-level work. Construction should
be fire re resistant (60min) and opening directly off the Prep Room,
adjacent to the chemicals preparation zone. There should be no direct
heating by the sun – i.e., not south, east or west-facing and no fl at roof
directly above the ceiling. No heating pipes or radiators should be
installed, and no holes or voids communicating to surrounding rooms.
No drains or windows should be fitted.
– Ventilation: This should provide ≥ 2 air changes per hour , usually
by forced extractionwith quiet operation (≤ 65bD at 300mm), at top
and bottom, with auto-control. Sufficient intake air is required for the
extraction.
– Door: This door should open out into the Prep Room and should
contain a view panel, fi re stop and lock which opens from inside
without a key, for user safety. The door should be locked at all times
when not in use, and closely supervised when open. Hazard signs
should beclearly visible on the door.
– Floor: The Prep Room floor should be impermeable to water,
chemical resistant and non-slip. It should slope to the back, or provide
a slight hump by the Prep Room door (all this is needed to contain
hazardous spills, therefore no drain in floor). The floor should be able
to take loading, especially of rolling storage. Stepladders of height
equal to shelves should be provided, with grab rails and a stable
platform.
– Fixtures and fittings: Light fittings should be flameproof, with
metal cabinets provided for flammables.
Shelves should be narrow, made from wood with no lips and fitted at ≤
2m high, stable and secured to the walls.
• Radioactive sources:
Radioactive sources must be stored in a secure metal cabinet, fastened
to the wall, in a secure general store, at least 2m (ignoring walls, floors
and ceilings) from a place where anyone spends extended periods of
time. (Not in the chemicals store.)
• Gas cylinder storage: These cylinders should be kept in separate
secure storage away from flammables (i.e., not in the Chemicals Store)
and away from Radioactives. Chained racks or specialist trolleys should
be utilized for storing the gas cylinders.
 SCIENCE LABORATORY TECHNOLOGY
Prep Room furniture
Appropriate furniture should be provided in order that Science
Technicians responsible for preparation of science materials and
equipment can work efficiently and effectively. Furniture requirements
include:
• Benches, ≥ 900mm high, to suit adults, with sufficient clearance
underneath for fridges, dishwashers, etc. A measurement of 240-
270mm from the top of the stool to the underside of the worktop allows
sufficient upper leg clearance for the technician to sit comfortably at
the work surface
• Chairs and Desks for administration.
• Notice boards and whiteboards for administration
• Equipment trolleys (for mobile storage and preparation) – these
should be the same height as benches.
• Shelving should be ≤ 2m high, stable and secured to a wall.
Floors should be designed to cope with maximum loads, especially
where rolling storage is installed. Stepladders should be provided:
height equal to the top shelf, with grab rails and platform.

Equipment
Equipment used in Prep Rooms can be extensive, and it should be
considered carefully at the design stage, especially because of the space
needed, the dimensions of furniture surrounding it, and the range of
services required to make it work. The main types of equipment
include:
• Fume cupboard: this should be installed within the chemicals
preparation zone. The fume cupboard should be ducted, with gas, cold
water, drainage and electrical services all required .
• Washing up machine: Positioned in the wet area with electricity
feed plus hot and cold water (anti-siphon) and drainage.
• Still, for producing distilled water: Wall mounted, with 13 amp
electrical sockets (often two), cold water feed and drainage.
• Drying cabinet: Located near the wet area, with electricity supply
and bench space.
• Fridges: One fridge should be supplied for experimental material
with an additional fridge provided for staff use (food storage, etc). A 13
amp socket will be required for each fridge, and the fridges should be
positioned under a bench of suitable height to accommodate them.
 SCIENCE LABORATORY TECHNOLOGY
• Freezer(s): Again, one or more, with corresponding electrical
sockets, fitted under a bench.
• Ice maker: These can be benchtop models or floor-standing.
Electrical feeds from 13 amp sockets will be needed for both options.
• Emergency eyewash: This should be located by the chemical and
wet preparation areas, and should provide a constant fl ow of clean,
cold water.
• Computer and printer: For administrative use. This equipment
would require mains electricity, plus local area networking links to the
Internet and the school’s intranet.
Services
Safety and security concerns are just as important in the Prep Room as
they are in the science lab. Prep Rooms require:
• Master controls (emergency shut-off s) for gas, electricity and water;
plus zoned controls for isolation and maintenance
• Colour-coded pipes, with direction of flow clearly marked.
• ICT: Internet/intranet access, with a telephone connected to the
school telecommunications system.
Fire, Health & Safety
As with the science lab, Prep Rooms require:
• Noise, fire, smoke and fumes to be contained.
• Fire extinguishers (CO2 – not water based and preferably not
powder) and fire blankets.
• First aid kits.
• Emergency Eyewash.
• Emergency alarms points in the Prep Room, or close to it.
• Telephones, to reduce the safety risks of working alone.
Environment
Prep Rooms have a particular need for natural light (as Science
Technicians work within the Prep Room environment for most of their
day) and good ventilation (≥ 6 air changes per hour).
Other science areas for consideration
Learning areas
For example: demonstration theatres, individual learning pods, group
discussion areas, student resources bases, study areas.
All of these need good mains electrical provision and ICT access.
Where gas, water, electricity services are provided, all master controls,
fire and Health and Safety provision are required. Security and
supervision also need to be addressed.
 SCIENCE LABORATORY TECHNOLOGY

Greenhouses, environmental areas, ponds, etc.

These should be sited near, or within, science areas for better security
and supervision, and be able to receive plenty of sunlight as well as
protection from the wind.
• Greenhouses will also need water and drainage, plus mainselectrical
power and lighting. Electrical services should be water-proofed.
• Ventilation and heat control should be operated by roller blinds and
automatic windows.
• Heating should be via a thermostatically controlled fan, independent
of school heating system.
• Watering system – automatic.• Lighting – standard 300lux for
working; 11000-21500 lux for plant growth
Support areas
• Management and administration areas should be sited adjacent to the
Prep Room and science lab.
• Work stations for teachers, technicians and support staff should be
supplied, each with secure personal locker, table, office chair, filing
cabinet, ≥ 4 electrical sockets and ICT access (Internet and school
intranet).
• Meeting rooms should be provided for interviews, counselling, etc.,
separate to other rooms. Electrical sockets should be available in each
room, along with telephone and ICT access, chairs and tables.
• Secure stores for paper, textbooks, exam papers, etc., with stable
shelving ≤ 2m height.
• Furniture: This should consist of easy chairs, coffee tables, etc.
Toilets: Staff , pupils and disabled toilets should be provided with
toilets , with easy access from science areas. Note: staff toilets must be
separate from pupils’ toilets, and male toilets separate from female.

Cleaning and maintenance: Stores for cleaning and maintenance

materials
should be completely separate from labs, Prep Rooms and science
stores.

LABORATORY FITTINGS
Laboratory fittings include
(a) Benches
(b) Water taps
 SCIENCE LABORATORY TECHNOLOGY
(c) Gas taps
(d) Sinks
(e) Electric gadgets
(f) Fume chambers
(g) Fire extinguishers
(h)Shelves

(a) LABORATORY BENCHES

There are two main patterns of laboratory benches i.e
(i)Permanent assembly benches
(ii)Unit assembly benches
Generally, laboratory-working benches should be atleast 36 inches
high.
(i) Permanent assembly benches
Permanent assembly benches are permanently fixed hence don’t allow
future modification or else they must be destroyed incase necessary
changes are to be made . they are designed for specific purposes
.Laboratory services e.g. water ,electricity and gas taps should not be

placed on them.
Permanent assembly bench
(ii) Unit assembly benches They are temporarily fixed , they are flexible
and movable and therefore can allow future modification.
 SCIENCE LABORATORY TECHNOLOGY

Unit assembly bench

Material used for making

laboratory benches

Wood
Advantages
Cheap and easily available
Poor conductor of electricity
High mechanical strength
Disadvantages
Easily attacked by fungi, pest and bacteria
Easily distorted if not well treated
Easy to burn
Easy to be cut
Place of use- Physics and chemistry Laboratories

Metal e.g. stainless steel, aluminum.

Advantages
High mechanical strength
Water resistant
Free from shrinkage and distortionDon’t burn easilyProne to chemical
attack
Disadvantages
Conduct heat and electricityExpensive
place to use Food ,medical ,biology and radiology laboratories
 SCIENCE LABORATORY TECHNOLOGY
Concrete
Advantages
Free from vibrations ,
High mechanical strength
Heat resistant
Disadvantages
Easily attacked by acids Poor performance if not properly finished
Place to use
Balance rooms

Plastics
Advantages
Resistant to fungal and bacterial attack
Chemical resistant
Poor conductor of heat and electricity
Easy to be cleaned
Disadvantages
Wear out easilyNot heat resistant
Easily damaged by laboratory equipment’s
Very expensiv
Place to use
Chemistry, biology physics and medical laboratories

Fume chambers
Fume chamber is designed to prevent contamination by drawing out
poisonous or dangerous fumes from the laboratory . They should be
designed in such a away that will only allow fumes to be drawn out of
the laboratory and not inside.Fume chambers consist of transparent
panels made of sliding glass that provides a working space for
operation.
 SCIENCE LABORATORY TECHNOLOGY

Fume chamber
Some also have a fan connected to a motor
There are two types of fume chambers
(i) Direct syste
(ii) Indirect system

(i) Direct system

The motor is placed directly above the fun in line with the fumes from
the lab .The motor is small and cheap but wears out easily due to
direct exposure to fumes
hence needs constant replacement

(ii) Indirect system

The motor is placed separately from the fun and is not in line with the
direction of movement of fumes from the laboratory. the motor is big
and do not wear out easily but it is however expensive.
Before using a fume chamber ,its important to ensure its in good
condition , switch on the fun and adjust the vertical sliding glass to
give a reasonable working space ,then close tightly ensuring that no
fumes escape out of the fume chamberNB/ All fuming chemicals must
be opened inside a fume chamber

( c) Taps, sinks and traps

The position of taps and sinks is very important and should be
considered during laboratory planning and designing stages. These
 SCIENCE LABORATORY TECHNOLOGY
should be in accordance to the type of work to be done on a bench.Taps
should be placed at the center of the bench or on the sides so as to
allow for communal work. They should not be placed next to electric
sockets
Traps receives waste from the sink and disposes it into the main
drainage system .
The water seal prevents foul smell from getting back into the laboratory
There are two types of sinks .
(i) Rimless sinks
(ii) Rimmed sinks

(i) Rimless sinks

They are fitted, tightly on the bench
(ii)Rimmed sinks,
They have rims that lie over the bench surface. There are two types of
rimmed sinks i.e. externally rimmed sink and internally rimmed
sinkThe fittings of the sinks should be such that should
not allow leakage or flooding of the water. Rimless and internally
rimmed sinks are recommended for use in laboratories because they
are easier to wipe out dirt from the bench into the sink .
Materials used for making sinks include

(i) Metals e.g. stainless steel

Resistant to chemical attac
Easy to install
Disadvantages - Expensive
Ceramic
Resistant to chemical attack
Plastic
Relatively cheap and affordable in many grades
Easy to installResistant to chemical attack

FIRE EXTINGUISHERS
These are equipment’s used for fire fighting.
Types of fire extinguishers
(i) Water type fire extinguisher
 SCIENCE LABORATORY TECHNOLOGY
Produces water to fight fire , its conventional fire code color is
normally red . it is suitable for class A fires and fires caused only by
some inflammable solvents which
are soluble in water .should not be used to extinguish fires caused by
insoluble solvents , metals and electricity

(ii) CO2 Fire extinguisher

Contain CO2 at high pressure and it fight fire by settling on the
burning object forming a blanket of CO2 over it because it’s denser
than air and does not support combustion . it is suitable for
extinguishing al classes of fires . Its conventional color code is black

(iii) Dry powder fire extinguishers

It have a dry chemical powder e.g. NaHCO3 and it function by cutting
down air supply on a burning object i.e. NaHCO3 decompose on
burning to give CO2 gas which covers the burning object . It’s an
improved version of dry sand method . It is suitable for all classes of
fires . Its conventional color-coding is blue

(iv) Vaporizing liquid

These is a highly vaporizing liquid which fights fire by covering the
burning object thereby excluding oxygen supply . These chemicals
include ; bromotrifloromethane ,tetrachloromethane and
bromochlorodifloromathane .
They are suitable for class D and C fires . They should not be used in
confined places
because it produces phosphogens which is a colorless heavy gas with
a choking and nauseating gas. Their conventional color code is gree
(v) Foam
Its color code is cream . it works by excluding oxygen supply from the
burning object it is suitable for fires caused by flammable solvents.
(vi) Fire blanket
These are made from asbestos material. It fight fire by cutting of
oxygen supply from the burning object it is used for extinguishing fire
from a burning equipment or a person on fire .
NB; Fire extinguishers should always be held vertically along the wall
in an upright position .They should be located in positions where
chances of fire risks is high.The right type of fire extinguishers must be
placed in the right place. They should never be pulled or dragged on
 SCIENCE LABORATORY TECHNOLOGY
the ground because these may cause pressure build up which might
result in an en explosion.
To use a fire extinguisher
(i) Remove it from the wall
(ii) Hold it in its upright position
(iii) Remove the safety pin
(iv) Aim the nozzle at the base of the fire
(v) Press the knob
(vi) Try to isolate the fire

Emergency showers
Emergency showers are very important because they are used to
extinguish fires when somebody is on fire .Emergency showers must be
placed in an easily accessible positions and should be connected with a
water storage tank and must be regularly tested and properly
maintained .

LABORATORY FLOOR SURFACES

Just like laboratory benches , various materials can be used for making
the surfaces of the laboratory floors, e.g. wood ,concrete
,terrazzo,linoleum. plastic ,PVC tiles etc. But before any of which is
used , there are some factors that should be satisfied for it to be
considered appropriate for use .i.e. They should be
Resistant to chemical,
Cheap and easy to clean
Offer maximum safety , comfort ,beauty to the laboratory users .
They should also not be noisy

VENTILATION IN THE LABORATORY

Ventilation simply means free circulation of air in and out of the
laboratory
Good ventilation is important in the laboratory , these is in order to rid
the laboratory from dangerous fumes . there are two types of
ventilation
(a ) Natural ventilation ; These include all ventilation that allows in
fresh air e.g. through windows, doors etc
(b) Artificial or mechanical ventilation. These is achieved by means of
fans , fume chambers etc
LIGHTING IN THE LABORATORY
 SCIENCE LABORATORY TECHNOLOGY
Lighting in the laboratory is also very important so as to enable the
occupants to easily see and work comfortably without straining .
Lighting also provides an agreeable working environment and warmth.
There are two types of lighting in the laboratory i.e.
(a) Natural light which includes day light passing through the
laboratory windows and doors
(b) Artificial lighting which includes use of electric bulbs and
fluorescent tubes.Good lighting can also be achieved through painting
of laboratory walls with bright or light reflecting paint

CHAPTER THREE

LABORATORY SAFETY
AND HEALTH
Laboratory safety is concerned with all safe practices in the
laboratory .Failure to observe laboratory safety practices will result in
risks and eventually accidents . Accidents can be defined as the
unexpected occurrence that causes or leads to injury to a laboratory
user or damage to property. Risks can be defined as anything that is
likely to cause an accident to occur Accidents in laboratories can
result in
 SCIENCE LABORATORY TECHNOLOGY
Injury to laboratory users
Work being disrupted
Loss of equipment’s ,supplies and records
Contamination of laboratory reagents
Damage to adjacent building environment
Good laboratory management is that which always strive to minimize
chances of risks in the laboratory environment

RISK ASSESMENT
Risk assessment is one of the laboratory management practice which
involves
(a) Identifying the hazards i.e. knowing what are the common causes
of accidents.
(b) Deciding what are the common actions to be taken so as to
minimize or remove the hazards e.g. by adopting safe storage of
chemicals and other safe working practices or simple structural repair.
(c) Evaluating for those hazards that cannot be removed so as to know
the harm that they can cause and to what extend .
(d) Deciding on what safety precautions , regulations and safety
awareness measures needed to minimize the risk and prevent
accidents from occurring.
(e) Writing codes of practice based on findings of the risk assessments
and putting them in place to be followed
.(f) Training all laboratory personnel on how to apply the safety codes
in their place of work.
OBJECTIVES OF SAFETY PROGRAMES
Good laboratory management is that which always strive to minimize
chances of risks in the laboratory environment The main objectives of
laboratory safety programs is ;
(a) Identify the hazards and asses the risk to the staff and others.
(b) Prepare and implement an effective code of practice .
(c) To check whether health and safety codes are being followed .
(d) To ensure that staff knows how to work safely , what to do when an
accident occurs and how to carry out emergency first aid .
(e) To ensure all laboratory accidents are reported informatively and
investigated properly.
(f) To promote safety awareness.
Laboratory hazards are grouped according to their causes i.e.
Physical or mechanical hazards
 SCIENCE LABORATORY TECHNOLOGY
Chemical hazards
Biological hazards
Radiation hazards
Electrical hazards
Fire hazards

A. PHYSICAL OR MECHANICAL HAZARDS

These includes all those physical injuries e.g. cuts , bruises , fractures ,
dislocations , back injuries etc caused by external objects /
equipment’s or machinery’s
Causes of physical injuries
Physical hazards can be caused by ;
Poor or careless handling of articles
Poor use of hand tools
Fall of the person on hard floor or on sharp objects
Heavy objects falling on the person
Stepping on or knocking against sharp objects

Prevention of physical hazards

Careful handling of hand tools by using gloves especially when
washing glasswares, avoid using excessive force when clamping or
corking glasswares or turning burette
taps ,cutting or bending glass tubes ad being careful when transporting
glasswares ( use laboratory trays and trolleys to carry glass wares) ,
Proper training and following instructions
Ensuring the laboratory floors are not slippery and avoid carelessly
placing obstacles on the way or leaving items lying on the floors and
ensuring good lighting in the laboratory
CHEMICAL HAZARDS
These are hazards associated with transportation, storage and
handling of chemicals . Such hazards includes ;
(i) Fire –caused by flammable chemicals
(ii) Poisoning by toxic, harmful or irritating chemicals
(iii) Corrosion caused by chemical burns
(iv) Cancer due to radioactive mutagens
(a) Flammable and inflammable chemicals
Flammable and inflammable substances are substances which readily
catches fire and burns. These chemicals have low flash point i.e. low
 SCIENCE LABORATORY TECHNOLOGY
temperatures at which the vapour above the liquid can be ignited in air
.they therefore have high risk of catching fire

Classification of flammable chemicals

(i) Extremely flammable chemicals
These are liquids with a flash point below 0oc and a boiling point of
35oc or below e.g. acetone and diethyl ether
(ii) Highly flammable chemicals
They are liquids with flash points below 21oc or solids which readily
ignite after a brief contact with a flame or which evolve highly
inflammable gases when in contact with water or moist air e.g.
absolute ethanol and methanol
(iii) flammable chemicals
They are liquids with flash points of between 21oc and 55oc e.g. glacial
acetic acids ,xylene or acetic hydride

Safety storage and use of flammable chemicals

Keep only small quantities of flammable chemicals on laboratory
benches and shelves .
Store stock supplies particularly those that are highly flammable in a
closed steel or thick plywood box at ground level shelves preferably in
a cool and well ventilate outside locked store .
Do not store oxidizing and reducing chemicals together .
The store room should have adequate, suitable and well-maintained
fire extinguisher . The lab should also be well ventilated.
Avoid naked fires near the store or when opening the container
containing flammable chemicalsFlammable chemical containers
should be always tightly sealed to discourage escape of vapor Place a
NO SMOKING label on the door so as to discourage smoking around
the flammable store
Use tray to hold the container so as to prevent the liquid from
spilling ont he floor incase of spillage
Use water bath to heat flammable chemicals

Oxidizing chemicals
Are substances that evolve O2 when they come in contact with other
substances and may cause them to burn strongly or become explosive
 SCIENCE LABORATORY TECHNOLOGY
in presence of heat e.g. peroxides , dichromate’s , KmnO4, chlorates
etc.
Oxidizing chemicals can increase the speed and intensity of a fire by
adding to the oxygen supply, causing materials that would normally
not burn to ignite and burn rapidly. Oxidizers can also:
a) React with other chemicals, resulting in release of toxic gases .
b) Decompose and liberate toxic gases when heated .
c) Burn or irritate skin, eyes, breathing passages and other tissues
Precautions to follow when using and storing oxidizers in the
laboratory include the following:
 Keep away from flammable and combustible materials
 Keep containers tightly closed unless otherwise indicated by the
supplier .
 Mix and dilute according to the supplier's instructions
 To prevent release of corrosive dusts, purchase in liquid instead of
dry form .
 Reduce reactivity of solutions by diluting with water
 Wear appropriate skin and eye protection
 Ensure that oxidizers are compatible with other oxidizers in the same
storage area .
(a) Toxic or poisonous chemicals
A poisonous chemical is any substance which when taken into the body
in insufficient quantities is capable of injuring or causing death to the
casualty . All chemicals in the laboratory should be regarded as
poisonous including distilled water.
Chemicals can gain entry into the body by:
(i)Inhalation of gases, vapours and particulate material (e.g. mists,
dusts, smoke, fumes)
(ii) Absorption through skin of liquids, solids, gases and vapours
(iii) Ingestion of chemicals directly or indirectly via contaminated
foods and beverages and contact between mouth and contaminated
hands (nail-biting, smoking)
(iV) Injection of chemicals through needles and other contaminated
laboratory sharps

Threshold limit value (TLV) and permissible exposure limits

(PEL)
These are terms used to refer to the level of a chemical above which a
worker should avoid repeated and prolonged exposure. TLVs and PELs
 SCIENCE LABORATORY TECHNOLOGY
are structured guideline which provide the guidelines for workers in
any industry including laboratories to follow in order to minimize
chances of hazards. They are very much applicable in the labs as they
also provide guidelines about which chemicals to use and when and
how to take precautions.
Many substances have effects which are acute and fast acting . But a
ceiling limit should be established on how to use these substan so
that never should one be allowed to be exposed beyond these limit
even for an instant. For those chemicals that are acute and fast acting
through skin absorption , gloves must be provided and worn, the
substance must either be handled in a fume chamber or in a ventilated
room.
Thresholds are normally set below that of the TLV e.g. if the lowest
level that is known to cause harm ( TLV ) is 0.05mg then the threshold
should be set at 0.01mg. i.e. the threshold of smell of many chemicals
is normally below that of TLV. Strong smell may act as a warning that
the TLV is being approached. But since human sense organs can be
impaired, one should not depend on his senses alone as a reliable
indicator of hazard.
The best approach and practice in the laboratory which will minimize
hazards is to maintain concentrations of all atmospheric
contaminants to the lowest practical level. There are three primary
steps that can help reduce chances of exposure to volatile or airborne
chemical substances.
(a)Ventilation
these could be by use of a fans (purge ventilation) or fume chambers.
(b)Substitution
in these case a more hazardous substance or chemical should be
substituted with a less hazardous chemical in a chemical reaction or
procedure
(c)Microscale
these refers to scaling down the quantity of materials used in order to
reduce the amount of volatiles that shall be released into the
atmosphere
Any substance can be armful to living organism, but however it is the
dose that frequently determines the extent of damage . Many other
factors including the time of exposure , route of exposure ,age .sex,
lifestyle , allergic factors , previous sensitization and genetic
 SCIENCE LABORATORY TECHNOLOGY
disposition may impact on the overall effect that a chemical has on an
organism.
Toxicologists have developed several tools to determine the toxicity of
chemicals . Acute toxicity which is immediate effect of a single dose is
easier to study than chronic toxicity which results from small doses
over long period of time. Toxicologist use LD50 value to estimate the
acute toxicity of a chemical on humans . LD means the lethal dose and
is expressed in milligrams of the chemical per kg of the body weight.
The 50 in the LD50 is the mortality rate (50%) of the test animal.e.g.
Aniline LD50 oral- rat: 250mg/kg.
Can be interpreted as an oral dose of 250mg /kg will kill 50% of the
test sample of rats.
NB/ it should be noted that no LD50 data exist for humans . instead
data from test animals is used to estimate the acute toxicity of
chemicals on humans . The lower the LD50 the more toxic the
substance.
There are three forms of poisons i.e. solid , liquid and gaseous poisons.
Toxic chemicals are substances that can couse a serious, acute or
chronic effect even death when inhaled , swallowed or absorbed
through the skin e.g. mercury .
Harmful chemicals only cause limited effect on health if inhaled ,
swallowed or absorbed through the skin e.g. benzoic acid , iodine or
xylene

Corrosive chemicals
Corrosives are materials, such as acids and bases (caustics, alkalis)
which can damage body tissues as a result of splashing, inhalation or
ingestion.
Also:
 They may damage metals, releasing flammable hydrogen gas
 They may damage some plastics
 Some corrosives, such as sulphuric, nitric and perchloric acids, are
also oxidizers; thus they are incompatible with flammable or
combustible material
 They may release toxic or explosive products when reacted with other
chemicals
 •They may liberate heat when mixed with water
 Precautions for handling corrosive materials include:
 Wear appropriate skin and eye protection
 SCIENCE LABORATORY TECHNOLOGY
 Use in the weakest concentration possible
 Handle in a chemical fume hood .
 Use secondary containers when transporting and storing corrosives
 Always dilute by adding acids to water
 Dilute and mix slowly
 Store acids separately from gases
Reactive chemicals
Reactive chemicals are those chemicals that may;
 Be sensitive to jarring, compression, heat or light
 React dangerously with water or air
 Burn, explode or yield flammable or toxic gases when mixed with
incompatible materials.
 Vigorously decompose, polymerize or condense
 Be toxic, corrosive, oxidizing or flammable
NB; Some chemicals may not be dangerous when purchased but may
develop hazardous properties over time (e.g. diethyl ether and
solutions of picric acid).
Follow these precautions when working with dangerously reactive
chemicals:
 Understand the hazards associated with these chemicals and use
them under conditions which keep them stable
 Store and handle away from incompatible chemicals
 Keep water-reactive chemicals away from potential contact with
water, such as plumbing, fire sprinkler heads and water baths
 Handle in a chemical fume hood
 Wear the appropriate skin and eye protection
 Work with small quantities
 Use up or dispose of these chemicals before they attain their expiry
date
Nerve poisons
Nerve poisons are those that when they are absorbed into the body
,they upset the nervous system e.g. morphine, opium etc
Nerve poisons are divided into two i.e.
sedatives - they cause the nervous system to relax
Stimulants – cause the nervous system to be excited

Irritating chemicals
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Irritating chemicals can cause inflammations and irritation of skin ,
mucus membranes and respiratory tract following immediate or
prolonged exposure e.g. ammonia, potassium dichromate etc
All chemicals in the laboratory should be regarded as poisonous
including distilled water.
Carcinogens
Chemical carcinogens are chemicals that have ability to induce
cancer . Carcinogenic effects takes long before its symptoms appear
unlike toxic chemicals
Mutagens
Mutagens are chemicals which produce mutation of the germ cells
leading to generally induced malformation , spontaneous abortion or
death of the offspring’s upon exposure
Allergens
Allergens are chemicals which causes allergy or hypersensitivity
reaction when they come into contact with the skin causing dermatitis
,when inhaled they cause asthma

General safe use and storage of toxic substance

 Highly toxic , irritating and harmful chemicals should be stored
under lock and key .
 Only authorized persons should handle toxic chemicals .
 highly toxic chemicals should always be handled with care and
hands should be washed after handling them also wear protective
clothing’s when handling them.
 Fuming chemicals should be handled in a fume chamber .
 Never taste or mouth pipette any chemical in the laboratory or use
any laboratory wear for food as utensils .
 Avoid using carcinogenic substances in the laboratory instead seek
alternative safer substitute for them
 Young children and expectant mothers should not come closer to
carcinogenic substances .
 Corrosive chemicals should be stored at lower level shelves .
 Never store NaOH and KOH in a ground glass stopper .
 Always pour corrosive chemical at below eye level,slowly and with
great care to avoid splashing
 When opening a corrosive chemical, place a cloth over the neck of
the bottle
 SCIENCE LABORATORY TECHNOLOGY
 Exercise great care when diluting or dissolving corrosive chemicals
e.g. Na,K,
 Never add water to an acid but add acid to water slowly by the edge
of the bottle

CRYOGENIC FLUIDS AND COMPRESSED GAS HAZARDS

Cryogen’s are fluids at extremely low temperatures e.g. liquid oxygen
=-182oc and nitrogen at –195oc.Cryogens are normally kept in a
metallic thermosflask called Dewar which are never sealed because if
they are sealed , they will develop a pressure build up which could
lead to an explosion.

Physical hazards associated with cryogenic fluids

Liquid nitrogen is normally used as a coolant in chemical reactions
and most often causes explosion due to blockage of tubes or pipes due
to formation of ice where moisture from the air is frozen . Also
cryogen’s are transported in glass vessels , these is normally a very
delicate endeavor because the glass may fall or be subjected to thermal
shock which could lead to spillage

Chemical hazards associated with cyogenics

Some cryogenic fluids e.g. oxygen are fire risk because they support
combustion and
incase of any leakage they can ignite and support fire

Physiological hazards associated with cryogens

ryogens cause cold burns if they spill on the skin ,also continuos
production and leakage of some cryogenic fluids e.g. oxygen can
accelerate growth of infectious microorganisms.
Safe use of cryogen’s
 Avoid all body contact with cryogens by wearing protective clothing .
 Ensure proper ventilation so as to discourage pressure build up .
 Use the right containers which can sustain and withstand the rapid
changes in temperature .
 Never mix different cryogens together
 Never use oil or grease on the equipment containing liquid oxygen.
 Keep all organic compounds away from liquid oxygen.
 Ban smoking in the immediate area.
Hazards of compressed gases
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Inert non toxic gases like Helium, Argon and even Nitrogen reduces
the concentration of oxygen in the air below the safe level especially in
confined areas . these may slowly lead to drowsiness to the laboratory
staff leading to doing mistakes and accidents .
Toxic gases e.g. Cl, NH3, SO2 etc can lead to toxic anoxia especially
when released in large quantities also even small leakage of these
gases can lead to irritation .
Flammable gases e.g. methane , acetone and ethylene etc may cause
fires and explosions especially when they are in contact with some
metallic elements Decomposing gases e.g. ethyne releases carbon and
hydrogen accompanied by release of large quantities of heat .
Cylinders carrying compressed gases may explode due to exposure to
heat and if dropped carelessly .

Safe use of compressed gases.

Gas cylinders are color coded for easy identification ,the actual names
of the gases should be given on the cylinder together with other
information should be recorded on the neck of the cylinder e.g. date of
filling , pressure and capacity of the cylinder etc. the color codes are as
follows ;
Hydrogen cylinder – red
Ethylene acetate cylinder – maroon
Normal air cylinder- gray
Oxygen – black
When cylinder are received in the laboratory, they should be checked
immediately for any damage and for correct identification . it is
important to ensure that all cylinders and cylinder fittings are not
damaged.
Storage areas for gas cylinders should be fire proof and well
ventilated , dry and away from heat source. Its infact advisable to keep
flammable gas cylinders separately.
They should be fixed in an upright position .
Never drop , drag or roll gas cylinder but use trolleys to transport
them .
Never temper with cylinder heads , valves or pressure control gauges
or any cylinder safety device.
Avoid inhaling gases and ensure that you are well informed of the
chemical properties of gases and the associated hazards before
 SCIENCE LABORATORY TECHNOLOGY
opening the gas cylinders and when in doubt, always ask for
assistance from senior members of staff.

RADIATION HAZARDS

Radiation is caused by disintegration of radioisotopes. These are heavy

unstable metals which breakup into small into small particles that are
more stable hence giving out energy which is part of the
electromagnetic spectrum i.e. radio waves , microwaves, infrared ,
visible light , UV light , x-ray and gamma rays

Types of radiation’s
(a) Alpha radiation
These are radiations associated with streams of helium which are
normally positive charged . They are heavy and can ionize other atoms
but have low penetrating power
b) Beta radiation
They are associated with streams of electrons emitted from nuclei of
radioactive isotope . They are negatively charged and can also ionize .
they have moderate penetrating power

c) Gamma radiation
These are radiation without charge , they have very high ionization and
penetrating power
Hazards of radiations could be acute or chronic , immediate or long
term . Radiation’s induces changes in genetic makeup (mutation) and
can also cause cancer . They can be prevented by using proper
protective clothing , radiation indicators e.g. pocket dosimeters which
measures the amounts of radiation one have taken or gadgets which
are photographic films placed on the laboratory coats or by simply
avoiding being near a radioactive material e.g. microwave oven , lesser
beams and UV sources . The pocket dosimeters and gadgets are then
taken for development in special laboratories in order to determine
the amount of radiation one has taken in from the laboratory.
Only well trained laboratory personnel should be allowed to operate in
such radiation laboratories and should use protective clothing and
must always undergo medical examination.
The use of radioactive materials should be carefully controlled.
 SCIENCE LABORATORY TECHNOLOGY
Radioactive materials must be stored in a restricted access conditions
in a block of lead that is surrounded by a thick wood and be clearly
labeled .
There should be radiation warning signs placed on the lab coats

ELECTRICAL HAZARDS AND SAFETY

Electricity is the flow of electrons in a closed circuit. Electricity can
cause electric shock , direct burn and secondary injury from non-
lethal shock , it can also cause fires and explosions
(a) Electric shock- electrocution
Alternating current cause tetany ( muscles become locked in a
contracted state). There are three mechanisms through which
electrocution can occur i.e.
(i) Respiratory arrest - in these case , the brain part that controls the
diaphragm muscle become stormed
(ii) Ventricular vibration – in these case , the heart muscle develop
tetany and hence stops pumping blood
(iii) asphyxiation - the lung muscle contract permanently leading
to lung failure
(b) Non lethal shock
Uncontrolled muscular spasm produced when one comes into contact
with electricity can result into cuts , bruises , falling from heights and
knocking over equipment’s

Static electricity
Static electricity is produced when electricity is displaced on the
surface of a material , it occurs as a result of friction when two surfaces
move over each other .
These can cause high voltage to be created on a material which can
be discharged as sparks at critical level . the energy of the spark is
enough to initiate dust or vapor explosion
Prevention of electrical hazards
Electrical hazards can be prevented in various ways ;
 The electrical equipment must be earthed so as to discharge .
 Electrical appliances must be carefully selected , correctly installed
and adequately maintained .
 Wiring should be done by qualified persons and be inspected by
acredited company on regular basis . Warn out cables should be
replaced immediately.
 SCIENCE LABORATORY TECHNOLOGY
 Always switch off the power before removing the plugs.
The color-coding of the wire should follow the conventional color code
i.e.
Red or brown – live
Black or blue – neutral
Green or Yellow – earth
Install a voltage stabilizer between the supply and the consumption
point and circuit breakers should be used to cut off the supply incase
too much current shall pass through . These prevents the main switch
from being blown off.
All conducting wires must be properly insulated.

BIOLOGICAL HAZARDS
These are caused by-
poisonous plants and microorganisms
· Laboratory animals

Hazards caused by plants are known as botanical hazards and those

caused by animals are zoological hazards

Botanical hazards
These are specific infections from microorganisms e.g. algae , fungi,
bacteria, virus and protozoan and plants e.g. datura . These pants have
alkanoid substances which
are very poisonous

Zoological hazards
These are infections caused as a result of poor handling of laboratory
animals e.g
worms , rabies ,chicken pox etcPants transit their infection through
ingestion , skin
and nose while animals transmit them through bites stings and
scratches .
Protective methods for biological hazards
 Wear protective clothing when handling lab animals and plants .
 Clean or disinfect tables using methylated spirit before and after
experiments .
 Incinerate used materials as soon as possible .
 SCIENCE LABORATORY TECHNOLOGY
 Use protective clothing only in the lab and never use it outside the
laboratory –leave them in the laboratory.
 Never smoke or eat anything in the laboratory.
 Do not store any material in a food refrigerator or mix with other
food utensils.
 Wash your hands thoroughly with an appropriate antiseptic after
every practical .Avoid body contact with laboratory animals and
plants .
 Ensure maximum care when handling and storing carcinogenic
chemicals.
 Stock of fresh animals must be obtained from a reliable source .
infected animals should never be used for any practical

FIRE HAZARDS
Fire is simply heat + light that is evolved during a chemical reaction
called combustion .It relies apon the presence of oxygen from the air
reacting with flammables . The combination of oxygen , fuel and heat
source provides three basic components of combustion which can be
represented as a fire triangle

Fire triangle
If the triangle is incomplete , then fire cannot take pace and therefore
fighting of fire is based on the breaking of the fire triangle e.g. oxygen
can be isolated by use of co2, isolation of fuel is extremely hard
because of wide spread and availability of flammable material but the
risk of fire can be reduced by keeping small quantities of flammables
at a time in stock. Removal of heat source is the most important aspect
of fire fighting e.g. hazards from electric sparks are removed by use of
flame proof fittings and equipments
 SCIENCE LABORATORY TECHNOLOGY
Flammable substances should be stored in safe containers and rooms ,
glass bottles should be stored where sunrays may not concentrate and
careless disposal of naked flames must be discouraged .
There are always two factors that must be considered when about to
fight fire i.e. can the fire be put off ? If it is not possible to put off the
fire , then how can its spread be slowed down or stopped ?

CLASSIFICATION OF FIRE
Classification of fire depends on the type of fuel used by the fire

Class A fire
Caused by Solid organic carbonaceous materials e.g. wood ,paper,
textiles ,etc
The most appropriate fire extinguishers to be used for this type of
fires include ; Water,CO2, foam, dry powder etc

Class B fire
caused by Liquids or easily melted solid organic compounds e.g. petrol,
ethanol , plastics etc . The most appropriate fire extinguishers to be
used for this type of fires include ; CO2 , Foam,, dry powder but not
water

Class C fire
Caused by flammable gases e.g. petroleum gases
The most appropriate fire extinguishers to be used for this type of
fires include ; CO2 , Foam and dry powder

Class D fire
Caused by Metals e.g. Na, Mg etc
The most appropriate fire extinguishers to be used for this type of
fires include ; Dry powder ,C02

Class E fires
This are firs causd by Electric faults '
The most appropriate fire extinguishrs to be used for this type of fires
include ; CO2, dry powder but not water .

Safety precautions for fire hazards

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Every work place must have a fire extinguisher , wide enough exit door
opening outwards or sideways but not inwards , the door should be
marked red and no obstructions should be on the way.
Regular inspection of fire safety should be in any workplace and
records kept. Regular fire drills should be held in which the staff and
the public should be informed and made familiar with the emergency
routine to be followed incase of fire outbreak.There should be a final
point of meeting from the adjoining fire risk areas where everybody
who may have escaped the fire tragedy in the building should assemble
and their names taken so as to easily identify those that could still be
trapped inside the building. These point should be easily accessible
and protected from fire risk

NB/ Incase of fire , the personnel inside the building should be

immediately evacuated from the fire risk area while trying to slow
down the spread of fire , all doors and windows should be closed to rid
off oxygen supply from the room , then call the fire brigades and raise
the alarm for every body to run to the point of meeting where the roll
call will be taken

BASIC SAFETY LABORATORY RULES AND REGULATIONS

(a) Preparing for laboratory work

Before starting to work in a laboratory, familiarize yourself with the
following:
 The hazards of the materials in the lab, as well as appropriate safe
handling, storage and emergency protocols. Read labels and material
safety data sheets (MSDSs) before moving, handling or opening
chemicals. Never use a product from an unlabeled container, and
report missing labels to your supervisor.
 The agents, processes and equipment in the laboratory. If you are
unsure of any aspect of a procedure, check with your supervisor before
proceeding.
 The location and operation of safety and emergency equipment such
as fire extinguishers, eye wash and shower, first aid and spill response
kits, fire alarm pull stations, telephone and emergency exits.
 Emergency spill response procedures for the materials you will
handle
 emergency reporting procedures and telephone numbers
 SCIENCE LABORATORY TECHNOLOGY
 (vi)designated and alternate escape routes

(b) During laboratory work

 Restrict laboratory access to authorized persons only. Children are
not permitted in labs.
 Smoking; eating; drinking; storing food, beverages or tobacco;
applying cosmetics or lip balm and handling contact lenses are not
permitted in laboratories.
 (iii)Wear lab coats (knee length) and safety glasses in laboratories
employing chemicals, biohazards or radioisotopes.
 Open shoes, such as sandals, should never be worn in the lab.
 Tie back or otherwise restrain long hair when working with
chemicals, biohazards, radioisotopes, or moving machinery.
 Keep work places clean and free of unwanted chemicals, biological
specimens, radios, and idle equipment. Avoid leaving reagent bottles,
empty or full, on the floor.
 Work only with materials once you know their flammability,
reactivity, toxicity, safe handling and storage and emergency
procedures.
 (vii)Prepare and maintain a chemical inventory for the lab.
 (viii) Never pipette by mouth; use mechanical transfer devices.
 (ix) Walk, do not run, in the lab.
 (x) Keep exits and passageways clear at all times.
 (xi) Ensure that access to emergency equipment (eyewashes, safety
showers and fire extinguishers) is not blocked.
 (xii) Report accidents and dangerous incidents ("near-misses")
promptly to your supervisor
 (xiii) Wash your hands thoroughly before leaving the laboratory.
 (xiv) Conduct procedures involving the release of volatile toxic or
flammable materials in a chemical fume hood .
 (xv)Perform procedures that liberate infectious bioaerosols in a
biological safety cabinet
 (xvi) Handle all human blood and body fluids as if potentially
infectious

(c) After laboratory work

It is important to Perform a safety check at the end of each experiment
and before leaving the lab. Make sure to:
 SCIENCE LABORATORY TECHNOLOGY
 Turn off gas, water, electricity, vacuum and compression lines and
heating apparatus.
 Return unused materials, equipment and apparatus to their proper
storage locations.
 Label, package and dispose of all waste material properly
 (iv)Remove defective or damaged equipment immediately, and
arrange to have it repaired or replaced .
 Decontaminate any equipment or work areas that may have been in
contact with hazardous materials.
 Leave behind protective clothing (lab coats, gloves, etc.) when
leaving the laboratory
 Close and lock the door to the laboratory if you are the last one to
leave

Routine laboratory safety checks

Even after you have identified and controlled all current risks, it is
critical that you remain open to the possibility that new unexpected
dangers can arise. Carry out weekly inspections on the condition of:
(i) Fire extinguishers
(ii) Emergency wash devices such as eyewashes
(iii) First aid kit contents
(iv)Fume hood and other ventilation devices
(v) Tubing for circulating water, vacuum, gases
(vi) Chemical storage compartments

Also, ensure that fire extinguishers and emergency showers are

inspected, tested and tagged annually.
NB;Working alone in the laboratory is an unsafe practice at any time.
However, if the nature of your work makes it unavoidable, take
measures to ensure that others are aware of your location and have
someone check in with you from time to time, either in person or by
telephone.
Before conducting any work alone in a laboratory go through this
checklist to determine if it is appropriate to proceed:
(i) Is your supervisor aware of your plans?
 (ii) Are there any hazardous experiments involved?
 (iii) Have you reviewed your procedure with your supervisor?
 (iv) Do you have a written operating procedure?
 (v) Are your apparatus and equipment in good working condition?
 SCIENCE LABORATORY TECHNOLOGY
 (vi) Are you trained to carry out the work?
 (vii) Do you have a check-in/check-out procedure?
 (viii) Do you have an emergency contingency?
 (ix) Does your door have a viewing window or other means of
indicating someone is inside?
 (x) Are you aware of the emergency evacuations procedure?
 (xi) Do you have access to a telephone in case of an emergency?
 (xii) Do you have access to a first aid kit?

FIRST AID

First aid is the immediate and temporary treatment given to a victim

of accident or sudden illness using facilities or materials available at
the time before disposal of the victim or casualty to the hospital if
necessary for medical treatment. the objective of first aid is to;
Prevent the conditions from becoming worse while still waiting for
professional medical assistance, to promote recovery and to sustain life
of the victim. The scope of first aid consist of four parts ;
(i) Assessment of the situation- finding out the nature of accident and
deciding quickly or prioritizing your operations with what should be
done first and last so as not to endanger your life or to intensify the
harm on the casualty

(ii)Diagnosis
Knowing what is wrong with the casualty basing on the history of the
case , symptoms and signs . symptoms are details of the casualty’s
sensations gathered from the conscious victim while talking to
him/her while signs are details obtained from complete examination
of unconscious casualty using your own senses to the maximum e.g.
by touching , determining the pulse rate ,body temperature etc
(iii) Treatment
Treatment should be done immediately and appropriately
 SCIENCE LABORATORY TECHNOLOGY
(iv) Disposal to the house for rest or hospital depending on the conditions
of the casualty and nature of accidents or illness. These will mean
calling for an ambulance NB/ but make sure the person you are
sending understood the massage very clearly about the nature of the
accident , number of casualties involved and the extent of damage ,
these person should come back and give the feedback. Incase of fire ,
call the ambulance

QUALITIES OF A GOOD FIRST AIDER

A good first aider
Must be courageous ,
 Should not panic
 Should inspire confidence to the casualty,
 Should think very fast and appropriately and
 Should be cautious not to do more than necessary until professional
help arrives

First aid for Wounds and fractures

A wound is a break in the continuity of the body surface (skin) which
allows escape of blood and entry of microorganism’s .there is many
types of wounds i.e.
(i) Incised wounds – these are caused by a cutting by a sharp
edged object e.g. razorblade or knife or broken glass
(ii) Lacerated wound - caused by rough object e.g. animal claws
or a saw cutting into the skin. Such wounds are normally irregular and
have rough edges and also usually not very deep
(iii) Contused wound - caused by heavy object falling on the part
of the body thus damaging the underlying capillaries and hence
causing bleeding these wounds are normally swollen and open .bruises
normally don’t show breakage on the affected skin surface
(iv) Punctured would - they are caused by a sharp pointed
instrument driven into the body e.g. a nail . these wounds are normally
deep with a narrow opening

General first aid treatment for wounds

 SCIENCE LABORATORY TECHNOLOGY
Try to stop bleeding and treat for shock
Clean the wound with antiseptic
Raise the bleeding part at least higher than the rest of the body
Keep the victim lying down

Fractures
A fracture is the break or crack of the bone caused by direct or
indirect force over the bone . Direct force results in the bone breaking
where the force landed while indirect force cause the bone to break at
another site away from the point of impact .
There are various types of fractures i.e.
(i) simple fracture – the bone breaks and remains under the
skin , usually there is a swelling a the place where the fracture has
occurred
(ii) open fracture - the bone breaks and protrude through the
(iii) complicated fracture
these is when a bone is broken and affect a different organ of the body
e.g. a fracture in the rib can cause injury to the lungs or liver
(iv) compacted fracture
these is when a bone breaks and it is driven into another bone
(v) commuted fracture
these is when the bone break into many smaller pieces within the body
Treatment for fractures

Put the broken part between two pieces of wood and then tie them up
to align the displaced bones
Make the victim lie down comfortably
Give painkillers
Avoid touching or squeezing the broken part and dispose the victim to
the hospital
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Injuries of the eyes

There are three main cause of eye injuries

Foreign bodies e.g. solids and insect
Liquids e.g. acids ,bases and organic solvents
Gases e.g. smokes , fumes etc

For solid foreign particles, never rub the eyes as they can cause further
injuries to the eyes instead use moisten linen or camelhair brush or
feathers to remove it . The movement of the brush should be towards
the final corner of the eye which allows the particle to be removed
easily.
To locate the particle, the lower eyelid is drawn gently and away from
the eye. If the eyes are damaged by direct thermal heat or hot metal , a
few drops of castor oil should be put in the eye . Incase of small metal ,
irrigate with plenty of water
Also incase of liquid and gaseous substances, irrigate with plenty of
water
Burns and scalds
A burn is an injury caused by dry heat e.g. fire , lighting ,hot metal etc
while a scald is an injury caused by moist heat e.g. steam and hot or
corrosive liquids
Burns can be classified into
(i) light burn – these is a burn where there is no general disturbance of
the skin
(ii)first degree burn - these causes the skin to become red and swollen
(iii)second degree burn - causes blisters to open up and release some
fluids
(iv)Third degree burn - these involves the destruction of the superficial
layer of the skin .
 SCIENCE LABORATORY TECHNOLOGY
First aid for burns and scalds

The general first aid treatment depends on the nature of the wound.
It involves treatment for shock,
Relieving the pain by dipping the affected area in cold water
Excluding air and infections due to air by covering the affected part
with clean cloth

Severe burns require immediate treatment for shock, its advisable to

remove the victims clothes so as to expose the area affected. The
exposed wound should be cleaned with an antiseptic and covered with
a sterile bandage and the victim disposed thereafter to the hospital.
For slight thermal burns , irrigate with plenty of water then wash with
a saturated solution of NaHCO3, cover the area with apiece of bandage
soaked in NaHCO3
Dispose the victim to the hospital
For acid, phenol burns and alkali burns, irrigate with plenty of water
preferably in a shower room and remove the clothes from the area as
possible
Bath the affected area with NaHCO3 (for acid and phenol burns ) or
5% NH4CL solution, boric acid or 2% acetic acid (for alkali burns) and
then a wet dress is applied over it .
Phosphorous burn is highly dangerous and only greasy dressings
should be applied , bath the place with 9% CuSO4 solution and
immerse the burnt part in water immediately . Soak in 2% NaHCO3 sol
and afterwards swab with CuSO4 followed by further washing with
NaHCO3
 SCIENCE LABORATORY TECHNOLOGY
Poisoning
Victims of poisoning due to corrosive chemicals will normally show
swollen and stained lips , the victim may vomit , suffocate or
experience shock
Victims of nerve poisons show convulsion, delirium and become
drowsy. The pupil of the eye becomes contracted and the face may
become flashed .
Terms used in first aid

Emetic these are substances given to induce vomiting and to rid the
stomach off the poison e.g. mastard seeds, salt water , soapy water or
form , or raw egg.
Antidote a substance administered to render the poison harmless or
to retard its absorption in the stomach e.g. milk of magnesia for acid
corrosion , vinegar or lemon juice for strong alkalis
Universal antidote

These consist of 2 parts of activated charcoal : 1 part of magnesium

oxide : 1 part of tannic acid mixed together . its best kept dry until
when required .it is given to the victim when the nature of the poison
taken cannot been established
Aperient or laxative

These is a substance given to a victim of poisoning so as to remove the

poison by diarrhea e.g. castor oil ,olive oil etc
Demulcent.
A substance given to a victim after the poison has been removed . it
sooths the inflamed membrane after the victim have been given an
emetic and a laxative to induce vomiting or diarrhea it includes milk,
water , raw white egg etc
General first aid for poisoning
 SCIENCE LABORATORY TECHNOLOGY
(a) gaseous and fuming poisons

Expose the poisoned victim to fresh air don’t let him stand since these will
cause poison to spread faster
Administer artificial respiration immediately if breathing have stopped
Keep the victim warm and quite

(i) halogen gases

apply general treatment
administer artificial respiration if breathing have stopped
allow the victim to inhale NHOH4 solution
treat eyes and skin for halogen burns
take the victim to the hospital

(b) Ingested poisons

Give an antidote e.g. milk of magnesia for strong corrosive acids ,and
vinegar or lemon juice for strong alkalis) immediately if the patient is
still conscious
Emetics e.g.. mustard seed, salt water, soapy water or foam or raw eggs
should not be given if corrosive poisons have been swallowed
For a victim who have ingested alkalis, caustics and concentrated
NH4OH, never give an emetic but give plenty of water to drink
followed by 5% acetic , citric or tartaric acids . give few mills of
mineral oils after every 15 min until four doses are administered . then
give an antidote e.g. milk of magnesia and lastly a demulcent
for victims who have swallowed an acid , don’t give an emetic but first
give plenty of water then an antidote , next give milk or an egg
albumen in cold water then dilute NaHCO3 , keep the victim warm
and dispose him to hospital
For victims who have ingested antimony , arsenic and mercury
compounds and other insecticides, apply the general treatment,, give
an emetic followed by 4g of tartaric acid in 50ml of water (for
antimony compounds), or 60g of MgSO4 in 50ml water and freshly
prepare FeOH (for arsenic) give white of egg or milk or cold water
and finally give strong tea as a stimulant
 SCIENCE LABORATORY TECHNOLOGY
SHOCK
Shock is a condition of severe depression of vital organs of the body
associated with poor blood circulation to the tissues . it normally
accompany severe injuries or emotional upset, it may result from
poisonous chemicals or food , pain , excessive bleeding ,stroke , heart
attack etc
Signs of shock include ; cold or clumsy skin , pale face , rapid or slow
pulse rate , chilled feelings , feelings of nausea or vomiting and shallow
breathing
Shock can kill and its therefore necessary to save life by preventing
shock though correcting the causes for that particular shock as much as
possible.
Its advisable to keep the victim lying down and warm with his head
lower than the trunk, his airways should be open.
Reasure the victim ,and give him tea or coffee if the victim is able to
swallow .then rush him to the hospital or let him have rest
ASPHYXIA
Asphyxia is simply the deficiency of oxygen in blood or the condition
in which there is lack of oxygen in the lungs and these arises from
lack of sufficient oxygen in the surrounding
Asphyxia is caused either by poisoning, drowning, hanging or
strangulation blocked throat or suffocation.
Its symptoms include ; rapid pulse rate which slows down thereafter ,
difficulties in breathing , dizziness and fatigue unconsciousness
swelling of the neck etc
Before treating for asphyxia , its important to first know the cause of
asphyxia and to remove the couse from the victim. Then administer
artificial respiration immediately and if incase the lungs are damaged,
send him to the hospital
ARTIFICIAL RESPIRATION
 SCIENCE LABORATORY TECHNOLOGY
Artificial respiration is simply a method by which oxygen is supplied to a
victim of asphyxia . there are three methods of ;
Mouth to mouth or mouth to nose (kiss of life)
Holger -Nelsen method
Sylvester method

(a) kiss of life

the victim is laid on his back and any debris is cleared from his mouth
or throat.
The head is tilted backwards and the first aider stands firmly with one
leg a head of the other one and then kneels near the victim’s head and
draws in a deep breath. He then pinches the victim’s nose using his
fingers and then tightens his lips over his mouth and blows air
through the victim’s mouth .
When the victim’s chest seems to rise , he removes his mouth to allow
the chest to relax . These process is repeated several times until the
victim improves
These method is however not applicable if the victim have taken
poison, or there are physical injuries on his face and mouth or when
the victim have poor dental hygiene or the mouth is infected with
pathogens.
(b) Holger - Nelsen method
These method is employed when the victim have facial injuries .
The victim is laid on his stomach with his arms across his chest and
head turned to one side
The first aider places his hands below the victims shoulder plate and
presses hard counting up to three times then lift the victim and
lowers him while pressing the fourth and the fifth time. These is
repeated until the victim improves

(c) Sylvester method

The victim is laid facing up and some soft material e.g. mattress is
placed under his shoulder so as to raise him up and allow the head to
drop backwards .
 SCIENCE LABORATORY TECHNOLOGY
The first aider kneels under the victims head facing the victim and
grasps the victims arms at the wrist crossing and pressing them
against the victims lower chest stretching and puling them outwards ,
upwards and backwards as far as possible. These is repeated 15 times
per minute until the victim recovers .
FIRST AID BOX AND ROOM
First aid box is a small portable box containing first aid materials . It’s
normally kept in readily accessible position and should be under
control of responsible personnel

It contains prescribed medical appliances like . first aid manuals,

bandages,,coton wool, plasters , various antidotes , emetics, laxatives
and demulcents ,pain killers , thermometers , forceps ,safety pins etc
First aid room is especially necessary where there is a large staff
working in an institution . It’s preserved and maintained for treating
serious cases . it should be centrally located so as to serve all the
laboratories around . the room should be quite , clean and well
ventilated and under care of a responsible personnel. The equipment’s
in a first aid room include examination couch, dressing table , cold and
hot water sink , medical cabinet oxygen cylinder , sterilizes , accident
report book etc
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER FOUR

LABORATORY WARES
Laboratory wares are apparatus used for general laboratory practice
e.g. for measuring ,containing ,preparation and analysis of solutions
and other chemicals
. These apparatus include -
Glass wares ,
Plastic wares
Ceramic wares
Platinum wares

A. GLASS WARES
Most apparatus found in the laboratory are made from glass. Glass
have several advantages i.e. ;
 They are transparent and easily graduated or calibrated
 They are inert towards most laboratory chemicals except
hydrofluoric , phosphoric acid and some alkalis
 They are bad conductors of heat and thus resist changes in
temperature
 They have smooth but hard surface which is easily cleaned
 SCIENCE LABORATORY TECHNOLOGY
 They can easily be molded into the required shapes
 They are light and easy to carry
They however have some disadvantages i.e. they are fragile and
therefore can break easily.

Classification of glass wares

Glass ware can be classified according to ;
(a) The type of material made from .
(b) The type of work its is purposed for .

(a) Classification according to material

it is made from
There are two types of glass i.e. soft glass and hard glass
Soft glass ( soda glass)
They have high percentage of sodium oxide and low percentage of
silicon oxide, it melts easily when heated and it is used to make
pipettes and glass tubings.

Hard glass (Borosilicate glass)

They have high percentage of silicon oxide and low percentage of
sodium oxide . it also have boron
oxide , they are highly resistant to heat hence do not melt easily. They
are used for making beakers , flasks and most laboratory apparatus
Borosilicate glass have high mechanical strength i.e. they do not beak
easily, fairly inert to chemical have high resistance to thermal shock ,
high melting point and softening point

(b) Classification according to purpose

Under these category, glass ware are classified as
i. General purpose glass ware
ii. Special purpose glass wares
iii. Standard joint glass wares
iv. Volumetric glass wares

General-purpose glass wares

 SCIENCE LABORATORY TECHNOLOGY
These glasswares are not calibrated. They are therefore used for
various jobs that do not require measurements e.g. test tubes , beakers
, flasks etc
Special purpose glass wares
They are designed to perform only specific jobs e.g. density bottles ,
condensers etc
Standard or jointed or quick fit glass wares
They are glasswares with interchangeable ground joints e.g. distilling
flask , soxhlet extractor’s etc. they are suitable for general use in
preparative organic chemistry

Volumetric glass wares

They include burettes , volumetric flasks and pipettes. They are
graduated either to deliver or to contain a specified volume i.e.
EX – to deliver
IN – to contain
The temperature of calibration is also shown is also shown on the
glassware ( usually 20 o c ).
Volumetric glassware are either classified as class A or class B
Class A have been verified to have a tolerance of + or – 0.001 cm3 at
the stated temperature while class B have a tolerance of 0.010 cm3 at
the stated temperature

GLASS WARES
Glass is a product formed by fusion of various oxides which have been
cooled down to rigid conditions by special freezing technique
whereby the liquid glass cools down without crystallization .
Types of glass
Glass is classified according to the parent oxide from which they were
formed . there are two types of glasses
(i) soft glass - soda lime glass or soda glass
(ii) hard glass – resistant glass or borosilicate glass

Effects of heat on glass

Glass is a poor conductor of heat, when its heated , the surface that is
near to the flame undergoes compression strains while the inner
surface undergoes tension strains . When it is removed from the flame,
 SCIENCE LABORATORY TECHNOLOGY
the air around it affects the outside layer fat and it therefore
undergoes tensile strength while the inner layer undergoes
compression strains . i.e. the outer layer cools faster than the inner
layer and hence enforces a strain within the inner layer , these causes
cracking .
To remove strains from glass , the glass is normally put into an
annealing oven at a certain temperature (annealing temperature )
and left for sometime . Eventually the temperature is reduced stepwise
at certain intervals until room temperature is reached . The starting
temperature should always be 5oc higher then the annealing
temperature .
Properties of glass
(a) soda glass or soft glass
 Soda glass melts on a Bunsen burner flame
 Its prone to diventrification (forms crystals easily ) there it needs
careful introduction on a flame
 They are very cheap and readily available in any quantity and size
 Give an intense and persistence yellow sodium flame on heating
 Its smooth on feeling with finger i.e. the edges will feel smooth on
scratching
 When viewed against a source of light , its edge will have a green
tinge
(b) Borosilicate glass
Borosilicate glass is a hard colorless glass with a low coefficient of
expansion it softens at high temperatures
 Can be put to the flame directly and requires less annealing than
soda glass
 ( Its rough on feeling or the edge will feel rough on scratching
 Its expensive than soda glass

Annealing
Annealing is the process of removal of strains from a fixed glass
product it’s a process of cooling down of the glass from its annealing
temperature in a slow , gradual and stepwise manner until the room
temperature is reached .
 SCIENCE LABORATORY TECHNOLOGY
BLOOM
A bloom is a ring formed on a glass product about 3mm from the
point of heating as a result of sodium ions in the glass coming into
contact with sulfur ions in the glass
There are two types of blooms
(a)Temporary bloom
These can be wiped out on cooling
(b)Permanent bloom
These is only removed by chemical means

Diventrification of glass
Diventrification is the process of formation of crystals in glass
(crystallization of glass )
Diventrified glass looks or appears translucent , it’s rough and cannot
be seen through easily .Its cause by ;
(i) Heating of a piece of glass on a hot flame for a very long time .
(ii) Heating a piece of glass and then suddenly introducing it to a cool
condition (rapid cooling of glass)
(iii) Chemical weathering (due to old age ) , the moisture collects on
glass and when it combines with atmospheric C02, it forms carbonic
acid which eats up the soft sodium ions of the glass and the hard ions
becomes crystallized or crystallizing on the surface of the glass
Devintrification can be eradicated by
(i) Pouring strong solution of NaOH on the glass surface and
leaving it on for sometime . These will etch up the ends of the glass
that are sticking out and a smooth glass will be produced , NB these
process may however result in the formation of thin glass
(ii) By fusion – in these case the piece of glass is put in an oven
until it softens out so that the crystals
ticking out of the glass will be fussed into the walls of the glass .these
method is not suitable for volumetric glassware , because when they
are heated to high temperatures , they don’t regain their original size
on cooling .

PLATINUM WARES
Apart from glasswares , platinum forms part of the laboratory wares .
they are used to make crucibles etc.
platinum wares are mixed with small quantities of rhodium , indium
or gold to give them strength.
 SCIENCE LABORATORY TECHNOLOGY
They are inert to most chemical reagents , have high melting point i.e.
1770 o c and have excellent conductance of heat and extremely small
adsorption of water vapor
Platinum crucibles are virtually indispensable for accurate
experimental analysis . They must be used with care because they are
expensive . They should be kept chemically clean and always avoid
reagents in which platinum is soluble e.g. aquaregia , HCL,liquids
mixtures which evolve bromine or iodine, concentrated H2SO4 and
H3PO4

When heating , its base should be above the blue core of the flame
otherwise the y will become brittle and break
Crucibles should be supported on clay triangles when heating
The process of cleaning crucibles is called tarring

PLASTIC WARES
They are generally made from polymers e.g. polythene , polyproprene
polytetrafloroethene .they have excellent chemical resistance at room
temperature , inert to acids , bases and peroxides but they are attacked
by organic solvents
Propylene is hard and more transparent , polytetrafloroethane is
extremely inert and can withstand temperatures greater than 250 o c .
Apparatus made from it are long lasting and more expensive . They
include evaporating dishes, beakers , burettes reagent bottles and
separating funnels.
When cleaning plastic wares , use warm soapy water, chromic acids or
nitric acid . avoid using scrappy objects but instead use warm alcohol
for obstinate dirt

CERAMIC WARES
They have very high mechanical strength and high resistance to
chemical attack . They are however heavy and expensive therefore
limited to making pestle and mortars.
Ceramic wares are cleaned using a detergent solution or abrasive
powder which do not scratch the surface
 SCIENCE LABORATORY TECHNOLOGY
COMMON LABORATORY APPARATUS
1. Spatula- to scoop small amounts of a solid substance and to scrape
something.
2.Glass Funnel- used to channel liquid or fine-grained substances into
containers with a small opening.
3.Stirring/Glass Rod- used to mix chemicals and liquids for laboratory
purposes
4.Thistle Tube- to add liquid to an existing system of apparatus.
5.Dropper/Pasteur Pipette- used to transport a measured volume of
liquid.
6.Volumetric Flask - used to measure one specific volume.
7.Mohr Burette- used to measure the volume of the liquid dispensed.
8.Geissler/Acid Burette- used especially in laboratory procedures for
accurate fluid dispensing and measurement.
9.Volumetric Pipette- a tool for measuring precise volumes of a liquid.
10.Serological Pipette- used in the same way as Mohr pipettes except
all the solution must be forced out in the receiving container to deliver
required volumes.
11.Graduated Cylinder- used to accurately measure the volume of a
liquid.
12.Beaker- Used to hold and heat liquids.
13.Florence Flask - used for heating substances that needs to be heated
evenly.
14.Erlenmeyer flask - used to heat and store liquids.
15.Iodine Flask - used for the wet chemical analysis16.Evaporating
Dish- used to heat and evaporate liquids.17.Porcelain Casserole
18.Watch Glass- used to hold solids when being weighed or
transported.
19.Ignition Tube- primarily used to hold small quantities of substances
which are undergoing direct heating by a Bunsen burner or other heat
source.
20.Porcelain Crucible- used to heat small quantities to very high
temperatures.
21.Crucible Tong– Used to hold the crucible
22.Distilling Flask - used for distillation processes.
23.Condenser- used in distillation
24.Adapter- a device that connects the condenser and the receiving
flask in a distillation process.
 SCIENCE LABORATORY TECHNOLOGY
25.Test Tube- used by chemists to hold, mix, or heat small quantities of
solid or liquid chemicals, especially for qualitative experiments and
assays.
26.Test Tube Rack - is used to hold test tubes while reactions happen in
them or while they are not needed.
27.Iron Stand– used to hold the iron ring and supports.
28.Iron Ring– used to hold or support beakers during experiments
while connected to the iron stand.
29.Tripod– three-legged support equipment used to place above the
bunsen burner in the science lab to heat/boil anything.
30.Burette Clamp- used to fasten glassware into place on a ring stand
31.Clay Triangle- used to hold crucibles when they are being heated.
32.Clamp Holder- used to secure an extension-type utility clamp to a
support stand (or ring stand)
33.Mortar & Pestle- used to crush solids into powders for experiments,
usually to better dissolve the solids.
34.Bunsen Burner- used for heating and exposing items to
flame.35.Alcohol Lamp– Used to heat things.
36.Wing Top/Fish Tail- used to bend glass as it spread out the heat
over a larger area,making it more uniform.
37.Wire Gauze- used to spread heat of a burner flame
38.Cork Borer- tool for cutting a hole in a cork or rubber stopper to
insert glass tubing.
39.Thermometer- used to take temperature of solids, liquids, and gases
40.Desiccators- used for preserving moisture-sensitive items.
41.Weighing Bottle- used when you're making up a standard solution.
42.Triangular File- used for many cuts, such as cutting angles less than
90 degrees.
43.Petri Dish- use to culture cells, which can be bacteria, animal, plant,
or fungus.
44.Spot Plate– Used for observing small amounts of solids.45.Test
Tube brush- used to easily clean the inside of a test tube
46.Pinchcock - used to regulate or close a flexible tube, especially in
laboratory
apparatus.
47.Rubber Aspirator– used for moving air,fluids, etc. by suction
48.Rubber Tubing- is used to connect two openings.
 SCIENCE LABORATORY TECHNOLOGY
49.Separatory Funnel- used in liquid-liquid extractions to separate
( partition) the components of a mixture between two immiscible
solvent phases of different densities.
Cleaning of laboratory wares

Cleaning Basics

It's generally easier to clean glassware if you do it right away. When

detergent is used, it's usually one designed for lab glassware, such as
Liquinox or Alconox. These detergents are preferable to any
dishwashing detergent you might use on dishes at home.

Much of the time, detergent and tap water are neither required nor
desirable. You can rinse the glassware with the proper solvent, then
finish up with a couple of rinses with distilled water, followed by final
rinses with deionized water.

How to Wash Out Common Lab Chemicals

Water Soluble Solutions(e.g., sodium chloride or sucrose

solutions) Rinse 3-4 times with deionized water then put the glassware
away.
Water Insoluble Solutions(e.g., solutions in hexane or
chloroform) Rinse 2-3 times with ethanol or acetone, rinse 3-4 times
with deionized water, then put the glassware away. In some situations
other solvents need to be used for the initial rinse.
Strong Acids(e.g., concentrated HCl or H2SO4) Under the fume
hood, carefully rinse the glassware with copious volumes of tap water.
Rinse 3-4 times with deionized water, then put the glassware away.
Strong Base(e.g., 6M NaOH or concentrated NH4OH) Under the
fume hood, carefully rinse the glassware with copious volumes of tap
water. Rinse 3-4 times with deionized water, then put the glassware
away.
Weak Acids(e.g., acetic acid solutions or dilutions of strong acids
such as 0.1M or 1M HCl or H2SO4) Rinse 3-4 times with deionized
water before putting the glassware away.
Weak Bases(e.g., 0.1M and 1M NaOH and NH4OH) Rinse thoroughly
with tap water to remove the base, then rinse 3-4 times with deionized
water before putting the glassware away.
 SCIENCE LABORATORY TECHNOLOGY
Washing Special Glassware

Glassware Used for Organic Chemistry

Rinse the glassware with the appropriate solvent. Use deionized
water for water-soluble contents. Use ethanol for ethanol-soluble
contents, followed by rinses in deionized water. Rinse with other
solvents as needed, followed by ethanol and finally deionized water. If
the glassware requires scrubbing, scrub with a brush using hot soapy
water, rinse thoroughly with tap water, followed by rinses with
deionized water.
Burets
Wash with hot soapy water, rinse thoroughly with tap water, then
rinse 3-4 times with deionized water. Be sure the final rinses sheet off
of the glass. Burets need to be thoroughly clean to be used for
quantitative labwork.
Pipettes and Volumetric Flasks
In some cases, you may need to soak the glassware overnight in
soapy water. Clean pipets and volumetric flasks using warm soapy
water. The glassware may require scrubbing with a brush. Rinse with
tap water followed by 3-4 rinses with deionized water.
Drying glasswares
It is inadvisable to dry glassware with a paper towel or forced air
since this can introduce fibers or impurities that can contaminate the
solution. Normally you can allow glassware to air dry on the shelf.
Otherwise, if you are adding water to the glassware, it is fine to leave it
wet (unless it will affect the concentration of the final solution). If the
solvent will be ether, you can rinse the glassware with
ethanol or acetone to remove the water, then rinse with the final
solution to remove the alcohol or acetone.
Rinsing with Reagent
If water will affect the concentration of the final solution, triple rinse
the glassware with the solution.
(ii) Drying Glassware
If glassware is to be used immediately after washing and must be dry,
rinse it 2-3 times with acetone. This will remove any water and will
evaporate quickly. While it's not a great idea to blow air into glassware
to dry it, sometimes you can apply a vacuum to evaporate the solvent.

Additional Notes
 SCIENCE LABORATORY TECHNOLOGY
Remove stoppers and stopcocks when they are not in use. Otherwise
they may 'freeze' in place.
You can degrease ground glass joints by wiping them with a lint-free
towel soaked with ether or acetone. Wear gloves and avoid breathing
the fumes.
The deionized water rinse should form a smooth sheet when poured
through clean glassware. If this sheeting action is not seen, more
aggressive cleaning methods may be needed.
Cleaning Plastic Laboratory Ware
Cleaning laboratory plasticware depends on the type and properties of
the plastic.
Temperature in the form of extreme heat or cold affect flexability and
strength.
Chemicals such as lubricants and oil cause cracking, and prolonged use
of oxidizing agents cause brittleness and breakage.
Laboratory ware made with glass, quartz, polyethylene and
polypropylene are subject to interaction between container and
sample,or with reagents and standards and can give incorrect results.
However, polyolefins and fluorinated hydrocarbons have excellent
resistance to high temperatures and chemical attack.
They have wettable surfaces and are easy to clean.

PFA Hydrocarbons
PFA has now become the plastic of choice for making laboratory
plasticware. It has the advantage of being formed by injection blow
moulding. It therefore has high transparency and ultrasmoothe
surfaces that do not interact with analytes, reagents or standards. It
has excellent resistance to conc acids, alcohols, bases,
aliphatic/aromatic/halogenated hydrocarbons, ketones, mineral and
vegetable oils. It is ideal for trace analysis in low concentration
determinations in ng/g and pg/g.

Cleaning methods
Acid bath
Immerse plasticware into a 1M nitric acid and allow to soak overnight
for mild contamination. Keep in bath for about one week for heavy
contamination. The cleaned plasticware is then taken out off the bath
and rinsed with distilled water and put to dry. A rinse with acetone or
placement in a glassware dryer at low temperature can be used.
 SCIENCE LABORATORY TECHNOLOGY
Note: Plastics tend to float when put into the cleaning solution and can
be sunk to the bottom using a pair of tongs
An acid bath is a container containing acid in which the plasticware is
placed and kept for some time until clean. The container should be
made of moulded plastic or pyrex glass with a loose fitting lid. The size
and shape may vary according to wash load and need.

Autoclave
Certain chemical contaminants on plasticware can be baked onto the
plastic at autoclave temperatures. Rinse thoroughly with distilled water
before autoclaving.
Autoclave within the tolerated temperature range of the plastic being
sterilized. Remove any stoppers, caps or fittings before autoclaving.
Plastic containers such as vials, sample tubes and bottles should be
autoclaved with their closures disengaged to avoid deformation.
Note: Nylon, polyurethane, polystyrene, polyvinyl chloride (PVC),
Acrylic, LPDE and HPDE must not be autoclaved under any condition.

CHAPTER FIVE
 SCIENCE LABORATORY TECHNOLOGY

LABORATORY CHEMICALS AND

REAGENTS
All chemicals and reagents in the laboratory should be regarded as
dangerous and therefore an orderly system must be designed to
handle , store ,prepare and dispose them away after use, chemical
store should be designed in such a way that all hazards associated
with the stored chemical are minimized .

Requirements of a chemical store

laboratory stores should be situated in the ground floor with an
emergency door opening on the outwards. It must be well ventilated
and specious enough to provide good clearance. It should have all the
necessary safety measures put in place e.g. fire extinguishers , shower
rooms , protective clothing and first aid facilities . it should be made of
fireproof materials and its shelves should be strong enough to be able
to withstand the weight of chemicals stored on them. It should have a
ladder and trolleys for reaching chemicals placed on higher shelves
and transporting them to and from the store . Smoking and fire
sources near the store should be discouraged

Requirements of a prep room

Prep rooms should be located at a central place for easy access by the
laboratory personnel. Entrance into a prep room should be restricted
to those persons whose presence is absolutely necessary . It should be
supplied with all main laboratory services e.g. water , gas and
electricity and fume chamber , it should also have good supply of
distilled water and atleast one large sink and a working table with a
cupboard , drawers and shelves for storage of apparatus and reagents
the prep room should always be kept clean and orderly . the purity of
the chemical used should not be doubted and if doubted it should not
be used .Prep room should have enough burettes , pipettes , volumetric
flasks etc to enable bulk preparation of reagents

LABORATORY REAGENTS
There are five main grades of laboratory chemical i.e.
(i) Aristar
 SCIENCE LABORATORY TECHNOLOGY
These are the purest grade of laboratory chemicals that is
commercially available . They usually carry full specification which
includes
The assay of the chemical
The molecular mass
The maximum and minimum limit of impurities
The physical constants e.g. the boiling and melting point
these grade is very expensive and mostly used for research purposes
(ii) Analar
These are the second purest chemicals available and it’s the
internationally recognized standard for laboratory chemicals . infact
some of them are used for standardizing other laboratory chemicals
They also carry full specification which may include;
 The assay
 The molecular weight
 Maximum and minimum unit of impurities
 Physical constants
(iii) General purpose reagents (GPR)
They are the third purest grades of laboratory chemicals and are used
for general analytical work , they also carry full specifications
(iv) Laboratory reagent
They are intermediate in purity between GPR and technical grade .
They sometime carry full specifications
v) Technical grade
these are reagents that are carefully selected for specific jobs and they
are subject to analytical control. They are mostly used for specialized
technical analysis , they carry no specification

SAFE STORAGE OF LABORATORY CHEMICALS

All chemicals prepared in the laboratory must be labeled . Labels alert
people to the dangers of the product and basic safety precautions.
label must contain the following information:
 Product name
 Information for the safe handling of the product
 Hazard symbols
 Risk phrases (words that describe the main hazards of the product)
 First aid measures (what to do in an emergency)
NB; for chemicals samples prepared in the lab , they must have the
following additional details
 SCIENCE LABORATORY TECHNOLOGY
 Date of preparation.
 Concentration prepared .
 Name of of the person who prepared it.
The singe largest chemical problem facing the laboratories is chemical
storage , handling and disposal. These is mostly due the uncontrolled
purchase of large chemical stocks and poor chemical inventory
management. The need for planning chemical stock and minimizing
chemical wastage is indispensable. Chemical disposal is also a
necessary part of laboratory activity.
Waste resulting from laboratories create serious problems to the air ,
water which can have negative impact to both animals and plants if
they are not properly disposed away . Proper methods must therefore
devised and employed for final disposal of laboratory waste without
endangering the personnel or polluting the environment
The following are steps that if carefully followed will help to reduce the
amount of chemical wastages and also enhance their disposal.
 Maintain upto –date inventory- these will eliminate buying execs or
unneeded chemicals
 Purchase chemicals carefully- purchase smaller size packages of
chemicals that is only enough for the next 2 year
 Date label your chemical and only by from chemical suppliers that
label their chemicals, these will help in determining the expiry of the
chemicals
 Use older stock first before they decompose
 provide good climate control in your store room i.e. the store should
be cool and dry so as to avoid deterioration of chemicals
 Ventilate the store room
 Label all chemicals and laboratory solutions
 Prepare only enough solutions for immediate intended use
 Never store chemicals or solutions in‘ home made‘ containers or
bottles, These will lead to shorter shelf life of laboratory chemicals and
these bottles may not be compatible with the chemicals stored in them
 Store hygroscopic and deliquescent chemicals in chemically safe
bags
 Follow good laboratory practice , never allow students to place
chemical back into the reagent bottles because these can result into
contamination
 SCIENCE LABORATORY TECHNOLOGY
 Never accept donation of chemicals because most donations are of
unknown age and unknown purity, instead by your own fresh
chemicals for best results and long shelf life
 Microscale your lab by only preparing the exact quantity of chemicals
you can handle and dispos
 Dispose of chemicals immediately after they are generated
 Keep waste solutions from every lab separate and do not mix them

General Storage Guidelines

 Do not block access to emergency safety equipment such as fire
extinguishers, eyewashes, showers, first aid kits or utility controls such
as breaker boxes or gas shut-off valves
 Avoid blocking exits or normal paths of travel: keep hallways,
walkways and stairs clear of chemicals, boxes, equipment and shelf
projections
 Ensure that the weight of stored material does not exceed the load-
bearing capacity of shelves or cabinets
 Ensure that wall-mounted shelving has heavy-duty brackets and
supports and is attached to studs or solid blocking. Regularly inspect
clamps, supports, shelf brackets and other shelving hardware
 Arrange items so that they do not overhang or project beyond the
edges of shelves or counter tops
 Do not stack materials so high that stability is compromised
 Leave a minimum of 18 inches (45.7 cm) of clearance between
sprinkler heads and the top of storage
 Use a safety step or stepladder to access higher items; never stand on
a stool or a chair
 Ergonomics
 Store frequently used items between knee and shoulder height
 Store heavy objects on lower shelves

Chemical Storage
 Store hazardous chemicals in an area that is accessible only to
authorized laboratory workers
 Minimize quantities and container sizes kept in the lab
 Do not store chemicals in aisles, under sinks or on floors, desks or
bench tops
 Store chemicals away from sources of heat (e.g., ovens or steam
pipes) and direct sunlight
 SCIENCE LABORATORY TECHNOLOGY
 Never stack bottles on top of each other
 Do not store chemicals above eye level/shoulder height
 Store larger containers on lower shelves
 Store liquids inside chemically-resistant secondary containers (such
as trays or tubs) that are large enough to hold spills
 Store chemicals inside closable cabinets or on sturdy shelving that
has 12.7 mm-19 mm (½ - ¾ inch) edge guards to prevent containers
from falling
 Ensure that chemicals cannot fall off the rear of shelves
 Store chemicals based on compatibility and not in alphabetical order
If a chemical presents more than one hazard, segregate according to
the primary hazard
 Designate specific storage areas for each class of chemical, and
return reagents to those locations after each use
 Store volatile toxic and odorous chemicals in a way that prevents
release of vapours (e.g., inside closed secondary containers, ventilated
cabinets, paraffin sealing)
 Store flammables requiring refrigeration in explosion-safe or lab-safe
refrigerators
 Label reactive or unstable chemicals (e.g., ethers) with the date of
receipt and the date opened
 Inspect chemicals weekly for signs of deterioration and for label
integrity
 Dispose of unwanted chemicals promptly through the Waste
Management Program
 Keep inventory records of chemicals, and update annually

HANDLING, STORAGE AND DISPOSAL OF LABORATORY

CHEMICALS

(a) Fuming acids and bases e.g. HCL, HNO3 ,NH4OH

Storage ;Protect container against physical damage ,Separate from
metallic powders , carbides , sulfides organic acids and combustibles
and other oxidizing compounds e.g.. Cl. Br I etc. Provide good
ventilation and away from direct sunlight
 SCIENCE LABORATORY TECHNOLOGY
Disposal;by dilutingwith alot of water and flushing out or
neutralization and flashing out
(b) Flammable liquids e.g. acetone ,petrol, di ethylether
storage:Protect container against physical damage, outside storage is
most preferable , store in cool well ventilated room away from any
sources of ignition and direct sunlight . isolate from other combustible
materials . place switches outside the store and use vapor proof bulbs
for lighting
disposal:Place in shallow metal trays and place in an open area where
they are left to evaporate or simply burn them by igniting them from a
safe distance

(c) Light sensitive materials e.g. silver bromide and hydrogen

peroxide
storage:Store in dark brown bottles with tightly fitting glass stoppers
and place them in dark cupboards or dark rooms
(d)Poisonous chemicals e.g. arsenic , cyanides etc
storage:Store under lock and key and keep details of the people
issued in a poison record book
disposal:Burning in an incinerator or by burrying
(e) Deliquescent and hygroscopic chemicals e.g. NaOH,
KOH,phenols
storage : Store in airtight containers . do not store NaOH and KOH in
glass stoppered bottles
Handling and preparation method:Use gloves. Dissolve NaOH and
KOH little by little in large amount of water to avoid overheating.
Disposal: By dilution and flushing out or neutralization and flushing
out.
(f) Radioactive substances
storage :Store in sealed lead container and placed in thick wood or
concrete and kept in a locked cupboard situated away from the main
building
methods of handling:Use proper protective clothing’s, radiation
indicators and gadgets e.g. pocket dosimeters and GM tubes. Regular
medical checkup is important.Disposal: Radioactive substaces are
ussually collected Its in stores then periodically shipped to disposal
centers.
 SCIENCE LABORATORY TECHNOLOGY
Care should be taken not to dispose in normal waste disposal systems.
They should be properly segregated and handled according to the
recommendation by experts
(g) Oxidizing agents e.g. chlorates , permanganates and
chromates Storage: Should be kept away from combustible materials
and in separate stores i.e. never keep both oxidizing and reducing
agents together
Disposal:They should be chemically converted into safe substances
which have low oxidation power by reacting them wit sodium
thiosulfate then Diluting and flushing out into the sewer system
( dispose by reacting with any reducing agent)
Handling:Use protective clothing’s when handling them . Be extra
careful when reacting them together
(h) Reducing agents e.g. alkali and alkaline earth metal ions
Storage: Should be kept away from combustible materials and in
separate stores i.e. never keep both oxidizing and reducing agents
together
Disposal:They should also be safely reacted with substances that will
destroy their reducing power e.g. sodium hypochlorite (dispose by
reacting with any oxidizing agent)Handling:Use protective clothing’s
when handling them . Be extra careful when reacting them togethe
(i) Corrosive acids e.g. H2SO4 , HNO3 H3PO4
Handling:Use protective clothing’s. Mix acid to water slowly through
the sides of the container to avoid splashing and not water to acid .
never mouth pipette
storage:Store in the lower shelves in closed bottles . use trolleys to
transport them,
Disposal:Diluting and flushing or neutralizing and flushing out into
the sewer system
(j) Alkali and alkaline earth metals compounds e.g. Na , K
,and Li, Ca, Mg etc
Handling:Use protective clothing . dissolve small amounts in water at
a time to reduce heat produce
Storage:Never store alkali metals in ground stoppered bottles because
they attack glass . store under paraffin and never expose them to air. Ca
and Mg can be store in ordinary closed containers .
Disposal:Dissolve in enough alcohol and leave them to stand until
bubbles cease to appear , add water and dispose into the sink with
 SCIENCE LABORATORY TECHNOLOGY
running tap water. Cal and Mg can be disposed by letting them react
with cold water then flushed through the sink.
(k) Explosive chemicals e.g. picric acid and related
compounds e.g. bouin‘s solution
Storage:Dry picric acid is very explosive , it react with metals to form
explosive metal picrates which are highly sensitive to detonation . it
explodes in the air and couse fire . Should be stored under water in
ground neck glass stoppered bottles. Avoid friction,shock or sudden
heating which can initiate an explosion. There containers should not
have metal caps since it vigorously react with it
Disposal;Only trained personnel (e.g. bomb squad) are allowed to
dispose picric since its very explosive Apply physical shock other
chemicals e.g. peroxides and ether are also shock sensitive and
therefore can be disposed in the same way and then transported to safe
disposal area.
(l)Phosphorous
Handling:Use gloves and chemical resistant apron.
Storage:Highly reactive and flammable also poisonous . Reacts with
air under controlled conditions to phosphoric acid and can
spontaneously ignite in air where it is converted to phosphorous
pentoxide .its therefore stored under water.
Disposal:Small quantities can be disposed by allowing them to ignite in
air in a controlled condition or letting it to react with 0.5 M H2SO4 and
KBr while stirring. Then add sodium metabisulfate and flush out.
(m)Hydrofluoric acid
Storage:Attacks glass hence they are stored in polythene bags or
plastic containers.
Disposal:Neutralization or dilution and flushing into the sewer.
(n) Non combustible organic solvents
storage: store safely in closed containers
disposal:Dissolve in flammable solvent e.g. alcohol or benzene and
place the solution in an open pan and burn them with due care
(o) Mercury metalDisposal:Only disposed off by returning to the
supplier for recycling . It should never be disposed of by burring,
burning or placing or through drainage system
(p)Heavy metals and their salts e.g. lead , antimony, cadmium,
chromium, cobalt and nickel
Storage: Are very soluble in water , extremely toxic and accumulates
in the body tissue.
 SCIENCE LABORATORY TECHNOLOGY
Disposal:They must be first converted into stable insoluble salts by
reacting them with sulfide or silicate ions then they can be buried in
approved land fills for disposal of hazardous waste

(q) Halogenated solvents

They are volatile, insoluble in water and cannot be burnt except in high
temperature incinerators
Disposal:Pour a shallow layer of the substance into the pan and place
it under efficient fume hood in appropriate outdoor site then allow the
material to simply evaporate.
(r)Inorganic sulfides:
They release highly toxic hydrogen sulfide gas on treatment with an
acid
Disposal:They should first be oxidized into sulfates using hypochlortes
as an oxidizing agent which forms a more stable salt . these can then
be flushed throughthe sink.
(s) Inorganic and organic peroxides
They are strong oxidizing agents, , flammable and explosive, sensitive
to heat , friction or contact with combustible materials.they are stored
in dark . sodium peroxide is stored in sealed containers to avoid
reaction with air
Disposal:Hydrogen peroxide is destroyed by reducing with sodium
metabisulfite or diluting it with water and flushing out. Organic
peroxides e.g. benzyl peroxide are reacted with bases which will cleave
between the two joined oxygen atoms and form sodium benzoate which
are insoluble in water and less harmful.
(t) Volatile hydrocarbons e.g. alcohols, ketones and esters
They have low toxicity but very flammable
Disposal:Remove any source of ignition and place on a shallow pan in a
fume hood and allow natural air to pass through it so as to evaporate.
(u)Non-volatile hydrocarbons
They are non flammable and easily converted to less toxic materials
Dsposal:Disposed by licenced hazardous waste company and by
burning.
(v)Silver compounds
They are very expensive but can be recovered after use by dissolving
the silver ions in the solution with NaOH and heating. then adding
sucrose . The sucrose will hydrolyze in a strong base to the
 SCIENCE LABORATORY TECHNOLOGY
monosacharides i.e. fructose and glucose which will reduce silver ions
to silver metal which will form as gray precipitated
Disposal:Disposed by precipitation into an insoluble silver chloride
using Nacl and disposed in a landfill.
(w)Microbiological cultures
Handling:Use protective clothing and sterilized equipments .
Storage:Never keep in food refrigerators or mix with other
instruments used for mouth pipeting.
Disposal :Sterilize instruments in autoclave or dispose them by
incineration.
(x) Carcasse
handling:Use sterilized equipments and wear protective clothing’s.
Storage:Keep in specimen bottles ,special polythene bags and under a
suitable preservative e.g. formalin.
Disposal:can be disposed by Burying ,burning or incineration.

SEGREGATION OF NON- COMPATIBLE CHEMICALS

Certain hazardous combinations can occur between incompatible
chemicals . it is therefore important to ensure that incompatible
chemicals are not stored in close proximity to each other.

Examples of incompatible combinations of some commonly used

chemicals.
CHEMICAL Keep from contact with:
Acetic Acid Chromic acid, nitric acid, hydroxyl
compounds, perchloric acid, peroxides, permanganate
Acetylene Chlorine, bromine, copper, fluorine, silver, mercury
Alkali Metals Water, chlorinated hydrocarbons, carbon
dioxide,halogens
Ammonia, Anhydrous Mercury, chlorine, calcium hypochlorite,
iodine ,bromine, hydrofluoric acid
Ammonium Nitrate Acids, metal powders, flammable liquids,
chlorates nitrites, sulphur, finely divided combustible materials
Aniline Nitric acid, hydrogen peroxide
Bromine Same as chlorine
Carbon, Activated Calcium hypochlorite, all oxidizing agents
Chlorates Ammonium salts, acids, metal powders, sulphur, finely
divided combustible materials
 SCIENCE LABORATORY TECHNOLOGY
Chromic Acid Acetic acid, naphthalene, camphor, glycerin,
turpentine alcohol, flammable liquids
Chlorine Ammonia, acetylene, butadiene, butane, methane,
propane , hydrogen, sodium carbide, turpentine, benzene, finely
divided metals
Copper Acetylene, hydrogen peroxide
Flammable Liquids Ammonium nitrate, inorganic acids, hydrogen
peroxide, sodium peroxide, halogens
Hydrocarbons Fluorine, chlorine, bromine, chromic acid,
sodium peroxide
Hydrofluoric Acid Anhydrous ammonia, ammonium hydroxide
Hydrogen Peroxide Copper, chromium, iron, most metals or their
salts, alcohols, acetone, aniline, nitromethane, flammable liquids,
oxidizing gases
Hydrogen Sulphide Fuming nitric acid, oxidizing gases
Iodine Acetylene, ammonia (aqueous or anhydrous), hydrogen
Mercury Acetylene, fulminic acid, ammonia
Nitric Acid Acetic acid, aniline, chromic acid, hydrocyanic acid,
hydrogen sulphide, flammable liquids, flammable gases
Oxalic Acid Silver, mercury
Perchloric Acid Acetic anhydride, bismuth and its alloys, organic
materials
Potassium Carbon tetrachloride, carbon dioxide, water
Potassium Chlorate Sulphuric and other acids
Potassium Permanganate Glycerin, ethylene glycol,
benzaldehyde, sulphuric acid

LABORATORY SOLUTION PREPARATION

Many of the reagents used in science are in the form of solutions which
need to be purchased or prepared. For many purposes,the exact value
of concentration is not critical; in other cases,the concentration of the
solution and its method of preparation must be as accurate as possible.
Professional quality solutions are possible when high quality and fresh
chemicals and solvents are used, and meticulous procedures are
followed.

Basic Terms and Concepts of Preparing Solutions.

 SCIENCE LABORATORY TECHNOLOGY
Normality: A concentration unit (N); defined as the number of
equivalents of solute per liter of solution. (e.g., 1 M H2SO4 = 2 N
H2SO4)
Saturated Solution: A solution that contains the maximum amount
of a particular solute that will dissolve at that temperature.
Solute: The substance which is dissolved, or has gone into solution
(typically a solid).
Solution: A uniform homogeneous mixture of two or more
substances. The individual substances may be present in varying
amounts.
Solvent: The substance which does the dissolving (typically a liquid,
such as water or alcohol). Must be greater than 50% of the solution.
Standard Solution: A very precise solution, usually to 3–4
significant figures, used in quantitative analysis or an analytical
procedure.
Supersaturated Solution: A solution that contains more solute than
equilibrium conditions allow; it is unstable and the solute may
precipitate upon slight agitation or addition of a single crystal.
Molarity
The most common unit of solution concentration is molarity (M). The
molarity of a solution is defined as the number of moles of solute per
one liter of solution. Note that the unit ofvolume for molarity is liters,
not milliliters or some other unit.
Also note that one liter of solution contains both the solute and the
solvent. Molarity, therefore, is a ratio between moles of solute and
liters of solution. To prepare laboratory solutions,usually a given
volume and molarity are required. To determine molarity, the formula
weight or molar mass of the solute is needed. The following examples
illustrate the calculations for preparing solutions.
If starting with a solid, use the following procedur
• Determine the mass in grams of one mole of solute, the molar mass,
MMs.
• Decide volume of solution required, in liters, V.
• Decide molarity of solution required, M.
• Calculate grams of solute (gs) required using equation 1. eq. 1. gs =
MMs x M x V
• Example: Prepare 800 mL of 2 M sodium chloride.
(MMNaCl = 58.45 g/mol)
gNaCl = 58.45 g/mol x 2 mol/L x 0.8 L
 SCIENCE LABORATORY TECHNOLOGY
gNaCl = 93.52 g NaCl
Dissolve 93.52 g of NaCl in about 400 mL of distilled water, then add
more water until final volume is 800 mL.
If starting with a solution or liquid reagent:
• When diluting more concentrated solutions, decide what volume (V2)
and molarity (M2) the final solution should be.Volume can be
expressed in liters or milliliters.
Determine molarity (M1) of starting, more concentrated solution.
• Calculate volume of starting solution (V1) required using equation 2.
Note: V1 must be in the same units as V2. eq. 2. M1V1 = M2V2
• Example: Prepare 100 mL of 1.0 M hydrochloric acid from
concentrated (12.1 M) hydrochloric acid.
M1V1 = M2V2
(12.1 M)(V1) = (1.0 M)(100 mL
V1 = 8.26 mL conc. HCl

Add 8.26 mL of concentrated HCl to about 50 mL of distilled water,

stir, then add water up to 100mL.
Percent Solutions
Mass percent solutions
are defined based on the grams of solute per 100 grams of solution.
Example: 20 g of sodium chloride in 100 g of solution is a 20% by mass
solution.
Volume percent solutions
are defined as milliliters of solute per 100 mL of solution.
Example: 10 mL of ethyl alcohol plus 90 mL of H2O (making approx.
100 mL of solution) is a 10% by volume solution.
Mass-volume percent solutions
are also very common. These solutions are indicated by w/v% and are
defined as the grams of solute per 100 milliliters of solution.
Example: 1 g of phenolphthalein in 100 mL of 95% ethyl alcohol is a 1
w/v% solution

Conversion Between Percent Solutions

You may wish to convert mass percent to volume percent or vice versa.
If so, follow this procedure:
A 10% by mass solution of ethyl alcohol in water contains 10 g of ethyl
alcohol and 90 g of water.
 SCIENCE LABORATORY TECHNOLOGY
1. The formula for determining the volume of the component(ethyl
alcohol in our example) is:
mass of ethyl alcohol
Volume = ——————————
density of ethyl alcohol
2. Determine the volume of the total solution by dividing the mass of
the solution by the density of the solution
3. Determine the percent by volume by dividing the volume of the
component by the volume of the solution.
Let`s solve 1, 2, and 3 above as follows:
1. Mass of ethyl alcohol = 10 g (given
Density of ethyl alcohol = 0.794 g/mL (from handbook
mass
Volume = ———
density
10 g
Volume of ethyl alcohol =——— = 12.6 m
0.794 g/mL

2. Mass of solution = 100 g (given)

Density of solution (10% ethyl alcohol) = 0.983 g/mL(from handbook)
100 g
Volume of solution = ————— =101.8ml
0.983 g/mL
3. Volume percent of solution
Percent = volume of ethyl alcohol
total volume of solution
12.6
= ———
101.8
= 12.4%

Calculating Molarity from Percent Solution

To determine the molarity of a mass percent solution,the density of the
solution is required. Use the following procedure:
1. Determine the mass of solution by multiplying the volume of the
solution by the density of the solution
mass = volume x density
 SCIENCE LABORATORY TECHNOLOGY
2. Determine concentration in percent by mass of the solute in
solution. Change to the decimal equivalent.
3. Calculate the molar mass of the compound, MM.
4. Multiply mass (step 1) by mass % (step 2) and divide by molecular
mass
(step 3) to find the number of moles present in the whole solution.
5. Divide the number of moles (step 4) by the volume in liters of the
solution to find the molarity of the solution.

Example: Determine molarity of 37.2% hydrochloric acid (density 1.19

g/mL)
1. Mass of solution = 1,000 mL x 1.19 g/mL = 1,190
2. Mass % = 37.2 % = 0.372
3. Molar mass of hydrochloric acid = 36.4 g/mo
4. mass x mass %
MMHCl
1,190 g x 0.372
= ————————
36.4 g/mol
= 12.1 moles
5. Molarity = moles/liters
= 12.1
moles/1 liter = 12.1 M
PREPARATION OF SIDE BENCH REAGENTS
Ammonium acetate.
Dissolve 231 g of the salt in 1 liter of water. (3M)
Ammonium carbonate.
The commercial salt is a mixture of bicarbonate and carbamate.
Dissolve 192 g of the salt in a mixture of 140ml conc. ammonia solution
and water to make up 1 liter of solution. (2M)
Ammonium chloride
dissolve 169 g of the salt in 1 liter of solution (3M).
Ammonium nitrate
dissolve 80 g of the salt in 1 liter of water. (1M)
Ammonium oxalate
dissolve 71 g of the crystaline salt in 1 liter of water.(0.5M)
Ammonium sulphatedissolve 132 g of the salt in 1 liter of water.
(1M)
Ammonium sulphide
 SCIENCE LABORATORY TECHNOLOGY
use the yellow commercial product
Ammonium thiocyanate
Dissolve38 g of the salt in 1 liter of water (0.5M)
Barium chloride
dissolve 61 g of the salt in 1liter of water.(0.25M)
Bromine watera saturated aqueous solution is prepared by shaking
35g or 11ml liquid bromine with water. Add more bromine if necessary
to a slight excess.
Calcium chloride(0.25M)
dissolve 55 g of the hydrated salt in 1 liter of water
Calcium sulphate (0.015M)
shake 3 g of the salt with 1 liter of water,
filter and decant the saturatrd solution after several hours
Chlorine water
saturate 250ml of water with chlorine.the chlorine may be prepared by
dropping conc. HCl upon KmnO4. Preserve in a dark coloured bottle.
(6.8g/l)
Cobalt nitrate(0.15M)
dissolve 44 g of the salt in 1 liter of water.
Copper sulphate
dissolve 125 g of the salt in 1 liter of water containing 3ml of conc.
Sulphuric acid.(0.5M)
Ferric chloride
dissolve 135 g of the hydrated salt in 1 liter of water containing 20ml of
conc. HCl (0.5M)
Ferrous sulphate
dissolve 140 g of the salt in 1 liter of water containing 7 ml of conc.
Sulphuric acid (0.5M)
Hydrogen sulphide
H2S generated from a Kipps apparatus ( ~42g/l)
Iodine solutiondissolve 12.7 g of iodine in a solution of 20g of pure
KI in 30 ml of water, and dilute to 1 liter with water.(0.05m)
Lead acetatedissolve 95 g of the salt in 1 liter of water (0.25M)
Magnesium sulphate
dissolve 62 g of the salt in 1 liter of water (0.25M)
Mercuric chloridedissolve 27 g of the salt in 1 liter of water (0.1M
Potassium chromate
dissolve 49g of the salt in 1 liter of water (0.25M)
Potassium ferricyanide
 SCIENCE LABORATORY TECHNOLOGY
dissolve 55 g of the salt in 1 liter of water (0.25M)
Potassium ferrocyanide
dissolve 53 g of the salt in 1 liter of water (0.125M)
Potassium iodide
dissolve 83 g of the salt in 1 liter of water (0.5M)
Potassium permanganate
dissolve 3.2g of the salt in hot water, dilute to 1 liter, and filter through
glass wool (0.01M)
Potassium thiocyanatedissolve 49g of the salt in 1 liter of water
(0.25M)
Silver nitrate
dissolve 17g of the salt in 1 liter of water (0.1M)
Silver sulphate
dissolve 8g of the salt in 1 liter of water. this is nearly a saturated
solution
(0.025M)
Sodium acetatedissolve 408g of the crystaline salt in 1 liter of water
(3M)
Sodium carbonatedissolve 430g of the crystaline salt in 1 liter of
water (1.5M)
Disodium hydrogen phosphate
dissolve 120g of the salt in 1 liter of water 0.34M)
Stannous chloride
dissolve with heat 56g of the salt in 100ml conc. HCl and dilute to 1
liter. Keep a few pieces of granulated zinc in the stored solution to
avoid oxidation. (0.25M)
Thiourea10g in 100ml of water (10%)
Titan yellow
dissolve 1g in 100ml of water (1%)

Zinc nitrate
dissolve 150g of the salt in 1 liter of water (0.5m

Aluminon
Use a 0.1% aqueous solution.
Alizarin
Make a saturated solution in ethanol.
Alizarin-S
Sodium alizarin sulphate
 SCIENCE LABORATORY TECHNOLOGY
Use a 0.1% aqueous solution.
1-Amino-1-Naphthol-4-sulfonic aci
Dissolve 0.2 g in 195 ml of sodium bisulphite solution (3 in20) and 5
ml of anhydrous sodium sulphite solution (1 in 5) and filter if
necessary. Stopper and store in a cool dark place. Use within 10 days.
Ammoniacal silver nitrate. (0.1M)
Add conc ammonia to bench silver nitrate until the initially formed ppt.
Just disappears.
Ammonium citrate, lead free
Dissolve 40g of citric acid in 100 ml of water and make alkaline to
phenol red with ammonium hydroxide. Remove lead by shaking with
small portions of dithizone extraction solution in chloroform until the
dithizone solution
retains its original green color.Discard the extraction solution.
Ammonium mercurithiocyanate
Dissolve 9g of ammonium thiocyanate and 8g of mercuric chloride in
100ml of water.
Ammonium metavanadate
Dissolve 2.5g of NH4VO3 in 500 ml of boiling water, cool, and add 20
ml of nitric acid. Dilute to 1 liter and store in polythene bottle.
Ammonium molybdate reagent
Method A: Dissolve 45 gms. of the commercial salt or 40 gms. of pure
molybdenum trioxidein a mixture of 70 ml.of concentrated ammonia
solution and 140 ml. of water; when solution is complete, add it very
slowly and with vigorous stirring to a mixture of 250ml.of concentrated
nitric acid and 500ml.of water, and dilute to 1 litre.Allow to stand 1 to 2
days and decant and use the clear solution.
Method B: Dissolve 45 gms. of pure commercial ammonium molybdate
in mixture of 40ml. concentrated ammonia solution and 60 ml. of
water, add 120gms. of ammonium nitrate and dilute to a litre with
water.Note: The alkaline solution of ammonium molybdate keeps
better than the nitric acid solution; there is little tendency for the
separation of solid. Before using the alkaline solution, it is important
that the test solution contains a slight excess of nitric acid.
Ammoniacal silver nitrateAdd ammonia dropwise to a 1 in 20
solution of silver nitrate until the ppt. that first forms is almost , but
not entirely, dissolved. Filter and store in dark bottle
Forms explosive compounds on standing! Prepare fresh.
Ammonium sulfanilate
 SCIENCE LABORATORY TECHNOLOGY
To 2.5 g of sulfanilic acid add 15 ml water and 3 ml ammonia and mix.
If necessary add more ammonia until the the acid dissolves. Adjust the
pH to 4.5 with dilute HCl and dilute to 25 ml.
Amylase
To 0.2 g of amylase crystal, add 100ml water, shake well and filter.
Prepare fresh.
Anthranilic acid
Dissolve 0.5g in 100 ml ethanol
AnthroneDissolve about 0.1 g in 100 ml sulphuric acid. Prepare fresh.
Aqua Regia
Mix one part by volume of conc. nitric acid with three (3) parts by
volume of conc. hydrochloric acid in a pyrex beaker and allow to stand
until a bright red color develops.
Barfords reagentDissolve 13.3gm.of crystalised neutral copper
acetate in 200ml.of 1% acetic acid solution. This reagent does not keep
well.
Bradys reagent (2,4 DNPH)
Dissolve 40g of 2, 4 dinitrophenylhydrazine in 80ml conc. Sulphuric
acid. Cool and add 900ml methanol and 100ml water.
Benedicts solution (qualitative)
Dissolve 86.5gm.of sodium citrate and 50gm. of anhy. sodium
carbonate in about 350ml. of water. Filter if necessary. Add a solution
of 8.6gm. of copper sulphate in 50ml. of water with constant stirring.
Dilute to 500ml. The resulting solution should be perfectly clear; if not,
filter through a fluted filter paper.
Benedicts solution (quatitative)
Dissolve 200g sodium citrate, 75g sodium carbonate and 125g
potassium thiocynate in about 600 ml water. Dissolve separately, 18g
copper (11) sulphate in 100 ml water. When the solutions have cooled,
mix them together with stirring. Now add 5 ml of a 5% potassium
ferrocyanide to the solution, and make up to 1 liter.
Benzidine
Dissolve 50 g in 10 ml glacial acetic acid, dilute to 100 ml with water
and mix. caution: Toxic!).
Benzodine
Dissolve 1g in 10ml acetic acid and dilute to 100 ml with water.
2,2-bipyridine
Dissolve 0.100 g in 50ml purified absolute ethanol.
Biuret reagent(qualitative)
 SCIENCE LABORATORY TECHNOLOGY
Take enough urea to cover the bottom of a test tube. Heat very gently ,
until the liquid which forms resolidifies. This white solid is biuret.
Dissolve the biuret in about 2ml. water, and use this solution for the
biuret test.
Or
Soln. A : 0.1m sodium hydroxide.
SoLn. B : 0.01M copper (11) sulphate solution. Add soln. A first to
sample, then add B. Pink or purple color confirms protein.
Biuret reagent (quantitative)
Dissolve in order, 3g copper (11) sulphate, 5g potassium iodide, 9g
potassium sodium tartrate, and 8g sodium hydroxide in about 600 ml
water. Make up the dissolved solids to 1 liter solution.
Brucine sulphate
Boil a 2 to 1 mixture of conc. sulphuric acid and water to to remove
nitrates, cool , and dissolve into it 0.6g of brucine sulphate and dilute
to 1 liter.
Carr-Price reagent
Weigh an unopened bottle of antimony trichloride. Open the bottle and
empty the contents into a wide mouth glassstoppered amber bottle
containing about 100 ml of chloroform. By difference, obtain the
weight of antimony trichloride and then add sufficient chloroform to
supply 100 ml for each 25 g. Dissolve by warming or shaking for
several hours and filter through sodium sulphate into a dry clean
amber bottle with ground stopper.Store at room temperature and keep
in dark when not in use. Rinse all glassware coming into contact with
this reagent wit chloroform or a mixture of ethanol and ether, since the
antimony oxychloride which forms is insoluble in water.
Cacothelinea-nitroso-b-naphthol reagent.
Dissolve 0.25 g in 100 ml of water.
Cadion 2B reagent
4-nitronaphthalene-diazamino-azobenzene
Dissolve 0.25 g in 100 ml of water.
Chloroplatinic acid
Dissolve 2.7 g in 10 mls. of water.
Chromic acid
Weigh out 10 g of sodium dichromate crystals, make it into a slurry
with a few mls of watwer, then dissolve in 250mls conc. sulphuric acid
with stirring and cooling (ice-bath), to give a thick syrupy dark brown
mixture.(very corrosive!)
 SCIENCE LABORATORY TECHNOLOGY
Chromotrope 2B
Use a 0.005% in conc. sulphuric acid.
Chromotropic acid
Dissolve 1g of 4,5-dihydroxy-2,7-naphthalenedisulfonic acid in 100 ml
water.
Cincholine
Dissolve 1g in 100 ml hot water containing a few drops of nitric acid,
cool, and add 2g of KI.
Cobalt-uranyl acetate
Dissolve with warming, 40 g of uranyl acetate in a mixture of 30 ml
acetic acid and 470 ml water.Similarly, prepare a solution containing
200 g of cobaltous acetate in a mixture of 30 ml acetic acid and 470 ml
water. Mix the two solutions while still warm, cool to room
temperature to separate xs salts from the solution and filter.
Cupferon
Dissolve 5 g in 100ml solution.(5%) Does not keep. Add about 1g of
ammonium carbonate to stock to enhance stability.
Cupron
Dissolve 5g of a-benzoin oxime in 100ml ethanol
Deniges reagent
Dissolve 5 g of yellow mercuric oxide (HgO) in a mixture of 40 ml of
water,and while stirring slowly add 20 ml of sulphuric acid, then add
another 40 ml of water, and stir until complety dissolved <0.5N)
Dimethyl glyoximeDissolve 1gm. of the solid in 100ml. of 95% ethyl
alcohol.N,N-dimethyl-p-phenylenediamine
a.)Measure and pour into a 250 mL beaker 89 mL of distilled water.
Stir on a magnetic stirrer. Carefully add 15 mL of concentrated sulfuric
acid. Add and dissolve 1.0 g n,n-dimethyl-p-phenylenediamine sulfate.
Add 5 g of Florisil and stir the mixture until all is absorbed. Allow the
adsorbant to settle and decant the supernatant solution.
b.Add 200 ml sulphuric acid to 700 ml water, cool, and add 1g of N,N-
dimethyl-p-phenylenediamine (p-aminodimethylaniline) and dilute ti 1
liter.
Dinitro-p-diphenylcarbazide Dissolve 0.1g in 100 ml ethanol
Diphenylcarbazide
Use a saturated ethanolic solution; or dissolve 0.125 g in a mixture of
25ml acetone 25 ml water; or dissolve 0.2g in 10 ml acetic acid and
dilute to 100 ml with methanol
Diphenylcarbazone
 SCIENCE LABORATORY TECHNOLOGY
Dissolve an approximately 1% solution in ethanol.
Dipicrylamine
Dissolve 0.2g in 20 ml of 0.1M sodium carbonate solution, boil, cool,
then filter.

Dipridyl reagent
Dissolve 0.1g in 0.5ml ethanol,or in 0.1M HC
Dithiol reagent
4-methyl-1:2 dimercarpo-benzeneDissolve 0.2g in 100 ml of 1% NaOH
Dithizone solution
Dissolve 30 mg (milligram) of dithizone in 1 liter chloroform, add 5 ml
alcohol, and store in refrigerator.
Dragendorff reagent
Solution 1: dissolve 0.85g of basic bismuth nitrate in 10 ml acetic acid
and 40 ml water.
Solution 2: dissolve 8 g of potassium iodide in 20 ml water.
Mix 5 ml of Solution 1; 5 ml of Solution 2; 20 ml of acetic acid; and 100
ml of water before use.
Ethylenediamine reagent
Add copper sulphate to a solution of ethylenediamine until the color
becomes dark-blue violetp-EthoxychrysoidinDissolve 50 g in a mixture
of 25 ml water and 25 ml ethanol, add 3 drops hydrochloric acid, stirr
vigorously, and filter if necessary to obtain a clear solution.
Fehlings solution
Solution A; Dissolve 34.6gms. of pure copper sulphate in distilled
water and dilute to 500ml. ( blur )
Solution B: Dissolve 173gms. of sodium potassium tartrate and
30gms.of pure sodium hydroxide in water and dilute to 500ml.
Alternately, dissolve 121gms. of pure sodium hydroxide and 93.1gms.of
pure tartaric acid in water, then dilute the solution to 500ml.
( colourless ).Mix equal volumes of solutions A and B immediately
before use, and then use as the reagent.
Ferric thiocyanate reagent
Dissolve 1.5 g of ferric chloride and 2.0 g of potassium thiocyanate in
100 ml water.
Ferron reagent
7-iodo-8-hydroxyquinoline-5-sulphonic acidDissolve 0.2 g in 100 ml
water
Fluorescein
 SCIENCE LABORATORY TECHNOLOGY
Make a saturated solution in 50% ethanol.
Formaldehyde
Dilute the commercial 40% solution (1 part) with water (7 parts)
Fuchsin solution
Dissolve 0.15gm. of fuchsin in 100ml. water.
Furil-dioxime reagent
Dissolve 10 g in 100 ml ethanol.
Hydrazine sulphate
Dissolve 2 g in 100 ml water.
Hydrogen peroxide
Use the commercial 10 volume (3%) or 20 volume 6%) solution.
Hydroxylamine hydrochlorid
Dissolve 10 g in 100 ml water.
8-Hydroxyquinoline
Usea% solution in ethanol.
Indigo solution
Gently warm 1gm. of indigo with 12ml. of concentrated sulphuric acid,
allow to stand for 48hrs. and pour into 240ml. of water. Filter if
necessary.
Indole
Dissolve 0.15 g in 100 ml ethanol
Iodine reagent.
Dissolve 20g KI and 10g iodine cystals in 100ml water.
Jones reagent
Mix 25g of chromium trioxide (chromic anhydride CrO3) with conc.
sulphuric acid to a paste, then dilute with water to 75mls.
Karl Fisher Reagent
Dissolve 762 g of iodine in 2,420 ml of pyridine in a 10 liter glass
stoppered bottle, and add 6 liters of methanol. To prepare the active
stock, add 3 liter of
the foregoing stock to a 4 liter bottle, cool in ice bath. Add carefully 135
ml of liquid sulfur dioxide, collected in a calibrated cold trap,and
stopper the bottle. Shake the mixture until homogeneous, and set aside
for one to two days before use.
Magnesia mixture
Dissolve 100gms. of magnesium chloride and 100gms.of ammonium
chloride in water, add 50ml. of concentrated ammonia solution and
dilute to 1 litre with distilled water.
 SCIENCE LABORATORY TECHNOLOGY
Magnesium nitrate reagent
Dissolve 130gms. of magnesium nitrate and 200gms. of ammonium
nitrate in water, add 15-20mls.concentrated ammonia solution and
dilute to 1 litre.
Malachite green
A 1% solution of malachite green oxalate in glacial acetic acid.
Manganese sulphate
Dissolve 90 g manganese sulphate in 200 ml water, 175 ml phosphoric
acid and 350 ml of diluted sulphuric acid (1 in2). Add water to make
up 1 liter.
Mayers reagent
Mercuric-Potassium Iodide
Dissolve 1.358 g of mercuric chloride in 60 ml water. Dissolve 5 g of
potassium iodide in 10 ml water. Mix the two solutions and add water
to make 100 ml.
4-(methylamino)phenol sulphate
Dissolve 2 g in 10 ml water. To 10 ml of this solution add 90 ml of
water and 20 g of sodium bisulfite.
Millons reagent
Warm one globule of Mercury with concentrated nitric acid and dilute
the solution with twice its volume of water.
Molischs reagent.
20% soln. in naphthol. Dissolve 20g of 1-naphthol in 100ml ethanol.
Murexide
Add 0.4 g of murexide to 40 g of powdered potassium sulphate, and
grind in glass mortar to a homogeneous mixture.
Naphthalenediol
Dissolve 0.1 g of 2,7-dihydroxynaphthalene in 1 liter sulphuric acid and
allow the solution to stand in the dark until the yellow color has
diappeared (at least 18 hrs.)
1-Naphthol
Dissolve 1 g of 1-naphthol in 25 ml methanol. Prepare fresh.
alpha-Naphtholbenzein
A 1% solution in benzol.
a-Naphthalamine
Boil 0.3 g in 70 ml water, filter or decant, and mix with 30 ml of glacial
acetic acid.
Nesslers reagent
 SCIENCE LABORATORY TECHNOLOGY
Dissolve 10gms. of potassium iodide in 10mls. of ammonia-free water,
adding saturated mercuric chloride solution (60gms. / litre) in small
quantities at a time with shaking, until a slight permanent precipitate is
formed, then adding 80ml. of 9M sodium hydroxide solution and
diluting to 200ml.Allow to stand overnight and decant the clear
liquid.Nesslers reagent has been described as a solution which is about
0.09M in potassium mercuri-iodide and 2.5M in potassium hydroxide.
An alternative method is to dissolve23gms. of mercuric iodide and
16gms. of potassium iodide in ammonia-free water and make up the
volume to 100ml; add 100ml. of 6M sodium hydroxide. Allow to stand
for 24hrs. and decant the solution from any precipitate that may have
formed, the solution should be kept in the dark.Another method that
reacts promptly and consistantly is to dissolve 143 g of sodium
hydroxide in 700 ml water. Disolve 50 g of red mercuric iodide and 40
g of potassium iodide in 200 ml water. Pour the iodide solution into
the hydroxide solution, and dilute with water to 1 liter. Allow to settle,
and use the supernatant liquid.
Ninhydrin
A 0.2% solution of ninhydrin (triketohydrindene hydrate,
C9H4O3.H2O) in water. Prepare fresh.
Nitron reagent
Dissolve 5g nitron in5 ml acetic acid and make up to 100 ml with water
a-Nitroso-b-naphthol
Dissolve 10 g in 100 ml of either 1:1 acetic acid , ethyl alcohol or
acetone.
Nitroso-R-salt
sodium 1-nitroso-2-hydroxynaphthalene-3:6-disulphate
Dissolve 1 g in 100 ml water.
1,10-phenanthroline
Dissolve 0.1 g of the monohydrate in 100 ml water.
Orthophenanthroline
Dissolve 0.15 g orthophenanthroline (C12H8N2.H2O) in 10 ml solution
of ferrous sulphate, prepared by dissolving 1.48 g of ferrous sulphate in
100 ml water. THe ferrous sulphate solution must be prepared
immediately before dissolving the orthophenanthroline.

Phosphomolybdic acid
Dissolve 5 g in water, filter and dilute to 100 ml.
 SCIENCE LABORATORY TECHNOLOGY
Picric acid
Dissolve the equivalent of 1 g of anhydrous trinitrophenol in 100 ml of
hot water. Cool, and filter if necessary.
Potassium cyanide
Dissolve 10 g of KCN in sufficient water to make 20 ml. Dilute to 100
ml. Shake with dithizone solution to remove lead
Quimociac reagent
Dissolve 70 g of sodium molybdate (Na2MoO42H2O) in 150 ml water
(Slution A). Dissolve 60 g of acetic acid in a mixture of 85 ml nitric acid
and 150 ml of water and cool (Solution B) Gradually add Solution A to
Solution B, with stirring, to produce Solution C. Dissolve 5 g of
synthetic quinoline in a mixture of 35 ml nitric acid and 100 ml water
(Solution D) Gradually add Solution D to Solution C, mix well, and
allow to stand overnight. Filter the mixture, add 280 ml of acetone to
the filtrate, dilute to 1 liter with water, and mix. Store in polythene
bottle.(Caution: Flammable).
Quinaldic acid
Neutralize 1g of the acid with NaOH and dilute ti 100 ml
Quinalizarin reagent
Dissolve 0.02g in 100 ml ethanol, or dissolve 0.05g in 100 ml of 0.01M
sodium hydroxide.
Rhodamine B
Dissolve 0.01 g in 100ml water, or dissolve 0.05 g rhodamine B and 15g
KCL in a solution of 15 mls conc. HCl and 85 mls water
Rubeanic acid
(dithio-oxamide) - 0.05% in ethanol., a saturated ethanolic solution
Salicylaldehyde
A 20% solution in ethanol.
Schiffs reagent
Method 1: Dissolve 0.2gm. of pure p rosaniline hydrochloride in 20ml.
of a cold, freshly prepared, saturated aqueous solution of sulphur
dioxide; allow the solution to stand for a few hours until it becomes
colourless or pale yellow. Dilute the solution to 200ml. and keep it in a
tightly stoppered bottle.The solution keeps well, and should not be
exposed to light or air. Store in the dark.
Method 2: Add 2gm. of sodium bisulphite to a solution of 0.2gm.of p-
rosaniline hydrochloride and 2ml.of concentrated hydrochloric acid in
200ml. of water.
Silicotungstic aci
 SCIENCE LABORATORY TECHNOLOGY
Dissolve 10 g ofsilicotungstic acid (Si02.12WO3.26H2O) in water and
neutralize with 10% sodium hydroxide (pH 6)
Silver ammonium nitrate
Dissolve 1 g of silver nitrate in 20 ml of water.Add ammonia dropwise,
withconstant stirring, until the ppt. is almost but not entirely dissolved.
Filter and store in dark container.
Silver diethyldithiocarbamate
Dissolve 1 g in 200 ml of freshly distilled pyridine.
Sodium azide
A 5% solution of sodium azide in water.
Sodium borohydride
Dissolve 0.6 g sodium borohydride and 0.5g of sodium hydroxide with
stirring and dilute to 100 ml with water.Sodium cobaltinitrite solution
Dissolve 17gms. of the pure salt in 250 of water.Alternately, the
solution may be prepared as follows: Dissolve 7.5 gms. cobalt nitrate in
30ml. of water; dissolve 60gms. of sodium nitrite in 30ml. of water,
mix the two solutions with vigorous stirring and add 15ml. of glacial
acetic acid, stir, dilute to 250ml, allow to stand and filter.Make up new
solution every 2-3 weeks.
Sodium diethyldithiocarbamate
Dissolve 1 g in water and dilute to 1 liter.
Sodium ethoxide
Dissolve 10 g of sodium metal in 120 ml of ethanol using the following
method: remove surplus oil from sodium with filter paper, dry again on
filter paper, and cut the weighed metal into small pieces about the size
of a pea. pour the ethanol into a 500 ml flask cooled on ice bath, and
add one or two pieces at a time until dissolved.
Sodium hypochlorite solution
The commercial product contains about 10-14 per cent w / v of
available chlorine. Dilute with an equal volume of water.
Sodium nitroprusside solution
Prepare a solution as required by dissolving a crystal in 5 ml. of water
Sodium rhodizonate reagent
Dissolve 0.5 g in 100 ml water
Starch solutionTriturate 0.5gm. of soluble starch with a little cold
water into a thin paste and add 25ml. of boiling water.Boil until a clear
solution is obtained (5-mins.). This solution should be freshly prepared
as required. Amore stable starch solution is obtained by adding 0.5gm.
of potassium iodide and 2-3 drops of chloroform.
 SCIENCE LABORATORY TECHNOLOGY
A more satisfactory starch solution for use as an indicator is prepared
as follows:* Mix 5.0 gm.of powdered sodium starch glycollate with 1-2
ml. ethyl alcohol, add 100ml. of cold water and boil for a few minutes
with stirring.This 5% stock solution is stable for many months; it is
diluted to 0.1% strength when required for use.
Sulphanilic aci
Dissolve 1 g in 100 ml of warm 30% acetic acid
Tannic acid
Dissolve 1 g of tannic acid (tannin) in 1 ml ethanol, and add water to
make 10 ml. Prepare fresh.
Tartrate solution, alkaline
Dissolve 34.6 g of sodium potassium tartrate (rochelle salt) and 10 g of
sodium hydroxide in water, dilute to 100 ml, let stand for two days, and
filter through glass wool.
Thiourea
Dissolve 10g in 100ml of water (10%)
Titan yellow
Dissolve 1g in 100ml of water (1%)
Titanium tetrachloride
Cool separately in small beakers surrounded by crushed ice 10 ml of
20%
hydrochloric acid and 10 ml of clear, colorless titanium
tetrachloride.Add the tetrachloride dropwise to the chilled acid.Stand
at ice temperature until all the solid disolves, then dilute to 1 liter with
20% hydrochloric acid.
Tollens reagent.
Add sodium hydroxide soln. to silver nitrate soln. to form a ppt. then
add dilute ammonia soln. until ppt. Dissolves.
Triton X-100
20% solution: dissolve 0.20 g of Triton-X-100 (polyethelene glycol
ether of isooctylphenol) in water, and dilute to 100ml.
Uranyl magnesium acetate
Make up an aqueous saturated solution of the salt.
Xylenol orange
Make up a 1% solution in ethanol
Zirconyl nitrate reagent
( for fluoride test )Dissolve 0.1gm. of zirconyl nitrate in 20ml.
concentrated hydrochloric acid and dilute with water to 100ml.
 SCIENCE LABORATORY TECHNOLOGY
Zirconyl nitrate reagent( for phosphate separations )Heat 10gms.
of commercially pure zirconium nitrate and 100ml. of 1M nitric acid to
the boiling point with constant stirring.Leave to stand for about
24hrs.and decant the clear solution.
Zinc amalgam
Add about 10 g of granulated zinc to to 20 ml mercury, to produce a
liquid amalgam on cooling, and heat to 150 degrees with stirringuntil
the zinc is dissolved.
Zinc amalgated (Jones Reductor)
The zinc is amalgated by immersing it in a solution of mercuric
chloride in hydrochloric acid. A quantity of 250 g of 20 mesh zinc is
covered with water in a 1 liter flask, and a solution of 11 g of mercuric
chloride in 100 ml of hydrochloric acid is poured into the flask. The
system is slowly mixed and shaken for about 2 minutes. The solution is
poured off, and the amalgam is washed thoroughly with hot tap water,
then distilled water.
Zinc uranyl acetate
Dissolve 10gms. of uranyl acetate in 6gms. of 30% acetic acid, warming
if necessary and dilute with water to 50ml. (soln.A) In a separate
vessel, stir 30gms. of zinc acetate with 3gms.of 30% acetic acid and
dilute with water to 50ml. (soln.B) Mix the two solutions A and b, and
add a small quantity of sodium chloride. Allow to stand for 24 hrs. and
filter from the precipitated sodium zinc uranyl acetate.
Alternatively, a reagent of equivalent concentration may be prepared
by dissolving uranyl zinc acetate in the appropriate volume of water or
1M acetic acid
Zwikkers reagent
Mix 1 ml of pyridine with 4 ml of a 10% aqueous solution of copper
sulphate and 5 ml of water

PREPARATION OF SIMPLE CHEMICAL

REAGENTS
Name/ Formula Pb(C2H3O2)2 • 3H2O
Concentration g/L 379.3
Lead acetate 0.1 M Lead chloride
38.0 g saturated 12.0g
 SCIENCE LABORATORY TECHNOLOGY
PbCl2 Magnesium sulfate 0.5 M
278.12 123.3 g
Lead nitrate 1M MgSO4 • 7H2O 0.1 M
331.2 g 24.7 g
Pb(NO3)2 0.5 M 246.50
165.6 g Manganese chloride 0.5 M
331.2 0.1 M 99.0 g
33.1 g MnCl2 • 4H2O 0.1 M
Lithium carbonate 0.1 M 19.8 g
7.4 g 197.91
Li2CO3 Manganese sulfate 0.2 M
73.89 33.8 g
Lithium chloride 1.0 M MnSO4 • H2O 0.1 M
42.4 g 16.9 g
LiCl 169.01
42.40 Mercury(II) chloride 0.25 M
Lithium nitrate 1.0 M 67.9 g
69.0 g HgCl2 0.10 M
LiNO3 0.5 M 27.2g 271.5
34.5 g Mercury(II) nitrate 0.1 M
68.95 34.2 g
Magnesium bromide 0.1 M Hg(NO3)2 • H2
29.2g 342.63 HNO3*
MgBr2 • 6H2O Mercury(I) nitrate 0.1 M
292.25 56.2 g
Magnesium chloride 1.0 M Hg2(NO3)2 • 2H2O
203.3 g 561.22 HNO3*
MgCl2 • 6H2O 0.1 M Mercury(I) sulfate 0.1 M
20.3 g 49.7 g
203.33 Hg2SO4
Magnesium hydroxide 497.24
saturated 300 g Nickel chloride 0.25 M
Mg(OH)2 59.4 g
58.3 NiCl2 • 6H2O 0.1 M
Magnesium nitrate 0.1 M 23.8 g
25.6 g 237.72
Mg(NO3)2 • 6H2O Nickel nitrate 1M
256.43 290.8 g
 SCIENCE LABORATORY TECHNOLOGY
Ni(NO3)2 • 6H2O 0.2 M K3Fe(CN)6 0.1
58.2 g M 32.9 g
329.26
Potassium ferrocyanide 0.1
Name/ Formula M 42.2 g
Concentration g/L K4Fe(CN)6 • 3H2
Nickel sulfate 1.0 M 422.41
262.9g Potassium hydrogen 0.1 M
NiSO4 • 6H2O 0.5 M 20.4 g
131.4 g phthalate
262.87 KHC8H4O4
Potassium bromide 0.5 M 204.23
59.5 g Potassium iodate
KBr 0.1 M saturated 214.0g
11.9 g KIO3 0.2 M
119.02 42.8g
Potassium carbonate 0.5 M 214.01 0.1 M
69.1 g 21.4 g
K2CO3 0.1 M Potassium iodide 1M
13.8 g 166.0 g
138.21 Kl 0.5 M
Potassium chloride 0.5 M 83.0 g
37.3 g 166.01 0.2 M
KCl 0.1 M 33.2 g
7.5g
75 Potassium nitrate 0.5 M
Potassium chromate 1.0 M 50.6g
194.2 g KNO3 0.1 M
K2CrO4 0.5 M 10.1 g
97.1 g 101.1
194.21 0.1 M Potassium permanganate 0.2
19.4 g M 31.6 g
Potassium dichromate 0.1 M KMnO4 0.1
29.4g M 15.8 g
K2Cr2O7 158.04
294.22 0.01M 1.6 g
Potassium ferricyanide 0.5 Potassium phosphate, 0.1 M
M 164.6 13.6 g
monobasic
 SCIENCE LABORATORY TECHNOLOGY
KH2PO4 NaHCO3 0.1
136.09 M 8.4 g
Potassium phosphate, 84.01
0.1 M 17.4g K2HPO4 Sodium borate 4%
174.18 40.0 g
Potassium phosphate, 0.1 Na2B4O7 • 10H2
M 21.2 g 381.42
tribasic Sodium bromide 1.0 M
K3PO4 102.9 g
212.27 NaBr 0.1
Potassium sulfate 0.5 M M 10.3 g
87.1 g 102.90
K2SO4 0.1 M Sodium carbonate
17.4 g saturated 214.0 g
174.27 Na2CO3 1.0 M
106.0 g
105.99 0.1 M
10.6 g
Name/ Formula Sodium carbonate 1.0 M
Concentration g/L 124.0g
Potassium thiocyanate 1.0 M Na2CO3 • H2O 0.1 M
97.2 g 12.4 g
KSCN 0.5 M 124.0
48.6 g Sodium chloride saturated
97.18 0.1 390.0 g
M 9.7 g NaCl 1 .0 M
Silver nitrate 0.5 M 58.5 g
84.9 g 58.45 0.1 M
AgNO3 0.1 5.8g
M 17.0 g Sodium dichromate 0.1 M
169.87 29.8g
Sodium acetate 1 M Na2Cr2O7 • 2H2O
136.1 g 298.03
NaC2H3O2 • 3H2O 0.5 M Sodium fluoride 0.1 M
68.0 g 4.2g
136.08 NaF
Sodium bicarbonate 0.5 41.99
M 42.0 g Sodium iodide 0.5 M
75.0 g
 SCIENCE LABORATORY TECHNOLOGY
NaI 0.1 M Sodium sulfate saturated
15.0 g 600 g
149.92 Na2SO4 • 10H2O 1.0 M
Sodium nitrate 0.5 M 322.2 g
43.0 g 322.19 0.5 M
NaNO3 0.1 M 161.1g
8.5 g Sodium sulfate saturated*
84.99 260 g
Sodium oxalate 0.1 M Na2SO4 1.0 M
13.4 g 142.0 g
Na2C2O 142.02 0.5 M
4134.00 71.0 g
Sodium phosphate, 0.1 M Sodium sulfide 2.0 M
13.8 g 480.0 g
monobasic Na2S • 9H2O 1.0 M
NaH2PO4 • H2 240.0 g
137.99 240.18
Sodium phosphate, 0.5 M Sodium sulfite 1.0 M
134.0 g 126.1 g
dibasic 0.1 M Na2SO3
26.8 g 126.05
Na2HPO4 • 7H2O Sodium thiosulfate 0.5 M
268.07 124.1 g
Sodium phosphate, 0.5 M Na2S2O3 • 5H2O 0.1 M
71.0 g 24.8g
dibasic 0.1 M 248.19
14.2 g Strontium chloride 0.5 M
Na2HPO4 133.3 g
141.96 SrCl2 • 6H2O 0.1 M
Sodium phosphate, 0.1 M 26.7 g
38.0 g 266.64
tribasic Strontium nitrate 1.0 M
Na3PO4 • 12H2O 211.6g
380.1 Sr(NO3)2 0.5 M
105.8 g
211.63
Name/ Formula Tin(II) chloride 0.1 M
Concentration g/L 22.6 g
SnCl2 • 2H2O 1 M HCl*
 SCIENCE LABORATORY TECHNOLOGY
225.65 F.W. 36.4 1M
Tin(IV) chloride 0.1 M 83m
35.1 g 37.2%, 12.1 M 0.5 M
SnCl4 • 5H2O 3 M HCl* 41.5ml
350.61 sp. gr. 1.19 0.1 M
Zinc chloride 0.5 M 8.3ml
68.1 g Nitric Acid* 6M
ZnCl2 380 ml
136.29 HCl* 0.1 M HNO3 1M
13.6 g 63ml
Zinc nitrate 0.5 M F.W. 63.01 0.5 M
149.7 g 32ml
Zn(NO3)2 • 6H2O 0.1 M 70.0%, 15.8 M 0.1 M
29.7 g 6.3ml
297.49 sp. gr. 1.42
Zinc sulfate 1.0 M Phosphoric Acid* 6M
287.6 g 405 ml
ZnSO4 • 7H2O 0.1 M H3PO4 3M
28.8 g 203ml
287.6 g F.W. 98.00 1M
68m
ACIDS 85.5%, 14.8 M 0.5 M
Name/ Formula 34ml
Concentration vol sp. gr. 1.70 0.1 M
Acetic Acid* 6M 6.8ml
345 ml sulfuric Acid* 9M
CH3CO2H 3M 500ml
173ml H2SO4 6M
F.W. 60.05 1M 333ml
58ml F.W. 98.08 3M
99.7%, 17.4 M 0.5 M 167m
29ml 96.0%, 18.0 M 1M
sp. gr. 1.05 0.1 M 56ml
5.8ml sp. gr. 1.84 0.5
Hydrochloric Acid* 6M M 25.
500 ml
HCl 3M BASES
250ml Ammonium Hydroxide* 1M
68ml
 SCIENCE LABORATORY TECHNOLOGY
NH4OH 0.1 F.W. 56.11
M 6.8 ml Sodium Hydroxide 1M
F.W. 35.05 40g
Potassium Hydroxide 1 M NaOH 0.1 M
56g 4.0g
KOH 0.1 F.W. 40.00
M 5.6g

Preparing Sodium Hydroxide Solution?

A great amount of heat is liberated when sodium hydroxide and water
are mixed. The temperature of the solution may rise very rapidly. In
fact, the temperature may rise so fast that the solution may boil and
possibly spatter a hot, caustic solution. Immerse theflask or beaker in
an ice–water bath to control the solutiontemperature. In addition, pay
special attention to the condition of the beaker or flask, you use to
prepare these solutions. If you use a glass vessel it must be borosilicate
glass and it must be free of any scratches, chips or breaks. Inspect the
vessel carefully before use. Add ingredients slowly with continuous
stirring.

General Solubility Rules for Inorganic Compounds

Nitrates (NO3–): All nitrates are soluble.

Acetates (C2H3O2–): All acetates are soluble; silver acetate is
moderately soluble.
Bromides (Br–) Chlorides (Cl–) and Iodides (I–): Most are
soluble except for salts containing silver, lead, and mercury
Sulfates (SO42–): All sulfates are soluble except barium and lead.
Silver, mercury(I), and calcium are slightly soluble.
Hydrogen sulfates (HSO4–) : The hydrogen sulfates (aka
bisulfates) are more soluble than the sulfates
Carbonates (CO32–), phosphates (PO43–), chromates
(CrO42–),silicates (SiO42–): All carbonates, phosphates,
chromates, and silicates are insoluble, except those of sodium,
potassium, and ammonium. An exception is MgCrO4, which is soluble
 SCIENCE LABORATORY TECHNOLOGY
Hydroxides (OH–): All hydroxides (except lithium,
sodium,potassium, cesium, rubidium, and ammonium) are insoluble;
Ba(OH)2, Ca(OH)2 and Sr(OH)2 are slightly soluble.
Sulfides (S2–): All sulfides (except sodium, potassium, ammonium,
magnesium, calcium and barium) are insoluble.Aluminum and
chromium sulfides are hydrolyzed and precipitate as hydroxides
Sodium (Na+), potassium (K+), ammonium (NH4+): All sodium,
potassium, and ammonium salts are soluble. (Except some transition
metal compounds.)
Silver (Ag+): All silver salts are insoluble. Exceptions: AgNO3 and
AgClO4; AgC2H3O2 and Ag2SO4 are moderately soluble

CHAPTER SIX

LABORATORY ANIMALS
Most laboratory experiments especially in biology involves working
with animals and plants .laboratory animals should be handled with
care and any experiments dealing with them should be under control
 SCIENCE LABORATORY TECHNOLOGY
making sure that no stipulated regulation and legislation stipulated
concerning them is infringed .
Laboratory animals include generally any small animal that can be
easily handle in the laboratory environment e.g. rats ,mice , rabbits ,
guinea pigs , hamsters , amphibians , reptiles, and insects like locust ,
mosquitoes ,and even some microorganisms like protozoa and bacteria

Care and handling of laboratory animals

Caging
Since many animals spend most of their lives in cages, these enclosures
are an important aspect of the laboratory animal environment.Animal
cages should be kept clean and cleanness should be done on regular
basis , its floors and walls should be sprayed and disinfected . The
animal cage should be sited in secure and easily accessible places , well
drained and away from direct wind and rain . The place should be cool
and also silent. Cages must provide the animals with comfort and
safety. they should nevertheless be well ventilated so as not to
predispose these animals to infections and other effects caused by
poor ventilation . The optimum humidity in these cages should be
between 45-65% , the cages should also be adequately lighted .
Materials for making cages include , wood , iron sheets wire mesh ,
plastic , glass etc . The choice of which material to use will depend on
many factors such as the cost , availability , durability, comfortability
and whether it is easy to be destroyed by the animals( e.g. hamsters
gnaw plastic cage materials and should therefore be kept in wooden or
metallic cages but must be provided with wood to gnaw otherwise their
incisors might overgrow until feeding is impossible ) cages should be
labeled and the materials used for labeling should be those that are not
easily chewed by the gnawing animals and insects.
Environment
Within an animal facility, environmental conditions such as
temperature, humidity, light, noise, and air exchange are regulated and
evaluated for proper level and consistency. The animal’s environment
affects the animals’ health, and therefore potentially affects the
outcome of a study in which they are used as models. Control of
environmental variable ensures that laboratory animals are provided
with the stable living conditions necessary for well-designed
experimental research projects. A consistent environment in rooms
and cages is important in research facilities. For animals housed
 SCIENCE LABORATORY TECHNOLOGY
outdoors, protection from heat, cold, and other environmental
extremes must be provided.
Two terms commonly used to describe animal housing are
conventional and barrier. In conventional rooms, no special
precautions are taken to prevent the introduction of disease into the
colony. You house the animals in a conventional laboratory facility.
You feed them, groom them, and vaccinate them against common
diseases, but you don’t keep them in a special room or pen to isolate
them from the rest of their environment. Most of the larger laboratory
animals, such as dogs, cats, pigs, and sheep, are housed in conventional
cages and rooms.
Barrier housing involves special cages and procedures, like sterilizing
the bedding and passing the room air through special filters. A barrier
can be a cage, a room, or even an entire facility. The object is to guard
against infection or, if the protocol involves the study of diseases that
infect humans or other animals, to keep the disease-causing organism
from getting out.
Bedding
Bedding is part of a laboratory animal’s environment, and thus must
meet certain criteria. It must not contain nutrients or be treated with
deodorizers, disinfectants, or other chemicals, as these could affect the
nimals and ultimately the research.
Changing to a different bedding material in the middle of an
experimentcould also affect the experiment’s results. Bedding must not
create levels of dust that irritate the animal’s lungs and eyes;
additionally, contact bedding must be free of sharp, splintery edges or
other defects that could injure animals and make nest-building difficult
There is no ideal bedding material for all species in all applications.
Wood shavings, compressed paper and straw (for large animals) are
the most common types of bedding.
The bedding materials should be provide maximum comfort to the
animals , easy to clean and sterilize and should not be contaminated
with pathogens , parasites e.g. mites and flees or with animal
droppings . Bedding provides the nesting materials for the animals
especially pregnant animals and prevents the animals from developing
sores on their feet’s.
Feeding
 SCIENCE LABORATORY TECHNOLOGY
Laboratory animals should be feed on good balanced diet and on daily
basis and drinking water should always be made available regardless
of the type of food supplied .
Hygiene
In a laboratory animal facility there are generally four definable and
achievable levels ofhygiene:
1.Cleaning: The complete removal of all visible soil from a surface.
2.Sanitization: The process by which the number of bacteria and other
organisms living on inanimate objects is reduced enough to prevent
disease. Sanitization does not necessarily totally eliminate all
microorganisms, but is aimed at reducing total numbers of organisms.
3.Disinfection: A more intense form of sanitation which is designed to
reduce to a harmless level the number of a specific type of organism,
specifically pathogenic organisms (but not necessarily spores), on an
object.
4.Sterilization: The process of rendering an object totally free of all
living organisms, including spores.
The food and water troughs ,bottles and other pieces of equipment
should be cleaned and regularly disinfected so as to keep them
contamination-free.
Disease outbreak is minimized in clean healthy cages but incase of
disease outbreak , only experts should be allowed to diagnose and treat
the diseased animals , sick and diseased animals should be culled ,
isolated and humanely killed and the cages sterilized . laboratory
animals should be obtained from accredited suppliers so as to avoid
chances and risks introducing epidemics in the cages .
Environmental Enrichment
Environmental enrichment is an attempt to reduce stress in laboratory
animals by allowing them to do the same sorts of things they would do
if they were living in the wild. Enrichment can be as simple as
providing frogs in a tank with a piece of plastic pipe in which to hide, or
providing paper material for mice to build a nest..another way is by
exercise, exploration, and social interaction with handlers.

General care of laboratory animals

Mice
They mate when at the age of between 6-8 weeks old .
Mice should be kept in threes (trios) i.e. one male : two females
Avoid inbreeding as these may result in drop in average litter size
 SCIENCE LABORATORY TECHNOLOGY
Their oestrius circle last between 4-5 days
They have a gestation period of 19 –21 days
They give birth to a litter size of between 7-8 and have a litter
frequency of 5 times yearly
The young ones should be weaned at 21 days

Rats
Rats mate at the age of between 10 – 12 weeks old
They have an oestrius circle which last for between 4-5 days
They have a gestation period of between 21-23 days .During this time
it is advisable to isolate pregnant rats and provide with nesting
materials to give birth to between 7-8 Litters and can give birth up to
7-9 yearly .This litters (young ones ).should be left in densely populated
cages for at least one week after weaning

Rabbits
Rabbits mate at the age of between 6-9 months . they have no definite
cycle Its advisable to introduce the doe (female) into the buck’s (male )
cage and not vis versa then transfer the doe to larger cage with nesting
materials after 24hrs
They have a gestation period of between 28-31 days. During this
period it is advisable to isolate pregnant rabbits and to provide with
nesting materials
They give birth to up to 4 litters and for up to 4 times yearly

Guinea pigs
They Mate at the age of between 12-20 months
Male to be placed in the cage with 5-10 females during mating.
They have a gestation period of between 59- 72 days .During this time
Pregnant females should be isolated and placed in separate cages with
nesting materials ,and fed with carrots,cabbages etc apart from pellets
they give birth to up to 3 young ones and up to 3 times yearly
Isolate pregnant rats and provide with nesting materials
Their oestrius circle can last for 16 days
Hamsters
Mating should be done at the age of between 7-9 weeks and should be
done on the table under supervision to avoid female injuring the male
genital after copulation
 SCIENCE LABORATORY TECHNOLOGY
They have a gestation period of between 16 –17 days
Their oestrius circle last between 4-5 days
They give birth to( Litter size) 6 young ones in one birth and give birth
up to 3-4 times yearly( Litter frequency)
Weaning should be done after 3-4 weeks .
They Should be kept in metal cages because they gnaw plastic cages.
But should be provided with wood to gnaw otherwise their incisors will
over grow hence making it difficult to feed
Xenopus frog (African clawed toad)
They are easy to maintain in the laboratory and breeding can be
induced at any time of the year, but they don’t breed until they are 2
years old. They are maintained in aquariums made from old plastic or
metal tanks with minimum size of 50X40X15 cm for stock and 30
x20x20 cm for breeding. The tanks should be carried with transparent
sheet and reinforced with wire
Adult stock can be maintained at ambient temperatures so long as it
does not fall bellow 10oc but for breeding and raring the larvae , the
temperatures should be raised and maintained at 23 oc using an
aquarium heater
Adult toads should be feed on earthworms and fine chopped liver
atleast twice a week and the water should be exchanged afterwards .
The toads should be marked by clipping one or two claws.
Xenopus toads can be induced to breed by injecting them with
pregynyl hormone which will cause them to begin to sprawn, if
sprawning does not occur , then temperatures in the aquarium need to
be raised to 28 oc. after sprawning , the adults should be removed and
the eggs distributed between containers so that atleast each egg is in
contact with the surrounding water and no eggs are left in the
surrounding solid mass . Aeration of the tank is done by means of
aquarium pump fitted to a diffuser block , these must be done gently
The newly hatched tadpoles may be feed on dried nettle powders .
Metamorphosis occurs approximately after seven weeks , during these
period , the larvae may be fed on microforms , daphnia etc but after
wards they can be feed on liver
Locust
Locust is also relatively easy to maintain in the laboratory as
permanent stock. Cages should be made of glass fronted containers of
approximately 15 x15x 20 inches with a false floor of perforated zinc.
The cages should be heated by means of electric bulbs one below the
 SCIENCE LABORATORY TECHNOLOGY
false floor and one above the floor i.e. in the locust compartment. The
floor should be arranged such that containers of sand for egg laying
may be placed underneath. The minimum size of the egg containers
should be 4inches deep and 1.25 inches diameter
It is essential to provide each cage with twiggy branches or a cylinder
of large mesh wire netting to provide perching space so as to reduce
chances for deformed adults i.e. unless the instars can hang freely
when molting , most of them will deform
Locust can tolerate a wide range of temperature but should be within
the range of 28- 34 oc. Excess humidity should be discouraged because
these can favour diseases . Excess humidity can be noticed if
condensation appear on the cages or if feces are soft or hard .
Locust should be provided with grass and sometimes wheat bran to
feed on in cage . do not overfeed them because these makes cleaning
difficult
Eggs are laid in frothy pods in holes excavated in moist sand each pod
contain between 30-300eggs .The sand must be soft and sterilized in
ovens and should always be sterilized after every period. It should have
good moisture content and should be 4inches deep beneath the floor of
the cage . Do not use porous containers as they lose water easily .after
checking for the presence of pods, cover them to avoid excessive
evaporation and set to incubate at 28- 32 oc where they will hatch after
11-17 days
NB. Some people are allergic to locust therefore avoid overexposure to
them

Earth worms
Earthworms are obtained by watering the ground with KMnO4 (aq)
and allowing it to spread . these will attract earthworms . Collect the
worms which come to the surface and rinse them before placing them
in a wormery. The wormery is made to act in the form of a typical soil
profile

Drosophila
Drosophila are grown in media which is prepared by adding about 6g
of agar to 35g black treacle and 75g of oat meal to 560cm 3 of distilled
water and boiling while stirring . nipagin is added which inhibit
 SCIENCE LABORATORY TECHNOLOGY
mold growth. These quantity is distributed into about 60 specimen
bottles and allowed to cool
When the culture media have set ,add few drops of bakers yeast
suspension , plug the container with cotton wool as soon as steam
escapes . a piece of folded filter paper or non-medicated paper should
be placed in the container before the introduction of the flies .
Optimum temperature for experimental mating is 25oc, prolonged
exposure at 10 oc or below will kill the flies and a temperature of 28-
30oc will result in sterile progeny.
Parasitic mites and molds are the major problems but they can be
completely controlled by regular subculturing if the flies are kept at 25
o
c because at these temperature , the life cycle of the flies is shorter
than that of the pest

Insect larvae
Take about 1kg meat and tie it using a string to hang on a tree branch
for about 1 week until it rot , insects will feed on it and will deposit
their eggs in it which shall hatch into larvae

Amoeba
Amoeba is cultured in shallow glass containers containing about 2cm
deep culture solution e.g. chackleys media which is prepared as a stock
solution and diluted for use . it contains
Nacl =16g
NaHCO3 = 0.8g
Kcl =0.4g
NaHPO4=0.2g
NB never use deionized water because it contain phenol which is
harmful.also extreme cleanness should be emphasized
For use , take 5cm3 of stock solution and dilute to 1dm3 with distilled
water . distribute the medium to culture dishes and about four boiled
wheat grains in each dish and allows bacteria and moulds to grow and
finally colpedium which will become food for Amoeba . now inoculate
with Amoeba
Paramecium
Add 30g of boiled wheat grain to about one liter of chackeys media and
inoculate with Bacillus subtilis then inoculate with paramecium 24
hours latter
Restraint and Handling of  Animals
 SCIENCE LABORATORY TECHNOLOGY
Animals can inflict serious injuries to humans and to themselves as a
result of improper handling.
If a study will involve significant handling of animals it is
recommended that the animals be
acclimated to the handling. Most animals, e will respond positively to
handling and will learn to recognize individuals.
The use of proper restraint and handling techniques reduces stress to
animals and also to the researcher. Animals experience stress as a
result of transportation from one laboratory to another. it is important
that the newly introduced animals must be allowed to acclimate to the
facility for three days before any experiment is done on them .
Do not make loud noises or sudden movements that may startle them.
Handle animals gently but firmly to avoid escaping especially during a
prolonged or potentially painful procedure .
 Rats are handled by grasping the tail base.
 Hamsters do not have tails, they must be grasped firmly by the loose
skin of its back, .
 Guinea pigs rarely bite, but are very easily frightened and will
vocalize and squirm to avoid restraint. The hind limbs must be
supported at all times to prevent the animal from injuring its back.
 SCIENCE LABORATORY TECHNOLOGY

 Rabbits are very susceptible to lumbar spinal luxation, resulting in

paralysis. It is necessary to support the animal's hindquarter at all
times. Although rabbits seldom bite, they can inflict painful scratches
with their hind legs. One way of lifting a rabbit is by grasping the skin
over the shoulder with one hand and gently lifting it with the other arm
cradling the body, the head nestled in the crook of your arm. Rabbits
must never by lifted by the ears.

Restraint and Handling of  Animals

Animals can inflict serious injuries to humans and to themselves as a
result of improper handling.
If a study will involve significant handling of animals it is
recommended that the animals be
acclimated to the handling. Most animals, e will respond positively to
handling and will learn to recognize individuals.
The use of proper restraint and handling techniques reduces stress to
animals and also to the researcher. Animals experience stress as a
result of transportation from one laboratory to another. it is importants
the newly introduced animals must be allowed to acclimate to the
facility for three days before any experiment is done on them .
Do not make loud noises or sudden movements that may startle them.
Handle animals gently but firmly to avoid escaping especially during
aprolonged or potentially painfull proceedure .
Rats are handled by grasping the tail base.
Hamsters do not have tails, they must be grasped firmly by the loose
skin of its back, .
Guinea pigs rarely bite, but are very easily frightened and will
vocalize and squirm to avoid restraint. The hind limbs must be
supported at all times to prevent the animal from injuring its back.
Rabbits are very susceptible to lumbar spinal luxation, resulting in
paralysis. It is necessary to support the animal's hindquarter at all
times. Although rabbits seldom bite, they can inflict painful scratches
with their hind legs. One way of lifting a rabbit is by grasping the
skin over the shoulder with one hand and gently lifting it with the
other arm cradling the body, the head nestled in the crook of your
arm. Rabbits must never by lifted by the ears.
 SCIENCE LABORATORY TECHNOLOGY
Humane killing
Humane killing is a scientifically approved method in which an animal
is killed without infringing or causing the to feel any pain whatsoever.
the choice of the most
humane method of killing an animal also requires consideration of
impacts the method could have on research results.Whenever an
animal’s life is to be taken it should be treated with the highest
respect.The method causing the least animal pain and distress should
be used.
The killing method should:
• Cause rapid loss of consciousness, followed by cardiac or respiratory
arrest and ultimate loss of brain function
• Require minimum restraint of the animal
• Be appropriate for the species, age and health of the animal
•Death must be verified following humane killing
• Personnel should be trained and competent
• Animal care committees should be responsible for approval of the
method of humane killing
NB; Humane killing should not be confused witheuthanasia killing
which means the act of killing n animal or somebody out of or because
of sympathy (e.g. normally done in rare cases in hospitals so as to
relieve the patient from excessive untreatable or unhealing pains due to
terminal diseases) euthanasia killing has not been made legal due to
ethical reasons.
Humane killing methods
There are many methods of humane killing.
Breaking of spinal cord
The animal is held by its hind legs and its cervical region or back bone
is cracked sharply across the edge of a table . It is done on Rats, mice ,
hamsters and rabbits.
Pithing
The soft skull of the animal is pierced using a sharp needle then
stirring up the brain. mostly done on frogs and toads
Knocking the head
The animal is held and the hind or fore head is knocked off with a
hammer. done on rabbits
Twisting the head
The animal`s head is restrained between the fingers and then twisted
by turning it backwards so as to break the spinal cord
 SCIENCE LABORATORY TECHNOLOGY
Overdosing with chloroform,Nitrogen or carbon monoxide
or Suffocating with CO2
The animal is placed in a closed vessel or killing jar and the chemical
substance is soaked in cotton wool and placed inside and left for
some minutes . done on rabbit , rat , mice ,hamsters etc

Emmersing the animal in the already boiled and cooled

water

The animal is dropped into oxygen free water and therefore the animal
will die slowly due to lack of oxygen.done on frogs , fish reptiles etc.

DISEASE FREE ANIMALS (SPF ANIMALS)

Specific pathogen free animals are animals that are free from specified
microorganisms and parasites but not necessarily free from other non
specified microorganisms. Such animals are normally obtained though
hysterectomy (cesarean or direct removal of the animals from the
fetus) and maintained in controlled sterile environment .such animals
are sometimes referred to as clean animals, disease free animals,
pathogen free animals.

SPF animals are therefore disease free animals that have been sought
for many years by drawing them from stocks that had been originally
established from hysterectomy and maintaining them within a barrier
that have been designed to discourage the entry of undesirable
microorganisms , these animals are normally maintained in a diet ,
nesting and bedding materials that are sterile and are usually cared
by a skilled technical staff .

The key to these development is the aseptic removal of fully

developed foetus (which have always been protected from the external
pathogens by placenta ) from the mother by heterectomy and there
introduction and maintenance in a sterile environment .however it
 SCIENCE LABORATORY TECHNOLOGY
should also be noted cautiously that some pathogens might gain entry
into the foetus through the placenta and these may cause these
animals obtained through these method not really disease free .its
therefore necessary that heterectomized or caesarian derived young
animals be quarantined in a germ free isolator until when rigorous
tests will be conducted to show that they are absolutely pathogen free
before they are allowed into an SPF building without risk of
contamination

Heterectomy must be done in a suitable room that is outside the SPF

building and the operator and their assistant must be in dressed in
protective clothing’s. The heterectomized animal should be shaved on
its ventral surface before she is humanely killed. The animal is then
pinned on a sterile dissecting board and its abdomen is covered with an
adhesive sterile tape. Surgery is done aseptically to remove the fetus
and the fetus should be protected from the unitarily outside surface of
the mother. It’s important to stress here that the se process should be
absolutely aseptically and any form of contamination either through
the instruments used or the protective clothing’s etc should be
minimized. Care should be taken so as not to puncture any part of the
mother or the fetus because these may allow entry of pathogens into
the fetus ( the foetus should be taken while still intact in or protected
by the uterus ) and should be immediately introduced while inside a
covered box(containing a long acting disinfectant) into an isolator or
in the SPF building

Inside the isolator or the SPF building, the uterus is opened with a
sharp sterilized scissors to remove the foetus. The conditions in the
isolator should be optimal for the foetus e.g. Temperature, humidity
and pressure should be favorable. The young animals should be fed
daily on sterile food directly or by fostering those on to a lactating
mother already inside the isolator or SPF built
 SCIENCE LABORATORY TECHNOLOGY
GNOTOBIOTIC ANIMALS

Animals are usually associated with a great variety of microorganisms

and parasites. A few of which may be acquired directly from the
parents during the embryonic or foetal state but majority of which
comes from the environment. Sterile techniques for rearing laboratory
animals can be provided through isolators. The demonstration that
laboratory animals can be reared and reproduce in a sterile
environment made it possible to consider the more extensive use of
this method. At present time, germ free rats, mice etc are reared in
many laboratories .infact colonies of such animals have been
maintained in a sterile environment since 1954

Such animals were earlier called microbe –free or germ free animals
but latter own these names were dropped and the name gnotobiotes
was used. Antibiotic animals are animals that are free from all
microbes or one that is associated with any combination of
organisms derived from a pure culture .i.e. these are animals that
have been isolated or are free from all microbes (but not free of
specified organisms ) . axenic gnotobiotes are those animals that are
totally free from all microorganisms , monoaxenic gnotobiotes are
those animals in which only only one particular type of microorganism
have been eliminated ,diaxenic animals are those that two types of
microorganisms have been eliminate whereas heteroaxenic are those
in which microorganisms affecting the animals have been modified
while those animals having unmodified microbiota are called
conventional animals

Uses of gnotobiotes

the use of sterile environment in maintaining gnotobiotes involves

considerable more expense and effort which must be equated with
the probable advantage of such a procedure some of the uses of
gnotobiotes are
 SCIENCE LABORATORY TECHNOLOGY
(a) they may be used in studies aimed at determining its
development and reactions in reaction to a diet that have been
chemically defined
(b) they also serve as sources of sterile organs and tissues
for cultivation for cultivation
(c) they provide good tools for research especially in
studies involving the study of immune system and the defence system
(d) when associated with one or more pure cultures , they
give an opportunity for a more critical study of the etiology of
infectious disease
(e) gnotobiotes give a more uniform susceptibility than
those that have been previously infected
(f) they are normally used in the production of colonies or
herds of animals that are free from one or more specific agent
Before maintaining or using gnotobiotes, it’s necessary to thoroughly
confirm their biological status so as to ensure that they are free from all
other living forms. Procedures for testing these should be modified
from time to time so as to reduce incidences of undetected
microorganisms. New methods should be used as soon as they become
available

Characteristics of gnotobiotes

Gnotobiotes have different characteristics front conventional animals.

they reproduce equally well without microbes , the growth rate is
within the normal range but however the humoral and cellular
defence systems is not as developed as in conventional animals .
However both systems respond in a characteristic fashion following
stimulation with antigens

Most gnotobiotes have distended caecum which may be about 30% of

the body weight .these can impair reproduction in these animals and
may become twisted in older rats and mice causing death . The content
of the enlarged caecum in large bowels have high moisture content in
gnotobiotes. These is mainly due to lack of microorganisms in the
mecum that are responsible for the degradation of endogenous
 SCIENCE LABORATORY TECHNOLOGY
proteins, mucopolysacharides and other substances which are
produced by cells lining the alimentary canal
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER SEVEN

LABORATORY EQUIPMENTS

A. BALANCES
Weighing is an essential part of almost any analysis used both for
measuring the samples and preparing the standards . Weighing must
be made to 3-4 significant figures e.g. 3.000g and therefore the
weighing device must be both sensitive and accurate .
Types of balances
(a) analytical balance
These are the most important equipment in any analytical laboratory.
They should be accurate and precise
. there are various types of analytical balances i.e.
- general purpose balance
- semi analytical balance
- micro-analytical balance
- other special purpose balance

(b) Double - pan balance

They consist of two arms of equal length LI = L2 . It also has two pans
each suspended on the either side of the arms . The masses to be
weighed MI are placed on the left-hand pan while a known weight M2
is placed on the right hand pan .weighing on double pan balance is
tedious and time consuming , it also have two
sources of errors i.e. rarely can the two arms be of equal length , any
slight difference in length between the two arms (even 1 / 10000 )
length can bring an error in weight being determined . and also the
positioning of the balance knife-edges is normally affected by the
additional load , these results in small-scale deflection which brings
about an error

(c) Single pan balance

 SCIENCE LABORATORY TECHNOLOGY

Single pan balance eliminates errors inherent in the double pan

balance . it have two knife-edges instead of three and the balance arms
are un equal in length i.e. it have a short arm and a long arm . The pan
balance and a full complement of weight are suspended from the short
arm and the longer arm has a constant counter weight together with a
damping devise built into the balance beam .hence the empty balance
is fully loaded. When the object to be weighed is placed on the pan , the
weights are removed from the shorter arm by turning the knobs on the
outside of the balance case . a set of dials shows the sum of the weights
removed . Thus weighing is done by substitution leaving the same load
on the beam at all times .
Constant loading of the beam provides constant balance sensitivity , a
characteristic not found in the double pan balance .
Once the weight of the weights removed is within
0.1g of the weight of the object , the beam is fully released and allowed
to come to rest . A scale inscribed in a glass plate is attached to the
longer arm of the balance , the deflection of the beam from its original
point of rest is greatly magnified by optically projecting the image of
these scale into a glass plate on the front of the balance . The scale
displacement in mg can be read directly and some devises such as a
vanier is employed to read to the nearest 0.1mg
Some commercial balances contain a device called tare which enables
the operator to set the balance to zero with an empty weighing bottle
 SCIENCE LABORATORY TECHNOLOGY
or beaker on a pan . the operator can then weigh the sample by
pouring
it directly into the container and hence avoiding the operation of
subtracting the weight of the container from that of the container plus
the sample .

Components of a single pan balance

The basic components of a single b pan balance are
(i) A pan on which the material being weighed is placed on
(ii) A beam which carries the sliding weight
(iii) A pointer at the end of the beam .it should rest at zero mark when
the beam is in horizontal position
(iv) A balance compensator screw for fine adjustment of the beam to
thehorizontal position

How to use a single pan balance

 Follow the manufacturers instruction
 Place the balance on a firm bench away from vibration and drought
 Place a filter paper or a small container e.g. weighing bottle or a
beaker on the pan
 Adjust the balance compensator screw if necessary until the pointer
of the beam is at zero mark i.e. the weight on the pan to read zero
 Slide the weights o the beam or adjust the dial knob (or both) so
that the balance is set to the required weight of the chemical s
 Place the chemical sample on the filter paper or in the weighing
bottle using a clean spatula . add the chemicals slowly and carefully as
the pointer approaches the zero mark
 (vii)Remove the weighed sample from the pan
(viii) slide the weights back to zero weights and clean the balance
with a soft brush , rubber bulb or with a gauze

Electronic balance
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Electronic balance
Electronic balance is based on the principle that when an electric
current is passed through a wire placed between the poles of a
permanent magnet , a force is generated which moves the wires
outside the magnetic air gap . in these system, the force associated
with the object being weighed is coupled mechanically to a
servomotor that generates the opposing magnetic force
The system contains a null detector which checks the position of the
wire in a magnetic field . These detector may be an optical device
consisting of a vane attached to the beam , a small lamp and a photo
detector .
When the two forces are in equilibrium, the error indicator is at the
reference position and the average current in the servo motor coil is
proportional to the resulting force holding the mechanism at he
reference position . When the beam is displaced from its balance
position , the amount of radiation reaching the detector changes and
causes rapid change in current through the coil.
An error signal is sent to the circuit that generates a correction
current . These current flows through the coil attached to the base of
the balance pan creating a magnetic field and restoring the indicator
to its reference position . The correction current needed to restore the
system is proportional to the mass of the balance pan . Calibration is
performed by placing a known weight on the balance pan and
adjusting the circuitry to indicate the mass of the calibrating weight.
There are many models of electronic balances available today with
numerous optional features ,but a typical electronic balance will have a
 SCIENCE LABORATORY TECHNOLOGY
way of indicating the zero setting with no load on the pan and a
digital display to show the weight of the mass .

Electronic balances however still have some errors but most of these
errors can be minimized by;
 Avoiding careless errors in weighing e.g. spillage’s and misreading
the values of the weight , the dial or the position of the vanier
 Ensuring that deliquescent and hygroscopic chemicals are weighed
immediately and in a closed containers or weighing bottles
 Glass vessels should not be wiped with a dry cloth before weighing as
these may acquire a static charge and couse an error in weighing
 The sample being weighed should be atleast at the same temperature
as the weighing balance. Heated crucibles and samples should first be
cooled to room temperatures before weighing
Electronic balance may also have other possible sources of errors i.e.
 Interference by ferromagnetism or magnetized samples
 Interference by electromagnetic radiation around the instrument
 dust which may lodge between the coil and the permanent magnet
of the servomotor
 Interference due to buoyancy and vibrations

Installation of balances
The correct positioning of balances depend a lot on the condition of the
balance room itself . The balance room should have the following
requirements
(i) Proximity – the balance room should be positioned in a
central location for easy access by many people
(ii) External environment – the balance room should not be
situated on the outside wall of the laboratory .
These is in order to avoid drought and fluctuation in temperature. It
should have only one
door opening into it and ventilation should be by means of air
conditioners and not windows
(iii) Vibration - the balance room should not be situated near
any source of vibration
such as moving machinery , passing traffic or movement of persons
near the vicinity of the apparatus
 SCIENCE LABORATORY TECHNOLOGY
(iv) Door – the door to the balance room should be fitted with a
spring to avoid vibration when it is slammed or banged humidity is
50oc and the temperature is 25 oc . constant temperature is very
important in order to prevent expansion of internal parts of the
balance .
Balance rooms should always be kept clean and the floor should be
mopped and not swept ,all the corners between the floor and the walls
should be covered to prevent accumulation of dust .
Effects of vibration on a balance
Vibration makes accurate reading on the balance to be impossible . it
also reduces the life span of the balance by causing excessive wear on
the internal parts i.e. the knife-edge . Vibration is caused by moving
machinery , traffic ,people etc
The basic method of overcoming vibration is by interposing a number
of dissimilar materials between the balance support and the source of
vibration . The materials used must be stable .if the amount of
vibration is small , the balance should be placed on a rubber stopper
and supported on a wooden support .

Care of a weighing scale

 The balance should be kept clean at all times

 Chemicals and dust should be wiped away with a soft cloth
 Always cover the balance
 Do not apply lubricants to the knife edges or bearings
 (iii) Servicing must be carried out by experts only

COLORIMETERS
Colorimeters are electrically powered instrument that are used to
measure the
concentration of the colored solution
 SCIENCE LABORATORY TECHNOLOGY

Colorimeter

Components of a colorimeter
All types of colorimeters consist of the following
(i) The light source consisting of ordinary bulb
(ii) The filter used to produce monochromatic light
(iii) Wavelength selector
(iv) Cuvettes to hold the colored solution being investigate
(v) galvanometer and absorptiometer

How to use of a colorimeter

 Switch on the colorimeter and let it warm for a duration that have
been
 recommended by the manufacturer . These is important for optimum
performance of filters and light detectors
 Set the wavelength to the correct wavelength or simply insert the
correct
 filter for the substance to be tested
 arrange the blank , standard and the test solution in the test tube
rack
 Clean the cuvettes using soft material e.g. lens cleansing tissue or
cotton
 gauze to avoid scratching them . do not touch the sides of the
cuvettes with fingers . instead hold them by the ground sides or sides
which do not face the directionof light
 Transfer the blank solution into the cuvette and place it into the
cuvette chamber with its aides facing the light path . close the cuvette
chamber and set the absorption to zero
 Remove the cuvette from the chamber , pour the blank solution back
into the test tube then pour the standard solution into the cuvette and
record the absorbenc
 repeat with the test solution and also record the absorbency
 rinse the cuvette with distilled water , drain off and wipe with the
sort material and store carefully in small boxes to avoid scratches and
dust
 calculate the concentration of the test solution as
 Conc. of test sol = abs of test sol X conc. std sol
 absorbency of standard sol
 SCIENCE LABORATORY TECHNOLOGY
Sources of error in Colorimetry
Colorimetry is subject to some errors which may be due t
 Use of faulty techniques e.g. failure to mix the solutions adequately ,
inaccurate pipetting etc
 Use of wrong filters for solution being tested
 Contamination of the reagents , standards or samples
 Use of scratched or dirty cuvettes
 Wrongly placing of the cuvettes in the cuvette chamber
 Use of cuvettes that are not recommended by the manufacturer
 Incomplete color development due to reading results early or fading
of color due to prolonged delay
 Reading solutions containing air bubbles
 Obtaining readings from colorimeters whose cuvette chambers are
open

Care and maintenance of colorimeters

 Keep them away from direct sunlight and near a power source
 Switch off the colorimeter after use.
 Clean the cuvettes and store carefully .
 Cover the colorimeter with a plastic cover to prevent dust and lock
it in a cupboard .Keep the cuvette chamber closed at all times.
 Protect the optical components from dirt and fingerprints .
 Wipe away immediately any liquid spilled on the colorimeter using
a clean gauze or tissue paper.
 Do not use organic solvent e.g. xylene , acetone , ether alcohol etc to
clean detachable filters instead use lens tissue .Store detachable filters
in a box or polythene bags .
 Replace the bulb carefully using the manufacturers instruction.All
servicing should be left to qualified experts .

C. CENTRIFUGES

Centrifuges are instruments used to hasten sedimentation of

suspended particles in liquids .

Components of a centrifuge
Centrifuge consist of the following basic components
 SCIENCE LABORATORY TECHNOLOGY
(i) central shaft spindle which rotates at high speed
(ii) head fixed to a shaft with its arms holding to buckets
(iii) mechanisms for rotating the spindle which could be
either manual rotating handle or electrical motor

Types of centrifuges
There are basically two types of centrifuges:
(i) Manual ( hand ) centrifuge
These are operated manually and cannot achieve very high speed of
rotation and are unsuitable for advanced centrifugation e.g. of blood
cells from plasma or serum. They are instead used for sedimentation
and concentration of cells and organisms in liquids e.g. urine , water
etc

Manual centrifuge
 SCIENCE LABORATORY TECHNOLOGY
(ii)Electrical or electronic centrifugeThey use electric power and
therefore can achieve high supersonicspeed and are used for advanced

separation procedures .
Electrical centrifuge

Centrifuges have maximum optimum speed by which they operate

called revolutions per minute( rpm).

Care and maintenance of centrifuges

Centrifuges require little maintenance if properly used .
They however Regular inspection and lubrication of the motor and the
spindle . to increase its lifespan and effectiveness
 Avoid uneven loading of the rotating head.
 Always ensure that the centrifuge are balanced before starting the
motor.
 Avoid spillage’s on the centrifuge .
 Avoid using centrifuge’s in corrosive atmospheres becomes these
will corrode and weaken the rotating head and motor ensure regular
inspections for worn out or loose electrical wiring
 Use appropriate size centrifuge tubes in the socket in the rotating
head
 SCIENCE LABORATORY TECHNOLOGY
 Do not open the centrifuge or stopping the machine mechanically
until when rotation have stopped by itself . these practice is dangerous
and also wears out the brushes.

REFRIGERATORS

Refrigerators are commonly used for preservation of foodstuffs ,

chemicals , biological materials , and for producing ice .They have a
liquid called refrigerant in a metal tube which is capable of being
converted into vapour and back to liquid by a device known as
compressor pump. The refrigerator has an evaporator at low pressure
and a condenser at high pressure.While the refrigerant is in the
evaporator , it boils at a temperature lower than that in the
refrigeration cabinet from which it draws warm air leaving the cabinet
cool . When the refrigerant is pumped into the condenser, it evaporates
at a temperature higher than that of the air outside the refrigerator and
therefore the warm air collected from the refrigeration cabinet
escapes to the atmosphere . The vapour condenses and becomes liquid
to circulate again.
The whole refrigeration process involves The continuos drawing of
warm air from the refrigeration cabinet and letting it escapes to the
atmosphere . The required temperature is controlled by a thermostat
which can be switched to achieve temperatures as low as – 15 oc
Refrigerators generally require little maintenance so long as they are
located in a clean atmosphere . They should be fitted with indicator
light Flammable substances must not be placed in open containers
inside the refrigerators because explosive concentration of vapor
mixtures can be formed from very small amounts of solvents which
may ignite sparks from thermostats or micro switches. The inside
should be kept clean and dry.
Avoid placing hot things in a refrigerator .Let them cool first.
Avoid packing things tightly into the refrigerator because they interfere
with free air circulation .
Defrost the deep freezer region to prevent building up of mountains
of ice which often breaks the delicate part of the refrigerator.Keep the
doors of the refrigerators closed except when putting or removing
materials from it . Frequent or prolonged opening of the refrigerator
doors interferes
 SCIENCE LABORATORY TECHNOLOGY
with internal temperatures .Repair and servicing should be done
regularly and by experts only .

AUTOCLAVES.
An autoclave is a closed vessel in which steam is subjected to pressure
that is greater than the atmospheric pressure . It is used for
sterilization

Autoclave(closed)

Components of an autoclave
 Autoclave chamber - these is made of a heavy cast metal
 Tightly fitting lid with clamps and rubber seals
 Metal packing drum which fits inside the autoclave chamber
 Pressure valve set to open at the desired pressure.
 Safety valve through which steam may escape if the pressure inside
the autoclave exceeds a set safety value
 Pressure gauge indicating internal pressure
 Temperature gauge indicating the temperature of the steam
 The air outlet used to vent air outside while heating and to also
introduce air after sterilization
 a built in heating and water cooling system
(x) Replaceable rubber seal
 SCIENCE LABORATORY TECHNOLOGY

Autoclave (opened)

How to use an autoclave

 Caps of containers to be sterilizedshould be loosen , other items
should be wrapped in porous brown paper and their edges carefully
secured with autoclave tapes
 (ii)Place the containers and other items in a wire basket or in the
autoclave drum.
 (iii)Pour distilled water or tap water into the autoclave drum to the
level indicated by the manufacturer.
 (iv) Place the basket or drum containing the items to be sterilized in
the autoclave .(v) Close the lid and screw the lid clamps firmly and
connect the horse to the air outlet.
 (vi) Switch on the autoclave electric or gas plug or place it on the a
suitable heat source e.g. charcoal
 jiko.
 (vii)As the water boils , a mixture of air and steam escapethrough
the air outlet . leave it to escape for about 5min.
 (viii) Test if all the air have been expelled out by placing the
other end of the horse inside a water bucket . the presence of air is
indicated by bubbles , pure steam produce cracking sound as it
ondense into water
 (ix) When all air have been expelled , close the air outlet and watch
the pressure and temperature gauge .when pressure reaches 15 psc ,
reduce the heat and leave it for the recommended sterilization time
using a timer clock
 SCIENCE LABORATORY TECHNOLOGY
 (x) Turn off the heat and wait until the pressure gauge reach zero,
open the outlet then open the lid and unpack .
 Some autoclaves have indicator strips on the autoclave tapes which
changes color after sterilization .
NB: indicator strips are sometimes not reliable.

Care and maintenance of autoclaves

(i) Follow the manufacturers instructions
(ii) Always fill the autoclave with the correct volume of water .
Irreparable
damage may occur if the autoclave boils when it is dry .
(iii) Do not use saltwater as these enhances rusting of the autoclave
(iv) Lubricate the screw s , clamps with oil
(v) Clean the autoclave immediately after use
(vi) Servicing must be carried out by experts only

WATER STILLS AND DEIONIZERS

Water for laboratory use must be free from contamination’s that could
interfere with the performance of the test . The quality of water
therefore depends on the type of the work to be carried out.

Water is required for the preparation of

(a) Reagents and stains
(b) For cleaning and disposal
(c) For preparation of culture media

The following methods can be used to obtain water of adequate purity

and quantity
(i) Distillation using water stills
(ii) Filtration using gravity filters
(iii) Deionization using mixed bed ion exchange resin
Distilled or deionized water is recommended for analytical work
(a) distillation
In these method impure water is boiled and steam produced is
condensed on a cold surface (condenser) to give chemically pure
distilled water from which volatile organic and inorganic matter are
removed . Distillation however does not remove dissolved gases e.g.
NH3 , CO2 and Cl.
 SCIENCE LABORATORY TECHNOLOGY
It also uses a lot of electricity and too much water goes to waste in
the process of cooling the distillate ,the condensed water is collected
when it is hot and will require cooling before it is used for analytical
work . Distilled water should be clear , colorless and odorless

Water Still
Distillation process is carried out in a water still . a considerable
volume of water is required to operate a still ,
the water feeding the still should not be heavily contaminated .The
condenser of the still should be preferably made of pure fussed quartz
. If really pure water is desired , then it may be advisable to b distill the
water for about three times . In regions where there is hard water , the
still elements and its walls acquire a scale and for effective distillation
to take place , these formed scales on the walls must be removed
regularly by using acids to dissolve it .A wide range of stills are
available, the automatically operated ones are the most expensive ones
and are capable of producing highly purified water for research and
specialized laboratory work i.e. grade 1 and grade 2 water quality ,

Use and care of stills

 Read and follow the manufacturers instructions carefully.
 Ensure there is a sufficient supply of cool running water to feed the
condenser.
 Do not allow the boiler to run dry
 Collect the distillate in a clean plastic or glass.
 Avoid storing the distillate in for more than few days , always keep
the
 container capped .
 Clean regularly the still , discale it incase you always hard water .
NB when buying a still , select one that can be easily be cleaned
without being
dismantled

DEIONIZATION
In these process impure water is passed through an ion exchange
resign to produce ion free water . Deionized water is more purer than
distilled water and it is sometimes called conductivity water because
it have such a low electric conductivity and is most suitable in
conductivity measurements , it have almost neutral pH and is free
 SCIENCE LABORATORY TECHNOLOGY
from water soluble salts , and sterile. it may however have some non
electrolyte contaminants and some organic extracts from the resins.

ION EXCHANGE RESINS

Resins are of two types :
(i) Cation exchange resin
(ii) Anion exchange resin.
De-ionizing resins are cost effectively used if already filtered water is
used . The use of unfiltered water will lead to rapid contamination
and exhaustion of the resin .Resin beds may also contain both cation
and anion resins and an indicator that changes color as the resin
becomes exhausted . The filtered water trickles through the resin and is
deionized and passes through the small holes in he bottom of the tube
into a collecting container .
Deionisation process s faster and a lot of water is produced within a
short period of time , deionizers are portable and cheap to operate. But
however the water may not be sterile and not all non-electrolytes are
removed

Maintenance of deionizer resins

 Keep the deionizer tube out of sunlight to avoid deionization of the
indicator
 When not in use , remove the deionizer tube and store in dark place ,
these will protect the indicator and also minimize contamination of the
resin column
 When first used after storage , discard the water passing through the
column for the first five minutes.
 Keep un used resin in an opaque sealed air tight plastic bag or tin .
NB deionizer resin can cause irritation if allowed to enter the eyes or
skin . it is therefore advisable to wear protective clothing e.g. goggles
when filling the plastic tubes .

NB; Distilled water is free of inorganic materials, suspended

impurities, and most organic contaminants. Deionized water is free
of inorganic materials and most suspended contaminants.

INCUBATORS
 SCIENCE LABORATORY TECHNOLOGY
Incubators are special ovens used to provide optimum temperatures
for living things so as to provide optimum conditions for their growth .
Its construction is finer than that of oven . The inside is lagged with
special materials which serves as insulators. The temperatures inside it
can be maintained between 0 – 80 oc with an accuracy of + or – 0.1oc.

Incubator

They have glass inner doors which allow the user to check the sample
without interfering with the actual conditions inside the chamber .
Anhydride incubators have pockets of desiccants e.g. silica gel which
absorbs moisture whereas hydride incubators have trays of water
which evaporates and supply the incubator with the necessary
humidity.
Incubators are electrically operated and have thermostats which
regulate and control the temperatures inside.

OVENS AND MUFFLE FURNACES

Oven are used for drying , sterilization and baking. The temperatures
inside range from 0- 250 oc. they normally made of two walls lagged
with glass wool. They also have thermostats which control the
temperatures and uses mercury thermometers. They also have other
accessories like ventilators to remove moisture , shelves etc.
 SCIENCE LABORATORY TECHNOLOGY

Hot air oven

Furnaces are used for ashing and fusion . Muffle furnaces are able to
withstand temperatures as high as over 1000 oc . They are made of
asbestos plates lagged with asbestos wool or powder . the
temperatures inside are measured by a pyrometer thermometer or any
resistant thermometer and the temperature control device is a
thermocouple thermostat. Accessories include chimneys to remove
smoke

WATER BATH
Water baths are special water troughs heated by electric coils below .
they provide working temperatures above the normal room
temperatures .it is constructed in such a way as to prevent heat loss by
conduction or convection i.e. they are made of double metals, plastic or
wood lagged with a poor conductor in between.
There are two types of water baths i.e.
(i) Thermostatic water bath -can measure and maintain temperatures
correct to 0.1oc , they use toluene , mercury or capsules to control
temperatures
(ii) The ordinary water bath which can measure and maintain
temperatures correct to 1 oc , they use bimetallic strips to control their
temperatures
 SCIENCE LABORATORY TECHNOLOGY

Water bath

HOT PLATES

Hot plates are used to provide moderate heating in the laboratory

especially .for long periods of heating . They are not very efficient since
they lose a lot of heat.
They have thermostats to regulate their heating temperatures.

Hot plate

THE MIXERS AND SHAKERS

These are special instruments used in the laboratory to provide
uniform or homogenous mixtures of liquids and solids. They have flat
basses onto which the substance to be mixed are placed and the
instrument is switched on , it provide swirling or shaking movements
that will cause the substances placed on it to mix substances placed on
it to mix.
 SCIENCE LABORATORY TECHNOLOGY
CHAPTER EIGHT

LABORATORY
ORGANISATION
Laboratory management is mainly concerned with the general
organization and safety of the laboratory , provision of materials for
laboratory work ,repair and maintenance of laboratory equipment’s
and maintenance of proper and up to date inventory for the
laboratory .
Poor laboratory organisation is the major cause many hazards and
accidents that would otherwise have been prevented. Most of this
accidents occur because of :
 Overcrowding and lack of laboratory space .
 Carelessness by the laboratory staff in organizing and planing the
laboratory area and to give proper instruction – the laboratory area
should be always clean and without obstacles on the way . the tables
should not be congested with practical experiments and should be
under supervision.
 Reluctance by the laboratory staff to ensure that the laboratory
service appliances e.g. electric switches and gas taps, instruments and
equipments e.g. vacuum chambers , ladders and shelves , stools and
benches are repaired and properly maintained
 Failure by students and staff to carefully read and follow laboratory
activity instructions and laboratory safety rules.
 Overloading of electric appliance
 Poor chemical storage and handling procedures
 Lack of proper disposal mechanism e.g. buckets should be provided
for keeping broken glassware, used reagents ,papers and specimens
 Lack properly laid down instruction as well as guidance for every
laboratory activity .
 the rules and instructions should be printed in legible writings and
placed in strategic positions where every student will be able to see
 Inadequate students orientation on techniques, procedures and
safety instructions during their first encounter in the laboratory
 SCIENCE LABORATORY TECHNOLOGY
 Disorderliness and insufficient cleanness in the prep, store and the
entire laboratory working area . The laboratory floors and benches
should not be slippery, should be tidy and not congested with
unnecessary chemicals and equipments
 Lack of adequate safety measures and protective clothing .
 Students should also be taught about how to use them every time
they are in the lab .

Ways to minimize accidents in the laboratory

 The laboratory staff should give adequate rules and instruction as
well as guidance for every laboratory activity . the rules and
instructions should be printed in legible writings and placed in
strategic positions where every student will be able to see.
 The students should be oriented and pre-tested on techniques,
procedures and safety instructions during their first encounter in the
laboratory.
 The laboratory staff should ensure orderliness and cleanness in the
prep, store and the entire laboratory working area . the laboratory
floors and benches should not be slippery, should be tidy and not
congested with unnecessary chemicals and equipments.
 The staff should ensure that all gas taps , electric switches, water
taps and vacuums systems are in proper working order i.e. they should
not be leaking or tempered with.
 All the laboratory equipments and instruments used should be
repaired first tested before giving it to students for practices.
 All chemical experiments done in the laboratory should be first done
in the prep room and procedures and guidance provided before
students are allowed to undertake the practice
 The staff should ensure that the lab is properly ventilated and
lighted
 The staff should ensure that all used chemicals and broken
glassware are properly disposed and that no chemicals whose reaction
is not known is undertaken in the laboratory.
 The store room should be safe and toxic , corrosive and flammable
chemicals locked into their safe cabinets and that no unauthorized
person is allowed in without permission.
 (x) Adequate safety measures should be put in place and at an
accessible location and students taught about how to use them incase
of an emergency.
 SCIENCE LABORATORY TECHNOLOGY
 Students should be provided with adequate protective clothing’s e.g.
goggles and gloves if the experiment shall deal with harmful chemicals
and microorganisms

Ways to improve laboratory organisation

The overall organization and safety profile of any laboratory can be
greatly improved if the entire laboratory was
properly designed in the first place .
The design should include :
 Provision for better working tables and floors .
 They should be easy to clean and relatively chemical resistant. The
lab furnitures should provide maximum student spacing and
movement in order to avoid overcrowding.
 Provision for good ventilation . Each laboratory should have atleast a
fan properly positioned .
 Provision for enough space to accommodate the required students
capacity and space for installing laboratory equipment’s.
 Provision and proper location of fume chambers.
 It should be located away from heavy students traffic areas and
away from main exit.
 Provision for laboratory safety requirements including fire
extinguishers , shower rooms, first aid rooms etc. all safety
equipment’s must be marked with location placards or signs
 Provision for adequate supply and proper location and installation of
all the required laboratory services including water , electricity , gas ,
vacuum etc
 Provision of maximum lighting that will not strain visibility
 Provision for adequate storage area for apparatus , hardwires and
equipment’s.
 Provision for adequate sinks and other places of disposal.
 Provision for preparation room with gas , electricity and a sink.
Prep rooms should also have fire blanket , eyewash , fire extinguishers
etc. the prep and storage rooms should have a vinyl tile or concrete
floor , their doors should have self return hardware with an automatic
lock and a fire rated door . The doors should swing out if it’s the only
exit door.

LABORATORY INVENTORY
 SCIENCE LABORATORY TECHNOLOGY
Responsible laboratory staff will agree that its absolutely necessary to
know exactly
what chemical, apparatus and equipments are present in the
laboratory and in what quantities. This is what is meant by the term
inventory. Inventories serve many valuable purposes such as
 To comply with the regulatory requirements
 To increase efficiency in the laboratory
 It rids the laboratory of acquiring excess or unused chemicals ,
apparatus and equipments.
 It enhances efficient implementation of storage of all remaining
chemicals in compatible chemical families and ensures isolation and
safety storage particularly of hazardous substances.
 It helps in creating and maintaining perpetual inventory of all
chemical substances.
 It helps to easily identify substances or items needed for
replacement and thus makes easier the requisition process. It also
serves as a reminder of items borrowed and owed.
 It helps to check and confirm substances expired and those that
need repair and hence enables proper disposal or maintenance.
Laboratory chemicals should not be purchased like any other routine
supplies i.e. their stock should not be bought in bulk. Because this will
result in purchasing of large quantities of chemicals. Most of them may
not be required at that particular time, and may end up being stored in
lab store rooms that were never designed to handle such quantities
and rarely equipped to meet even the minimum safe storage standards
such chemicals may end up expiring even before they are used .
It is infact wisdom to buy smaller quantities since most of them will
be used and there will be less hazards associated with their use and
storage .
There is great need to recognize the problems created by lumping
hazardous chemicals into the buying routine . These will aggravate and
perpetuate the problem of storage and will increase wastage. The
laboratory staff should adopts laboratory a chemical requisition policy
which should be aimed at completely eliminating or reducing the
quantity of some substances purchased and also oversee the
requisition process so as to ensure that proper chemicals in proper
conditions are bought and stored in proper conditions .
 SCIENCE LABORATORY TECHNOLOGY
Laboratory inventory taking is a task that requires teamwork and
cooperation between the laboratory staff and the administration but
students should not be involved in this process.
Each item in the store should be listed by name, the approximate size
of the container and the amount still left in the container and its expiry
date. If the shelves are loaded, Its is advisable to remove some
containers in order to see those at the rear end of the shelf . Its also
appropriate to isolate and scrutinize the bottles carefully as these will
enable you to decide which one should stay and which one should be
disposed. Disposal should be in accordance with the properties and
regulations concerning that particular substance.
Those items which are to remain on the shelf can now be reorganized
into their compatible chemical families.

Laboratory Store management

The specific objectives of laboratory store management are :
 To ensure the availability of
 chemicals and equipments when needed.
 To reduce the cost of storage as much as possible by avoiding
deterioration and wastages .
 To maintain accurate records and provide management data.
 To repair and maintain laboratory apparatus and items.
An ideal store is that one which can meet the following requirements
 It must economically provide its services in a manner that it will
economize funds allocated to it.
 It should simplify the problems encountered in classification and
identification of chemicals , apparatus and equipment’s .
 It must allow continuously flow of materials in and out of the
laboratory so that stock balances are adequate to support the current
rate of consumption by the laboratory . It must therefore adopt an
efficient and reliable system of receiving or giving out all materials ,
equipment and apparatus.
There is great need to recognize the problems created by lumping
hazardous chemicals into the buying routine because these will
aggravate and perpetuate the problem of storage and will increase
wastage .
The laboratory staff should adopts laboratory a chemical requisition
policy which should be aimed at completely eliminating or reducing
the quantity of some substances purchased and also oversee the
 SCIENCE LABORATORY TECHNOLOGY
requisition process so as to ensure that proper chemicals in proper
conditions are bought and stored in proper conditions .

Stock taking, Stock checking and Stock auditing

Stocktaking is the process of verification of quantities and conditions
of gods on periodic basis, laboratory stock levels should be appropriate
for the lab. They should not be too high or two low. High stock levels
will result in undue amount of money being spent, greater risk of
deterioration and damage , while low level will endanger supplies
needed immediately for use and may demand impromptu purchase
hence implying high prices .
Stock checking is similar to stock taking but it its mainly concerned
with the verification of the suitability of the chemicals and even the
operation of the equipments in the lab. Stock checking helps to confirm
whether the chemicals in stock are expired or deteriorating it also
checks whether the lab equipments e.g. balances , incubators ,
microscopes etc are working as desired or faulty.

Stock auditing is the actual verification of value of stock in monetary

terms . It’s a process that is normally done by hiring an external
agency. These helps in preparing auditing report .

Purchasing of lab items

After stocktaking and stock checking process, a requisition is normally
made for those items that need to be replaced .
Acquiring supplies and controlling the inventory may be an operations
function that occurs totally within the laboratory. However, external
departments are always involved, at least to the extent of receiving
incoming shipments, transferring goods to the laboratory, and in
payment through accounts payable. Staff members within the
laboratory must recognize their finite roles and establish clearly
defined procedures to be implemented by well-trained personnel.
Careful attention to the entire process is essential so that the system
works smoothly and does not burden the laboratory or external
departments.
Generally, the laboratory is responsible for specifications, as well as
storage and use of products. Most often, communication with the
vendor is required, especially concerning product improvements,
 SCIENCE LABORATORY TECHNOLOGY
method changes, and similar information that affects the test
procedure or quality control.
The laboratory must accept the responsibility for these functions and
provide accurate records to allow traceability for overall audit
purposes.
As a first step, each department head/supervisor should prepare a
rough draft listing the products used and how frequently they need to
be ordered specific information should include the name of the
product, catalog number, package configuration, average monthly
usage (AMU), unit of measure (U/M), normal lead time for delivery,
and storage area or single site use.
Unless the supply system is thoroughly understood and operating
smoothly, panic situations occur because of shortage or overstocking.
Obsolescence is also a strong possibility if expiration dates are
imminent or if improved technology changes the needs of the
laboratory.
Routine orders and standing orders should be reviewed every 6
months, at a minimum, to ensure the requirements, vendors, and
pricing are acceptable and correct.
It is important that managers and supervisors verify that pricing
changes are factored into the laboratory budget. Involving major
vendors in the process will help determine the likelihood of pricing
changes in the upcoming fiscal year.

Basic Function Definitions

Purchasing
The department or person authorized to purchase goods and services
for the laboratory.

Supplier/Vendor
That person or entity for whom the purchase requisition for goods or
services is directly intended, e.g., distributors and manufacturers.

Receiving

The department that handles the physical receipt of material; the

inspection of the shipment for conformance with the purchase order
(quantity and damage); identification of and delivery to laboratory
destination; and the preparation of receiving reports.
 SCIENCE LABORATORY TECHNOLOGY

Accounts Payable
The accounting function of comparing the receiving record with the
purchase order to ensure that the information agrees prior to payment
of the supplier.

Materials Management
The function that is responsible for knowing the unit cost of supplies,
the logistics to support receiving, distribution, and disposal of
materials.

Inventory Records
The management tools used to set up specifications, adjust to varying
usage, and provide a smooth flow of supplies and audit records. These
may include but are
not limited to traveling purchase requisitions, periodic count records,
perpetual inventory records, and specialized inventory records.

Supply Requisitioning
This function involves recommending when to order and the quantity
to order (based on usage, delivery time, quantity, packaging, available
storage space, and
similar factors). Because supply requisitioning is one of the most
sensitive parts of the inventory management process, training and
good judgement are required.
To ensure that there is a mutual understanding of expectations, any
change to orders, shipping dates, or pricing should be clarified between
the laboratory and
the vendor before the order is placed.

Purchase Orders
The official record sent to the supplier to authorize shipping and
billing. To ensure that there is a mutual understanding of expectations,
any change to orders,shipping dates, or pricing should be clarified
between the laboratory and the vendor before the order is placed.

Blanket Orders
A long-term (e.g., 6 months) commitment to a supplier to ship based
on delivery authorizations specifying delivery, often over a short period
 SCIENCE LABORATORY TECHNOLOGY
such as 15 to 30 days. This procedure is especially useful for shortdated
products.

Standing Order (Ordinary)

An order with a vendor to deliver a specific quantity at stated intervals.
Because changes in usage may increase or decrease needs, inventory
should be reviewed periodically (e.g., every 3 months) to keep standing
order quantity in balance with needs. At least 30 days notice should be
given to the vendor when a standing order is changed.

Standing Order (Protected

An order that covers a specific lot number to be delivered over a long

period of time, in which case the vendor sequesters inventory for a
specific customer.
Some vendors also protect the inventory designated for customers with
standing orders by declaring an item out of stock for other customers
for an agreed time interval, usually 60 days. Such arrangements with a
supplier may require a price adjustment for inventory carrying costs.

Backorder
An unfilled customer order or commitment that places an immediate
demand against an item which has inventory insufficient to
immediately satisfy the demand.

Confirming Order
A purchase order issued to a vendor, listing the goods or services and
terms of a verbal order or written order before issuing the usual
purchase document.

Delivery Schedule
The required or agreed time or rate of delivery of goods or services
purchased for a future period.

Order Preparation Lead Time

The time needed to analyze requirements and open order status (if
any) and to create and release a purchase requisition or a work order.
 SCIENCE LABORATORY TECHNOLOGY
SUPPLY ANALYSIS AND IDENTIFICATION
Laboratory Section Lists
Development of a classification and list of products used by the
laboratory, preferably by section (i.e., Chemistry, Cytology,
Hematology, Histology, Immunology, Microbiology and
Radioimmunoassay), is the first step in addressing any
inventoryproblem.

Basic Analysis
Begin with a basic analysis of items used . Use the form to list not only
the products used by the section but also to indicate the following
pertinent information:

(1) complete description of the item;

(2) unit of count;
(3) approximate (average) usage per month (variances may be useful);
(4) priority level: high, medium, low
(5) order lead time/delivery time;
(6) where stored and conditions of storage;
(7) comments such as supplier/catalog number.

Demographic Information
The form may be expanded to include most of the product
demographic information. The additional information is not needed for
the basic analysis. It would, however, expedite the development
process in future steps. As a minimum, include the specifications of the
product in and the following specifications:

(1) standard packaging and alternative configurations, if available and

known;
(2) part number or manufacturer's number;
(3) vendor (alternative vendors should be listed if available) and
notations of when no substitutions are permissible;
(4)shipping costs (important for determining budget allocations)
because some materials are expensive to ship.

Department Forms
As the forms are completed by each of the sections in the laboratory,
verify the products listed and list them alphabetically for each section.
 SCIENCE LABORATORY TECHNOLOGY

Form Review
Because one product might serve several sections, review and analyze
the lists from all laboratory sections to identify duplicate or similar
items.

Inventory Monitoring
Assign responsibility for monitoring the inventory in each of the
storage areas (i.e., hospital storeroom, laboratory storeroom, section
storage areas, and special storage areas for flammable supplies or
refrigerated supplies).
Different inventory control procedures may be needed for the various
storage areas. Traveling purchase requisitions may be convenient for
ordering and for special storage areas such as corrosive, flammable, or
refrigerated products not kept in individual section storage areas.
Weekly physical count records or perpetual inventory records may be
used for both hospital and laboratory storeroom products. A traveling
requisition system and a perpetual or a weekly count inventory record
are needed for section storage areas supervised by a section
supervisor. The specific steps are explained in the following sections.
Product Demographics
Product Listing
After all products are listed by laboratory section and storage area,
obtain the demographic information for each product.
As mentioned , much of the product description can be recorded at the
same time the product lists are generated.

Minimum Information
As a minimum, obtain the following demographic information for the
product:

(1) complete description;

(2) standard packaging and alternative configurations, if available and
known;
(3) part number or manufacturer's number;
(4) vendor (alternative vendors should be listed if available);
(5) price breaks (i.e., standing order);
(6) unit cost;
 SCIENCE LABORATORY TECHNOLOGY
(7) all special ordering instructions (i.e., required delivery schedule and
deadline for receipt; need for product to be all one lot number; need for
product to have long shelf life).

MONITORING INVENTORY
To ensure the efficient, uninterrupted, and economical operation of a
laboratory the following factors are evaluated:

Requirements
Requirements are demands on a unit/time basis that depend on
historical (past) and projected usage data. The rate of use can be
determined by checkingpurchase records over a 6-to-12-month period
and by reviewing specific laboratory test volumes. Measure the average
amount of stock to be maintained against a 30-day period.
Procurement Time
Procurement time, also known as order preparation "lead time," is the
total period of time necessary to obtain a new supply of an item or to
have any item replaced that may have been unavailable due to an
unforeseen variable.

Optimal Frequency
Once these factors are considered, determine the optimal frequency of
measure by evaluating the activity of the item. Assign a priority to the
actual frequency of review and measure as shown in the table on the
following page. Alternatively, the actual frequency can be inserted. The
frequency should be part of the procedures manual of the appropriate
section.

CONTROLLING INVENTORY
Effective inventory control is expected to increase efficiency by
providing an uninterrupted flow of materials needed to operate the
laboratory, while reducing both quantity of inventory and the physical
space needed for its storage. Analysis of the effects on use and on
storage can be used to develop master supply lists for inventory usage.

Inventory Levels

To save space and reallocate funds for other needs, inventory should be
maintained at a minimum level. Inventory is generally measured in
 SCIENCE LABORATORY TECHNOLOGY
"weeks' supply," or, the amount of a particular item used in a week's
time. Use a minimum of 2 and a maximum of 6 weeks' supply as an
initial guideline to establish inventory levels. Inventory level is the sum
of the minimum inventory plus safety stock.
Vendors can and should help in determining minimum and
particularly maximum inventory levels for the laboratory. If the vendor
will "sign up" for rapid shipment turnaround time (from receipt of
order) and is reliable (e.g., no or minimal backorders or delayed
shipments), maximum inventory levels can be reduced, which results
in real cost savings and improved cash flow to the customer.

Inventory Rotation
To rotate inventory, use older supplies before newer supplies.Place new
shelf items behind the older items so the older items will be used first.
Label the containers holding stores.

Minimum Inventory Level

Minimum inventory levels are the amounts necessary to support
normal operations until additional material can be supplied, either
from a vendor, an isolated storeroom, or general storeroom.

Safety Stock
Inventory maintenance depends on many factors that the laboratory
cannot control. Lead time is the total time between placing an order
and receipt of the product in the laboratory. Several departments may
be involved with order processing in addition to the manufacturer,
distributor, and shipper. Lead time is determined through analysis of
past history of supply and is a prime consideration in setting inventory
levels. Safety stock is that amount of inventory that allows for unusual
demand or usage plus the usual lead time for receipt of the item. In
setting safety stock, consider the product's importance in laboratory
operation.

Order Point

The order point is the inventory level at which the decision is made to
replenish stock. The order point is generally the minimum inventory
level.
 SCIENCE LABORATORY TECHNOLOGY
Handling/Storage
Receipt/Expiration Dating
Review manufacturers' expiration dates on laboratory material upon
delivery and determine the acceptability of the item. Shipments must
be reviewed by a person who has a knowledge of the product usage, so
that a decision can be made before the item is entered into inventory.
To prevent the possibility of disagreements with the vendor, it is
important that the laboratory establish an upfront understanding of
expectations with regard to delivery and expiration dates.
Arrange with the supplier a return mechanism for rejected shipments.
Advance arrangements will reduce negotiation with the supplier who
must take back material with short expiration dates. The supplier is
also alerted that dates are reviewed as products are received by the
laboratory, thereby reducing the likelihood that the supplier will ship
shortly-expiring material in the future.
Receipt-date all products upon entering them into inventory.

Product Changes by Manufacturer

Configuration Changes
The manufacturer/supplier should notify the user of package
configuration changes prior to shipment to allow for appropriate
adjustment for storage location and space.

Methodology Changes
To alert the user of changes in methodology, color or graphic changes
in packaging and product labeling should be used by the manufacturer.
Package inserts should clearly note the revision date and direct the user
to the specific change(s) involved. Laboratories should have a
documented protocol for ensuring that changes to the package insert
are incorporated into the current laboratory protocol and procedure
manual.

Change Notification
Those laboratory employees who participate in the inventory function
should formally acknowledge vendor change notification because it
may require attention by laboratory management.

Recalls
 SCIENCE LABORATORY TECHNOLOGY
Unsatisfactory products that must be returned to the vendor should be
announced to the laboratory administration. To avoid possible usage
before return shipment or on-site destruction, all merchandise to be
sent back or destroyed (as requested in some cases) should be
immediately isolated and appropriately marked.

Storage
Adequate space must be provided for inventory storage. The inventory
area may be an internal laboratory storeroom or part of general supply
area. There should be adequate space to stock inventory items and to
permit a clear view of all inventory. Unpack shipping cases and store
items in the unit by which they will be delivered to individual sections.
Certain items may fall into special categories and should be stored
outside the supply area, as appropriate.

Section Supplies
Store items that are purchased rarely, have a short shelf life, or in small
quantity for use by a single section within that section and requisition
them as needed. For example, radioimmunoassay (RIA) kits and blood
bank reagents have short shelf lives and are probably best controlled
within the section involved. Nevertheless, apply the same basic
principles of inventory control to these items as to general laboratory
inventories.

Refrigerated Supplies
Reserve a master storage area for inventory that must be kept under
refrigeration. Back up freezers may be required in addition to those
necessary for minimum inventories (i.e., minimum freezer space
should exceed the volume necessary to store minimum frozen
inventories).

Flammables and Hazardous Chemicals

Reserve specialized storage areas for flammable reagents and
hazardous chemicals (e.g., strong acids and bases).

Inventory Recording
Devise a system to maintain a written record of inventory levels and
usage. This record may be as complete as desired, but it should include
at least the item name, a minimum quantity that sets the order point,
 SCIENCE LABORATORY TECHNOLOGY
and a maximum quantity that sets the order quantity. Depending on
requirements, this system can be a weekly count record, a perpetual
inventory record, or a specialized inventory record. It is possible to
train clerical staff to be responsible for inventory recordkeeping.

Periodic Count Record

Where strict control of inventory is not necessary, make a weekly or
biweekly count of in stock inventory and simultaneously update written
records.

Perpetual Inventory Record

Where strict control of inventory is desired, maintain a perpetual

inventory record. Note the date and movement of individual items into
and out of inventory storage. To operate the system most efficiently,
limit the number of people who have access to inventory storage areas.
Accuracy of physical count and inventory record count is mandatory. A
comparison of actual stock on hand and the record count must be made
at least annually.
Specialized Inventory Record
Replacement parts for instruments are a good example of a slow-
moving, limited quantity inventory that is vital to laboratory
operations. Keep a record of replacement parts showing the name;
catalogue, model, and serial numbers; and manufacturer's name and
address. Provide adequate space for recording inventory periodically
(e.g., on a monthly or bimonthly basis).

ORDERING SUPPLIES
The manner in which inventory is ordered may vary depending on the
size and function of the laboratory and the requirements of the
institution's materials management or administrative departments. A
single person may be responsible for ordering in a small laboratory,
whereas a number of persons may be more practical in a larger,
departmentalized laboratory. It is important that the laboratory have a
protocol documenting the procedure to ensure that mutual
expectations between internal "customers" (e.g., the laboratory,
purchasing, and accounting) are clearly defined.

Specifications
 SCIENCE LABORATORY TECHNOLOGY

The basic information necessary for ordering inventory must be

provided in a thorough and explicit manner. As a minimum, include
the following product
specifications:

(1) Complete description;

(2) Standard packaging;
(3) Part number or manufacturer's number;
(4) Vendor (alternative vendors should be listed if available);
(5) Price breaks (i.e., standing order);
(6) Unit cost; all special ordering instructions (i.e., required delivery
schedule and deadlinefor receipt; need for product to be of all one lot
number; need for product to have a long shelf life).

Ordering Formats
The prescribed format for a requisition will be determined by the
laboratory's needs and organization and the institution's materials
management and administrative departments. In creating any ordering
system, attempt to limit the required paperwork by consolidating
several functions into one form.

Order Forms
In some institutional settings, the within-laboratory form is used to
prepare the institutional requisition , which is then used as the
reference for the purchase orderthat is sent to the
manufacturer/distributor.

Inventory Control Sheet

We recommend an inventory control sheet or stock inventory record
because it combines the inventory control and the laboratory ordering
functions.
Regardless of the format, each list must contain all the following
information : maximum/minimum stock levels; an area to record the
periodic inventory; an area to document that a product has been
ordered (including the date and quantity ordered); and for what and
where the product is used.

Periodic Inventory
 SCIENCE LABORATORY TECHNOLOGY
The actual periodic inventory can be taken by whomever is responsible
for a laboratory section, area, instrument, or analysis.

Review
The inventory control sheets are reviewed by the laboratory supervisor
and an order generated using the institution's requisition.

Signal Flags
Place signal flags on lists that contain items that have been ordered. If
the items are not received within the appropriate length of time, the
signal flags will alert the laboratory and a follow-up inquiry can be
made.

INVENTORY EFFECTIVENESS REVIEW

A review of the products for changes that might affect the entire
inventory control program is a fundamental but sometimes overlooked
aspect of laboratory inventory control. The inventory control program
must incorporate, where appropriate, an effectiveness check
mechanism to address these product changes. To assure continued
supply in the proper quantities, as well as continued proper use, early
recognition of product change is important. The following sections
discuss some product changes that can occur and that should be
identified via an inventory effectiveness check system.

Required Changes
Potential or required changes for the specific source of supplies and/or
changes in specific brand names. Changes in supply brand or
distributor are sometimes initiated from within the laboratory but may
originate in the purchasing department or even with the vendor.The
laboratory should let the purchasing department and the
vendor/distributor know what alternate brands are acceptable and any
that are specifically not acceptable. A vendor should not be permitted
to substitute a brand not on the list of acceptable alternatives without
consulting the laboratory.
InvestigationInvestigate changes in product source, i.e., vendors or
distributors, for impact on supply capability, delivery lead times,
differences in shipping and/or billing procedures, availability of
 SCIENCE LABORATORY TECHNOLOGY
discounts, and availability of the proper or required brand name
product.

Evaluation
Evaluate changes in specific brand names for their impact on
availability, suitability for direct substitution, and qualification of
specific and required performance characteristics.Known Changes
Any potential or known changes in product configuration, e.g., the
number of assays per unit (kit) should be routinely evaluated. The
knowledge of and record of package configuration on inventory records
can be helpful should a particular configuration be temporarily
unavailable or discontinued.

Effectiveness Check
Effectiveness checks with respect to the supplied directions for use
must also be a part of inventory records.

Effect on Inventory
Although major changes in package inserts are usually highlighted by
the product manufacturer, the laboratory must ascertain whether these
changes affect the required inventory supply.

Record Date

Publication and revision dates appear on package inserts and should be

entered into inventory records.

Additional Checks
Effectiveness checks are helpful in maintaining continuity in the use
and supply of laboratory products. When this pertinent information is
incorporated as part of the inventory record, the management of
inventory at the bench level is more effective because the information
is immediately available. The recognition of a change,evaluation of its
impact (including effects on laboratory safety, and cost), and review of
approval requirements to institute or accept the change will enhance
efficient inventory use.
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER NINE

LABORATORY AUDIO-
VISUAL AIDS

Audio-visual aids are those resources that should /are made available
to the teacher to help in explaining , demonstrating or interpreting
concepts to the students in class or in the laboratory .
Audio-visual aids are only effective if they are; -
(a) Carefully introduced to students who are aware of its
purpose
(b) Should be appropriate for the purpose for which they are
intended for
The ability to perceive visually presented information can improve the
process of learning ,but however perception of visual stimuli by
students should be accompanied by verbal communication . These will
help pupils to interpret the visual images correctly and will increase
the likelihood that they will remember what they have been taught
(a) Chalk boards
The commonest chalk bored is the black board , but other colored
boards are available . The characteristic of chalkboards surfaces are
important e.g. it must be sufficiently rough to allow chalk to write and
it must make rubbing easy . The cheapest chalkboard is thew wooden
board on which one of its side is coated with a suitable chalk paint .
Other types of boards are glass, rubber etc .most chalks used for
writing are known to produce chalk dust but these can be minimized
by using dustless chalks which produce large grains than the ordinary
 SCIENCE LABORATORY TECHNOLOGY
chalk s and hence the dust produce fall directly downwards on the
trough rather than becoming air borne .

The problem of dust can also be minimized by cleaning the board

down stroke rather than horizontal sweep and also by ensuring that
the duster is unsaturated with chalk dust . Chalk boards should be
painted regularly and should be sighted properly o that it does not
reflect light to students because these will prevent he students from
reading the materials written on it . They should not be placed adjacent
to windows but should also be adequately illuminated .
(b) Marker boards
The main advantages with marker boards are that it eliminates s the
chalk dust .these are boards on which
felt –tip pens with a variety of colors are used . It important to use the
correct type of marker when writing on the marker boards , water
based markers are recommended as they are easy to erase using a
dump cloth . But also sometimes permanent marker pens which are
spirit based are used , these surfaces are cleaned with dusters having
special solvents e.g. acetone
(c) Display boards
These are boards made of special materials used for displaying of
charts , notices and photographs , some are designed for indoor use
while others are for outdoor use . They are available in variety of sizes
and with a variety of frames
(d) Magnetic boards
Magnetic board s have a number of uses in science teaching
laboratories and are particularly useful in illustrating transfer or of
shift movement of electrons in bonding , ions and chromosomes in
cell division . They are made from a suitable sheet of mild steel
mounted on a wooden frame . Some are white plastic coated with
magnetic materials and mounted on the walls . Their surfaces permits
them to be written on with dry markers felt-pens which can be erased
easily with a soft cloth .

(e) Screens
These are plan surfaces or mats mostly coated with a white color .
They could be made of a mat , plastic coated screens , a silvered
surface , glass beaded surface etc . Screens are designed to reflect light
 SCIENCE LABORATORY TECHNOLOGY
from the projector s , they should therefore be bright / white or
refractive . Screens could be portable or permanent.

(f) Wall charts

Wall charts should not be left to stay on walls for too long because
students might after sometimes ignore them since they might
associate them as part of the furnitures in the room and hence they
loose their value and importance .
When planning or electing a chart for teaching purpose , the following
factors should be considered
(i) the content - the information contained in it should be relevant ,
upto-date and must be in appropriate form
(ii)teaching situation – they should be appropriate to a particular
teaching situation , they should:
- Provide initial stimulation to demonstrate a particular skill,
- Provide a visual description ,`give appropriate information
- Should highlight the main teaching points
- it should be visible at a distance
Storage of wall charts is most problematic, but small numbers should
be kept between two sheets of thick cards or hard boards held
together with tapes . For large numbers , the ideal system of storage is
to a system of shallow drawers . Charts should not be folded and only
those produced on linen material should be rolled
(g) Models
Models are often used to convey an appreciation of three dimensional
structure s. most biological models are made from plaster of Paris
while others from rigid or flexible plastics. Most models are often very
expensive . several models of atomic and molecular structure are
available which helps to simplify and explain concepts like atomic
structure and bonding

(g) The slide projectors

These are devices which consist of slides which are either fed manually
into a carrier which may either be by sliding or rotating process . These
type of projectors are adequate for viewing only few slides . its
mechanism of operation is simple and hence can be handled by
students without cause for worry . For more slides a good magazine
loading projector is used which normally consist of a rectangular box
shaped magazine each holding about 36- 50 slides which are fed
 SCIENCE LABORATORY TECHNOLOGY
horizontally into the projector position . Magazine loading projectors
may be manually or mechanically operated .in selecting a slide
projector , the following factors should be considered
 Flexibility - i.e. can it be used for also viewing filmstrips ?
 automation –i.e. how is it operated , manually or mechanically
 brilliance – in what size of the room is the projector to be used , these
determines the powerfulness of the projector
 projection lens
 portability - can the projector allow frequent movements from room
to rooms
 heat – in some projectors , excessive proportion of heat is
produced ,these may damage the projected slides therefore such
projectors requires an inbuilt cooling system
 After use, a slide projectors should be allowed to cool thoroughly
before being moved . It should also be covered to prevent dust
accumulation on the lenses . Slides must be stored properly in
transparent plastic view- packs or some specially designed wallets
(h) The film strip projector
Filmstrips are very light ,cheap and easier to sequence during
presentation . there are two types of film strips
(i) single frame / half frame (24x18 mm)
(ii) Double frame (36 x 24 mm)
Care must be taken in loading and using filmstrips . For long life span
of filmstrips , they should be prevented from crocking by passage
between two glass plates during projection . Failure to observe these
leads to the strip being scratched . They should also be scrupulously
clean . They should be handled by their edges and not on the surface
by fingers . They are best stored in drawers

(i) Overhead projectors

Overhead projectors have a main advantages of their images being
more brilliant , these permits them to be used even in full light . Also its
constructions anable the operator to face the class while using the
projector . Overhead projector consists of a lamp and a reflector
situated below a horizontal glass table or stage . The projection lens
assembly is placed above the stage and may be adjusted to bring
about forcasing .In most modern projectors , the light is reflected
 SCIENCE LABORATORY TECHNOLOGY
through the stage by means of frasnel screen consisting of a number
of concentric rings which have a prismatic effect .
Screens used in overhead projectors are tilted forward . This avoids the
keystone effect in which the top of the picture is wider than the
bottom.
The disadvantage of overhead projector is the comparatively short life
span of the lamp, these can be considerably extended by using an
efficient fanned cooling system .
The lamp should be switched on when the fan is already in operation
and should be left running after the lamp is switched off .when not in
use , the projector should be kept free from dust by covering it with a
plastic cover . The stage should always be clean and should not be
written on as these can couse scratches .
They should be conveniently mounted on trolleys to enhance their
portability . They should also not be left exposed to direct sunlight e.g.
near windows because these will cause charring of transparency
material and can also couse fire .

CHAPTER TEN

CRYOGENICS
Cryogens are gases or fluids at extremely low temperatures (below –
150 °C, –238 °F or 123 K). Cryogenic technology can be defined as the
study of the production of low temperature fluids , measurements at
low temperatures, behavior of these materials at low temperature and
the practical application of low temperature processes and techniques .
 SCIENCE LABORATORY TECHNOLOGY
Low temperature is the temperature obtained by liquefaction of gases
whose critical temperatures is below terrestrial temperature. The
common gases, which can be liquefied, are;Nitrogen, oxygen, air,
hydrogen and helium.
Cryogenic fluids are (or liquefied gases) are developed at extremely low
temperatures. These are generally fluids which at atmospheric pressure
has a boiling point below 25°C . These liquids are held in either special
containers known as Dewar flaskwhich are generally about six feet tall
(1.8 m) and three feet (91.5 cm) in diameter, or giant tanks in larger
commercial operations. These flasks are never sealed, otherwise there
would be a danger or a risk of explosion due to pressure build up as the
liquids inside evaporates to form gasses.

Uses of cryogens
Cryogens, like liquid nitrogen, are used for specialty chilling and
freezing applications. Nonetheless these cryogens have many
applications in the control and tampering with human beings. Some
chemical reactions, like those used to produce the active ingredients for
the popular "Statin" drugs, must occur at low temperatures of
approximately -100 °C. Special cryogenic "Chemical reactor" are used
to remove reaction heat and provide a low temperature environment.
The freezing of foods and biotechnology products, like Vaccine ,
requires nitrogen in blast freezing or immersion freezing systems.
Certain soft or elastic materials become hard and Brittle at very low
temperatures, which makes cryogenic Mill (grinding) an option for
some materials that cannot easily be milled at higher temperatures.

Examples of cryogenic fluids (liquid refrigerants)

(a) Liquid nitrogen – it have a boiling point at 760mmhg of 77.
3° k. its normally a safe refrigerant because its is chemically inner and
neither explosive or toxic. in the laboratory, its used for ;
 Maintaining an intermediate heat sink between the room
temperature and other parts that are suppose to be maintained at low
temperatures
 Providing pre-cooling in liquefies and refrigerants
 Pre- cooling apparatus that will later be used at lower temperatures
 Cooling adsorbents used for purifying gases e.g. cold traps
(b) Liquid oxygen – its boiling point at 760mmhg (at
atmospheric pressure) is 90.2°k. It can also be used as a refrigerant,
 SCIENCE LABORATORY TECHNOLOGY
but care should be taken when handling it because serious explosions
can occur due to its chemical actions with hydrocarbon substances e.g.
lubricating oil, grease etc .it also support combustion.
(c) Liquid hydrogen –its boiling point at 760mmhg is 20.4 °C .
It’s nowadays produced in large quantities as a rocket fuel.
It ignites so fast and these make it unsafe in the laboratories. It can also
be used as a pre-coolant.
(d) Liquid helium – it boiling point at 760mmhg is 4.2°k , it’s by
far the most commonly used refrigerant for works at temperatures
below that of liquid nitrogen . Its safe compared to liquid oxygen and
hydrogen.NB all liquid refrigerants can cause cold burns if they are
kept in contact with the skin and therefore put on protective clothing
and goggles

Storage of liquid refrigerants

Liquid refrigerants must be kept in vessels or Dewars whose
constructions are designed to minimize heat leaks into the refrigerants.
Heat can leak into the refrigerant by
(a) Thermal conduction down the material of the neck tube of
the dewar vessel
(b) Radiation of the outer shell to the inner shell of the dewar
vessel
(c) Conduction through the residual gas between the two shells
of the dewar flask
To minimize heat lose due to thermal conduction, the neck tube of the
Dewar is made long and of materials whose thermal conductivity is
poor. Whereas to minimize heat lose by radiation, the surface of the
two shells should be polished. Heat loses or leaks due to conduction by
residual gas are normally reduced by using a multilayer vacuum
insulation, which is referred to as supper insulation.
If any air is allowed to enter a dewar vessel containing a cryogenic
fluid, the air would solidify inside and as a result, these may cause a
blockage of the neck tube (causes gas- plug ) and prevents the escape of
boiled off gases. The gas –plug formed can be removed by directing a
steam of room temperature helium gas down through the on to the gas
–plug so as to dislodge it.
Ullage space is the region above the cryogenic liquid in a cryogenic
vessel, which contains air and vapour. This space provides room for
 SCIENCE LABORATORY TECHNOLOGY
change of liquid state to vapour state in the event of abrupt change of
temperature thus avoiding explosion

The Dewar flask

A Dewar vessel is a double walled vessel made of a highly polished
stainless steel. Its neck should be long and narrow and should be made
of materials with low conductivity.

Dewar flask

Transportation of cryogenic fluids

Cryogenic fluids are transported in cryogenic vessels which are well

insulated with vacuum jackets and poor heat transmission jackets e.g.
polystyrene. During transportation the containers are not filled to
capacity.
Dispensing of the liquids calls for safety precautions e.g. wearing safety
clothing and gloves. The liquid is normally dispensed in a well-
ventilated place .
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER ELEVEN

VACUUM TECHNOLOGY
The atmosphere is composed of various gases mainly nitrogen-78%
,oxygen 21%, carbon dioxide 0.03 % etc. Pressure exerted by these
gases is given in units called pascals , Torrs or atmospheres.
The atmospheric pressure is usually 760 Pascal’s . Existence of any
pressure below these 760 pascals is called a vacuum .Vacuum is a
Greek word that means‘ empty‘, indeed in practical sense , vacuum
means an enclosure where pressure is lower than the atmospheric
pressure.

Degree of vacuum
Vacuums are often classified according to their operating systems .
There are five categories of vacuum systems :
low vacuum =760 –25 Torr
medium vacuum = 25 – 10-3 Torr
high vacuum = 10-3 –10-6 Torr
very high vacuum =10 –6 –10-9 Torr
ultra high vacuum = 10 –9 Torr and above

The type of vacuum system is determined by ;

(i) The application it is to be put into .
(ii) The pressure range its to be operated in .
(iii) The pump down time .

Vacuum systems are divided into two:

 SCIENCE LABORATORY TECHNOLOGY
(a) Static systems
(b) Dynamic systems

Static systems
Static systems is one in which its made leak free, pumped down and
then thoroughly outgassed by heating or guttering and is often
sealed off from the pumping system. Such system must be kept clean ,
vacuum tight to the highest degree and be made of materials which
are not porous . Static vacuum systems are mainly made of glass ,
examples of static systems are
x –ray tubes
cathode ray tubes
radio and TV valves
thermos flasks

Dynamic systems
These is a system which is continuously pumped down in the presence
of the gases evolving from the walls of the apparatus or from possible
small leaks on the surface of the apparatus .
Examples are industrial and research vacuum systems.

How vacuum is created .

Vacuum systems can be created using a vacuum pump. The limit
and the choice of the vacuum system depends on the choice and the
design of vacuum pump components , care , fabrication and cleaning
techniques .
The major components of a vacuum systems are
(a) The vacuum vessel
(b) The pump
(c) The piping connecting the vessel and the pump
(d) The vacuum gauge
(e) The valves
(f) Baffles and traps
(g) Seals and other protective devises

Through-put
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In any vacuum system ,its necessary to define the measure of the rate
of flow of gas. The quantity of gas is a measure of molecules in a
gas ,it’s the product of pressure and volume . The rate of gas flow is
called through put .
Through-put is thus defined as the quantity of gas at a specified time
and temperature passing through an open cross- sectional area of the
vacuum system per unit area .
Thus

through put Q = P V Torr litres / sec

T
Where P = pressure (Torr)
V= volume (volume)
T = time (sec)

Vacuum production
Vacuum can be produced by either of the following ways;
(a) Compression or expansion of gases e.g.
(i) Rotary pump
(iii) Root pump
(iii) Piston pump
(b) Viscous drag e.g.. –vapour injection pumps
(c) Drag by diffusion e.g. – vapour diffusion pump
(d) Molecular drag e.g. – turbo molecular pump
(e) Ionization effect e.g. ion pump

(a) ROTARY OIL SEALED PUMPS

These are the most commonly used pumps . During its operation, air
from the vacuum chambers enter the pump through the inlet part and
passes into the space created by the eccentric mounting of the rotar
in the strator.
Two vanes mounted in the rotar sweeps the created space and the
trapped air is compressed to a pressure just above the atmosphere .
These causes the exhaust flap valves to lift so as to expel air through
the sealing oil –bath to the atmosphere . The process is repeated many
times until an ultimate pressure is reached . These type of pumps are
called single stage pumps ., they cannot attain an ultimate pressure
that is lower than 5 x 10 –3 torrs due to a back leakage of gas across
the top seal and the outgassing of the lubricating oil . such limitations
 SCIENCE LABORATORY TECHNOLOGY
of such pumps can be overcomed by providing a second pumping
chamber in series with the first type of pumps. The second stage is
backed by the first one via an internal transmission part .
In the two-stage design, the pressure difference across the top seal of
the stage connected to the vacuum system is eliminated and therefore
no back leakage could occur .
The lubricating oil is fed into a high vacuum stage (second from the
first stage ). These ensures that it is degassed before entering the high
vacuum stage .
Sometimes the system would contain condensable vapor e.g. water
vapour at the exhaust part , those vapours are compressed at their
saturation point before the discharge valves lifts thus causing liquids
to be ejected into the sealing oil . The liquids circulates into a
vacuum system causing a deterioration in the ultimate pressure
attainable by the pumps also impairing the sealing and the
lubricating properties of oil . Serious contamination of oils promotes
corrosion of the pumps interior .
The problems of condensable vapour can be minimized by
(a) Using condensers or traps which cool the vapor until their
pressure are very low before they reach the pump .
(b) by raising the temperature of the pumps so that the
vapours are not so readily condensed .
(c) By gas balasting – these is a method by which air at
atmospheric pressure is deliberately introduced through a one way
valve into the pump exhaust part while the trapped gas and vapours
are still at comparatively low pressure .
When the gas and the vapour is compressed , the discharge valve lifts
before the partial pressure of the vapour of the components reaches
the saturation point and condenses. Thus the vapour is ejected
together with the gas .
NB Gas balasting produces a deterioration of the ultimate pressure
attainable by the pump.
The oil in the pump area of the rotary pump fulfil the following
functions
a) it lubricates the moving part and takes away any heat
generated in the pump
b) it seals the clearance between the moving parts .
c) it fills the dead space between the exhaust valve where the
exchange holes passes through the pump body (strator)
 SCIENCE LABORATORY TECHNOLOGY
Rotary oil sealed pumps are used for.
 production of medium high vacuums
 roughing the system down to a pressure at which a diffusion pump
can start pumping
 breaking the diffusion pump

(b) VAPOUR PUMPS OR DIFFUSION PUMPS

Vapour pumps are commonly known as diffusion pumps . They are
commonly used for production of high vacuums i.e. 10-3 – 10-6 Torr .A
vapour pump can be described as any pump that employs vapour as a
pumping means .
Oil is placed in the pump at the base on which there is a heating
element , which heats the oil and vapour flows out through the jet
chimney and emerge from the nozzle at a supersonic speed.
The gas molecules present in the vessel above diffuse into vapour jet
and are forced downwards by the vapour jets . The gas is then
compressed to a relatively high pressure at which it can be removed
by some other types of pumps e.g. rotary oil pumps through the fore
vacuum connections.
The oil molecules condense on the pump walls which is either water or
air cooled and flow back to the boiler in form of oil film where its
reboiled and evaporated . The fore vacuum connection is similarly
cooled in order to condense the oil vapour ejected from the pump and
allowed to be drawn back into the pumps boiler s . These fore pump
cooling will prevent loss of oil from the pump back to the backing line.
Diffusion pumps can be a single jet stage or several jet stage in series
acting as a backing pump to each other . Therefore multistage
diffusion pumps have higher pumping speeds , higher critical backing
pressure and produce low ultimate pressure than single stage pumps .
Multistage pumps can be either fractionating or non fractionating .
Non- fractionating pumps are the ones that have one chimney while
fractionating pumps have individual jet chimneys for each nozzle.
When oil is heated and evaporated , the more volatile higher vapour
pressure oils constituents will evaporate first through the outer
chimney compartment connected to the lower nozzle , while the less
volatile low vapour pressure oils constituents will evaporate last
through the center chimney compartment connected to the top
nozzle .
 SCIENCE LABORATORY TECHNOLOGY
Fractionation is also aided by heat concentration at the center of the
heater falling off towards the sides ( mercury can also be used as a
pumping fluid ) but here , mercury being a pure element cannot be
broken into either constituents and therefore it cannot be used in
fractionating diffusion pumps as a pumping fluid .

Critical backing pressure

In any diffusion pump, gas or air being evacuated diffuses into the
pumping fluid vapour stream where it’s forced to move in the
direction of the stream , its compressed at the backing side of the
pump .
The backing pressure created must not be greater than a certain
critical value known as the critical backing pressure . Therefore the
critical backing pressure is the highest pressure which can be
tolerated in the backing line for the diffusion pump to function
properly.
If the backing pressure is greater than the critical pressure of the
pump , the pump would seize to function . The rotary pump backing
the diffusion pump must therefore be able to remove the gas at such
a rate that it always maintain a backing pressure less than the critical
backing pressure. The critical backing pressure varies with the pump
and the pumping fluid used .

Defects of diffusion pumps

(a) Back streaming - this is the process whereby vapour pumps allow
pumping fluids back into the vacuum system by direct flight of
vapour molecules scattered from hot vapour jet or evaporation at
the mouth of the inlet of the vapour pump .
These can be reduced by careful design of nozzles and proper cooling of
pump barrels or by addition of cooling baffles on top of the pumping
mouths or inlet .
(c)Back migration – this is the transfer of vapour to the higher vacuum
side by re-evaporation of moleculeswhich cling to the surface s within
the pump .
To avoid this , the oil used should have the following properties
 Have low pressure
 Have Suitable viscosity
 Have Suitable lubricating properties
 SCIENCE LABORATORY TECHNOLOGY
 Should not decompose on heating
 Should not be hazardous
The types of oils used include
 Mineral oil lubricants
 White oil
 Inert mechanical pumping fluid

Operation of diffusion pumps

 Initially all valves should remain open except the admittance valve
 The air admittance valve is closed and the main helping vacuum
pump e.g. rotary pump is switched on
 Isolation and roughing valves respectively are opened in that order
and gradually they bring down the pressure to within the diffusion
pump range . the gauge starts showing a gradual decrease in pressure
because the rouging pump has started the pump
 As the pressure reading in the gauge tends to show about 10-2 Torr,
a liquid coolant e.g. liquid nitrogen is poured in the cold trap of the
diffusion pump and the pump is switched on .
 The throttle and the roughing valves respectively are closed ( in
order to isolate the rotary pump) while the backing valves and the
baffle valves are opened . At the same time the main rotary pumps is
switched off and the small holding pump is switched on . These assist
the diffusion pump to discharge because as the vacuum is continued
to be created , the working speed is very slow.
 The throttle valve starts showing a gradual decrease in pressure ,
while the diffusion pump continues to evacuate the vacuum chamber
until the desired vacuum i.e. ultimate pressure is reached .
INSTRUMENTS USED FOR MEASURING VACUUM

MCLEOD GAUGE
its normally regarded as the standard gauge from which other gauges
are calibrated . The basic principle of these gauge is that it takes a
known volume of gas at a pressure that’s too low to be measured by
direct measurement then compresses it in a known ratio until the
pressure becomes large enough to be measured by ordinary
manometer .

VACUSTAT
 SCIENCE LABORATORY TECHNOLOGY

This is a specialized reading portable type of mcleod gauge .Its

normally kept horizontally when its not used and turning it vertically
when taking readings . The mercury inside traps and compresses the
gas from the system in a closed capillary . The height which mercury
rises indicates the degree of vacuum . Vacustat covers a pressure
range from 1 torr – 10-3 torr.

THE PIRANI GAUGE

It is also one of the most used gauge that measures within the range of
medium to high vacuum . Its action depends upon the variation in
thermal conductivity of gas with pressure .
A metal filament heated by an electric current reaches an equilibrium
temperature as heating generated by an electric current is balanced by
the heat loss due to conduction , convection and radiation .
At higher pressure , the number of gas molecules striking the heated
filament in unit diameter are large due to the intermolecular
conduction , while at low pressure, the mean free path is comparable
with the size of the vessel and therefore few gas molecules strike the
heated filament hence the measurement of the electric resistance of
the filament provides a means of determining the pressure .
Pirani gauge consist of a glass or metallic envelop containing a heated
filament made of a material of high temperature coefficient of
resistance e.g. platinum or tungsten .
The resistor of the filament is used as one arm of a whetstone bridge
inserted in a vacuum system . The bridge is balanced initially at
atmospheric pressure or at fixed low pressure (<10-3 torr) and any
change of filament resistance will cause a deflection of the null
indicator which can be calibrated in unit pressure .

Pirani gauge control circuit

The voltage is controlled by a variable resistor in the circuit with a set
of voltmeter to indicate correct adjustment . One of the other resistor
in the bridge is made variable in order to set zero in the galvanometer
or indicator .
External temperatures effects and differential radiation are minimized
in a Pirani gauge by making one of the bridge balancing resistor to be
of similar material to filament and then sealing it off in a vacuum .
 SCIENCE LABORATORY TECHNOLOGY
Pirani gauges have fairly rapid responses to change in pressure , their
electrical circuits are simple and they measure the total pressure or
permanent gases ie. both partial pressure of permanent gases and
vapor .They usually covers pressure ranges from 10 torr – 10-3 torr.
THERMOCOUPLE GAUGE
In this type of gauge , just like the Pirani gauge , a change in gas
pressure causes a change in temperature of a heated filament .
In thermocouple gauge, the temperature is measure by means of a
thermocouple fixed to the filament which is heated by a constant
supply of current.
The electromotive force (emf) generated can be used to operate a
micrometer calibrated in pressure units . The operating range of a
thermocouple gauge is from
1 torr – 10-3 torr and the filament can be heated by alternating current

COLD CATHODE IONIZATION GAUGE OR PENNING GAUGE

These depends upon ionization of gas molecules for its operation. The
gauge head consist of a pair of cathode in form of a wire loop placed
between them . A 2 KV direct current (DC) voltage is applied between
the anode and cathode . The cathode will emits electrons which are
attracted towards the anode .
A magnetic field (provided by a horse shoe magnet ) perpendicular to
the cathodes prevents the straight line motion of electrons from
cathodes to the anodes and instead cause a helical path to be taken .
The electrons pass through the anode loop and are repelled by the
cathode many times before they eventually strike the anode . These
action greatly increases the path which electrons take thus increasing
the number of ions formed as a result of collision with the gas
molecules in the vacuum system .
The collision of the ions at the cathode gives a current that is large
enough to be measure on a micrometer . The ion current is
proportional to the number of collisions and is thus a measure of the
pressure in the system . The meter can be calibrated directly in units
of pressure . The cold cathode gauge can measure from 10-2 –10-6 torr

LEAKS AND LEAKAGE DETECTION IN VACUUM SYSTEMS

The attainment of ultimate pressure or the long pump down time in a
vacuum system is delayed by continuos appearance of gas in the
 SCIENCE LABORATORY TECHNOLOGY
system . The general term ‘LEAK‘ is used to describe these
phenomenon . Leaks can be real or virtual .
A real or true leak is due to the gas entering the system through a hole
or any opening in the system , whereas a virtual leak is caused by the
outgassing (i.e. release of gases and vapours by the materials
used in the construction of the system ) of the inner surface of the
system .(Virtual leaks can only be minimized by proper fabrication
and cleaning techniques together with proper choice of materials used
)
Both real and virtual leaks produce the same results i.e. they cause a
rise in pressure in the system when it’s isolated from the pump or
limits the pressure attainable in a continuously pumped system . Leak
rate is defined as the quantity of gas which enters or appears to enter
the vessel in a unit time . It’s thus defined as the through-put .

LEAK DETECTION

Leakage detection can be divided into two broad groups

(a) High or over-pressure method
In this method , the system under test is filled with gas to slightly over
atmospheric pressure and the outflow is detected
(b) Low or under -pressure method
In this method, a special device in the system is used to detect the flow
of an externally applied gas

A ) OVER PRESSURE METHODS

(i) Wet testing
In this method , the vessel is emersed in water or liquid of low surface
tension such as alcohol , any bubble produced will indicate the
presence and origin of a leakage .
If the vessel is too large to be emmersed , the suspected area is painted
with a soap solution or any other search liquid available , the leakage
will be indicated by bubbles
NB ; The vessel must first be filled with pressure before the search
liquid is applied or before emersion
(ii) Sealing compound
In this method , suspected soldered joints are covered with plasticine
or a Q compound , if the leakage stops then that is exactly where the
 SCIENCE LABORATORY TECHNOLOGY
leakage is . The leakage is then sealed and the vessel pressure is
reduced
(iii) The sniffer technique
In this method , gas issuing from the leaks in a pressurized system is
sampled by being drawn through a flexible tube into a suitable
detector (sniffer) .Within this detectors head , electrons are emitted
and these in turn shall ionize any gas molecules present from the
system . This can be measured.
(iv) Spark coil
In this method , testa coils is used for locating leakages in a gas
vacuum system .The tip of the coil is passed over the surface of the
vessel and when it comes into contact with a leak , a high frequency
spark jumps from the tip of the coil to the leakage area .

B) LOW PRESSURE METHOD

In this method , the detector is usually a pressure gauge mounted
within a continuously pumped system . A leak will be indicated by an
apparent change in pressure when the leaking air is replaced by a
suitable probe gas . For sensitivity to be maintained, the following
must be taken into account .
 The pressure in the region of the detector head must be stable so
that small pressure changes due to the probe gas can be detected.
 The detector head must be sensitive to the probe gas.
 The detector head must be positioned so that the gas from all
possible leaks flow past it.

Detectors commonly used include

a ) Discharge tubes -this uses change of color when different probe
gases are introduced
(b)Pirani thermocouple gauge- Hydrogen gas is used as a probe gas
(c) ionization gauges – butane gas is used as a probe gas .its infact the
best gas to use due to its change in ionization potential and the change
in the electron emission characteristic of the filament.
(d) Palladium barrier leak detector – This is a form of filament
ionization gauge which is sensitive only to hydrogen . It depends upon
principle that in cold state , palladium is impervious to all gases but
under red hot conditions , it’s highly porous to hydrogen . The head is
normally evacuated to a pressure of the order 10 –1 torr . during
 SCIENCE LABORATORY TECHNOLOGY
operation , the palladium barrier is heated by electrons
bombardments . The electrons being produced thermally and
accelerated towards the positively charged palladium. Hydrogen probe
gas which enters the system via the leaks diffuse through the hot
palladium and is ionize by collision with electron streams . The
resulting positive ions are collected by an electrode which is at the
negative potential with respect to the cathode . These cause a current
to flow in the collector circuit . the current is amplified as in the hot
filament ionization gauge . The palladium barrier can detect leaks of
10-8 torr lit /sec
f ) Halogen leak detectors—This detector is based on the increase in
positive ions emissions from a hot platinum anode when it is exposed
to gas molecules
containing one or more of the halogen element i.e. chlorine , bromine ,
fluorine and iodine .
The instrument consist of two coaxial cylindrical electrodes . The
inner one is the
anode made of platinum which is heated to about 500°C by an
internally insulated platinum filament and the outer one is the cathode
.
A potential difference of 50 – 500 v is maintained between the
electrodes .The hot platinum anode emits positive ions which flow to
the cathode and therefore an ameter connected in series with a
voltage supply records the ion current. Freon is generally used as the
probe gas . The detector can be connected to the vacuum system in the
same way as a pirani gauge is connected and then the surface of the
vacuum system is probed with the probe gas in the usual way .
Halogen leak detectors can be used by probing the gas issuing from
the leak of a special detector if a specious detector head is pointed to
the surface of the vacuum system which had been filled with the
search gas to a pressure just above one atmosphere . On entering the
detector, the presence of the halogen at the heated anode surface
increases the positive ion emission from the platinum .An increase of
the emission current is therefore recorded. A minimum leak of 10 –8
torr can be detected
g) Mass spectrometer leak detector - This is the most sensitive and the
most expensive leak detector using a probe gas . It separates ions of
different mass from each other , ions of particular masses are selected
and measured . Helium ions are usually selected while helium gas is
 SCIENCE LABORATORY TECHNOLOGY
used as the probe gas but other probe gases e.g. argon , neon or
hydrogen can have their ions selected

Materials used in vacuum system construction

(i) Gasket – used for sealing all demonstrable joints .a gasket
seal in a closure is affected by mechanical pressure on a deformable
material . Most seals employ relatively s soft gaskets between hard
compressed flanges. Gaskets are made from artificial rubber also metal
gaskets e.g. tin , aluminum , copper , lead , nickel and gold are used at
high and low temperatures and at very high and ultra high vacuum
since they can be raised to fairly high temperatures for outgassing . for
medium and high vacuums , elastomers i.e. rubber gasket are
frequently used the shape of the gasket maybe circular or
rectangular , the circular one is the most preferred .
The cross-section may be circular , square , rectangular or L- shaped .
The most common shape being the O – ring which is circular in shape
with a circular cross section .
(ii) Greases –there are two types of common greases i.e.
silicone and apiezon grease . High vacuum greases are used to effect
seals in such devices as stop corks and gasket joints which may be
static, rotating or sliding .
Silicon high vacuum grease viscosity remains constant over a wider
temperature range than apiezon greases , its unattacked by many
chemicals and does no attack rubber .its vapour pressure is less than
10-6 torr.
Apiezon high vacuum grease are hydrocarbons and are found in three
types normally L,M and N. they are attacked by some chemicals and
the also tend to attack rubber gaskets . Their vapour pressure is as low
as about 10-6 torr.
(iii) Waxes – they are used to make temporary seals . Apiezon Q
is a plasticine compound with vapour pressure of 10-4 torr .it’s a semi
solid compound which is pressed by fingers around the joint to make
it vacuum tight . it becomes too soft for use above 30oc
. apiezon W is a hard substance with a low vapour pressure of 10-8 torr
and is applied at thejoint at 100 and is normally used where fusion
and welding are not possible due to high teperature requirements

(iv) Metals –the most commonly used metals in vacuum

systems are stainless steel - these is mostly used for construction of
 SCIENCE LABORATORY TECHNOLOGY
vacuum equipment and piping lines at low temperature .its advantages
are , their high mechanical strength , resistance to corrosion, and low
thermal conductivity.
Copper – these is used for vacuum system
lines .its properties are ; ease of handling , soft soldered , high thermal
and electricconductivity and resistance to serious oxidation under
normal conditions.
Brass – these is used for flanges construction and coupling joints in
avacuum pumping lines .
(v)Baffles - Baffles are used to prevent backstreaming of the
pumping fluids and thus helps to conserve pumping fluids . They are
normally fixed above the diffusion pump and are normally water
cooled although some may be refrigerated . baffles are arranged in
various forms
(vi)Cold traps – they are placed in between the diffusion pump and
the vessel being evacuated . A cold trap can be made of liquid
nitrogen , dry ice or mechanical refrigerators . Its used for trapping
and condensing all condensable vapours from the vacuum vessel .
Once the trap have been set ,i.e. made cold , it must be kept cold since
if the temperature is allowed to rise , the condensable vapours will re-
evaporate and re-enter the system.

Vacuum cleaning techniques

Contaminants in vacuum systems are
 water vapour
 greases
 fluxes
 oils
 all other visible dirt’s
These contaminants can be found on
 The surface of the vacuum system
 In holes and cracks
 Absorbed gases and vapours on the surface of the vacuum systems
 Absorbed gases and vapours in vacuum materials
 Chemically absorbed gases and vapours
The commonly used cleaning solvents include :
 Acetone – used extensively for cleaning and degreasing
 Ethyl alcohol – used as a cleaning and drying agent.
 Methyl alcohol - used for cleaning and drying , its very poisonous
 SCIENCE LABORATORY TECHNOLOGY
 Isopropyl alcohol used for cleaning ,in brazing and soldering
techniques
 Chloroethene – a solvent and a detergent for cleaning and degassing
diffusion pumps .
Cleaning techniques in vacuum systems
Cleaning is the physical or chemical removal of undesired materials
from the surface of apparatus . In vacuum technology , cleaning also
involves removing those contaminants physically stack on the surface
( e.g. oils , grease and dust) or resulting from a chemical reaction e.g.
oxides and sulfides .
There are several methods of cleaning and none of them is adequate
to achieve the desired degree of cleanness . In most case a
combination of two or more methods is required for proper
cleaning . These methods include
a) Mechanical methods –these is not specific for vacuum technology ,
its used for the purpose of leaning the seals ,rust etc , an abrasive or
wire brushing is usually utilized.
b) Pickling- these is the mechanical removal of oxides and other surface
layers leaving the cleaned part with a metallic appearance with
smooth or a rough finish depending on the concentration of the
pickling solution NB pickling solutions are virtually acidic hence the
pickling surface should be thoroughly rinsed and subsequently
neutralized in an alkaline bath and dried with hot oil.
c) Electrolytic etching and polishing –these is done in the anodic or
cathodic
d) treatment of metallic surface. NB ; The appropriate etching solution
(or electrolyte ) is of paramount importance . Polishing should be
done after the etching process
e) Alkaline detergent cleaning , these is performed by emmersion in
hot solvent (60-85oc )using cleaning agents e.g. NaOH, soaps and
wetting agents

Application of vacuums
vacuums are useful in ;
 Aluminizing of mirrors
 Production of optical films
 Production of electronic components e.g. capacitors , resistors and
integrated circuits (ICs)
 Production of thin films
 SCIENCE LABORATORY TECHNOLOGY
 Mounting of specimens in electron microscopy
 Production of tungsten filaments lamps , discharge lamps e.g.
fluorescent tubes etc
 Manufacture of X-ray tubes ,radio receiver valves voltage stabilizes
and cathode ray tubes etc
 In vacuum metallurgy i.e. production of alloys and pure metals of
varying strength and properties
 Simulation of outerspace and high altitude environments
 In freeze drying of food staffs and pharmaceutical products
 In filtration
 In evaporation
 In suctions
 In distillation
 In crystallization
 In cleaning

CHAPTER TWELVE

PHOTOGRAPHIC
TECHNOLOGY
The camera
A camera is one of the scientific most valuable tool. Cameras can be
attached to many other advanced optical instruments e.g. microscopes
in biology or telescopes used in astronomy. When fixed to microscopes,
cameras can reveal very fine details that the resolution of human eye
 SCIENCE LABORATORY TECHNOLOGY
cannot see. Infact such specialized photography is becoming essential
for scientific research as the information gathered by them can be
reproduced in magazines and books with great accuracy. These enables
scientist to share their experiences with others.
Remote control cameras can be used to take photographs from
positions that are too dangerous e.g. sites of atomic bombs or
explosions or even sites that are too small for man to go e.g. in the
throat and the stomach to give the doctor the detailed information
about these internal organs.
Photographic evidence is generally regarded as truthful and accurate
e.g. in any court of law when dealing with criminal cases and other
forensic matters. In fact no center of medical, industrial or scientific
research would be properly equipped without a photographic facility.

Principles Of Photography
Photography simply means writing, drawing or printing with light.
Light is therefore most fundamental in photography because without it
then no photographs will be produced.
Light is a form of energy, it’s a source of all colors and its composed of
different wavelengths, those that are of importance in photography are
those that fall within the visible region of the electromagnetic spectra
because they can be perceived by the human eye. These are compose of
different colors e.g. red, blue green yellow orange etc

Light is naturally ‘white light ‘but objects around it normally absorbs

some wavelengths and reflect others, transparent materials transmits
all wavelengths equally unless they are colored but colored objects
reflect some colors and absorbs others. This selective transmission of
light is important in photography, its infact the only way in which
different tones are produced and this normally determine the way we
perceive the shape and forms of objects.
Light, Lens And Images
 SCIENCE LABORATORY TECHNOLOGY
Light travels in a straight line, buts its often dispersed as it passes
through different surfaces and mediums. These causes light to produce
dim or sometimes ill-defined images. But in order to produce brighter
and sharper images, its necessary to gather more light and gather it or
converge the light rays at one point so as to focus the image. This
requires a lens. Lenses converge light by bending them (difraction) to
one point by simply transmitting light from each part of the object then
focusing it on a flat surface i.e. film which is always placed on the
opposite site of the receiving surface.
Light Sensitive Material
Basically, there are two essential stages in taking photographs;
(i) Focusing an image of the object
(ii) Recording the image of the object permanently on a surface
Light is a form of energy and it can cause chemical changes in
materials, its infact these changes which record the image. If a material
which is changed by light is exposed to light, it will change more where
the image have been exposed to more brighter light and less where it
have been exposed to a dim light.
Some salts especially silver salts (particularly the halides e.g. silver
bromide) are affected by light, these are indeed the salts used in
photography to record images. A layer of these light sensitive salts . A
layer of these light sensitive halides is coated over a flat surface and
placed where light from the image fell. By arranging the object directly
on a sheet of film and then exposing it for a specific time, a negative
image of the object is created in black and white. Such image created is
however not permanently fixed on the film and it may be easily affected
upon more exposure to light. For it to be permanently fixed, it have to
further undergo additional steps in processing which will make the
image resistant to any further exposure to light. This process is called
development.
The art and science of photography initially used to encounter three
main problems which had to be solved i.e.
(i) How to make the silver salt react to very short exposure to
light e.g. fraction of a second
(ii) How to stop the image formed to resist further darkening
each time its brought to light even after development
(iii) How to turn or convert the negative image formed by silver
salt to a positive image.
 SCIENCE LABORATORY TECHNOLOGY
However, modern silver-based salts have solved these problems. These
salts are able to pick light which is just brief enough to initiate the
darkening process of the image (at these stage, the image is not visible
to the eye). The silver-coated film is kept in total darkness and is
treated with a chemical solution, which develops and accelerates the
chemical change until when a strong visible black silver image appears.
These image is however not permanent and it have to be further fixed
using another chemical which makes the remaining silver salts to be
sensitive to any further exposure to light. These remaining silver salt
can now be washed away and a stable negative image is left behind
which can safely be left exposed to light without any further changes
occurring. Converting the negative image into a positive image is done
by shining a beam of light through the negative into another light
sensitive sheet of photographic paper. This paper is then developed
and fixed to give a positive image that can now be enlarged into various
desired sizes and many copies made from it.
The Simple Camera
A simple camera consists of;
(a) A light tight box with a lens
(b) A shutter to admit light briefly to the film
(c) A film cassette (a compartment for film )
Most simple cameras have a fixed forces lens that is build in front of
the camera. These is not variable and its only set maximum overall
sharpness of the image i.e. you cannot adjust the lens so as to move the
object closer or away from the point of focus.
Behind the lens is a shutter consisting of movable blades or rotating
disks which protects the film from light until when it’s necessary. They
open when the shutter is pressed and these when light is allowed to
reach or fall briefly on the film surface.
Simple cameras have a limitation, which includes;
(i) View finder error
(ii) Fixed distance of focus
images not being sharp due to their cheap or poor quality lens
(iii) Enlarged photographs are generally of poor quality

Direct Vision Rangefinder Camera

These are cameras, which have the viewing system, which is usually a
tube on top of the camera with a lens at each end. An object is looked at
directly and it appears reduced in size. They have a range finder for
 SCIENCE LABORATORY TECHNOLOGY
accurate focussing. The viewing system is separate from the taking
lens. These cameras are very quick and clear but however they do not
show how the image will actually record on the film since all the out of
focus parts of the background and fore ground always appear shar

Single Lens Reflex Camera (Slr)

They are designed so that the distance between the lens and the
focusing screen (via the mirror) is exactly the same as between the lens
and the film. Consequently, whatever parts appear on the screen will
also appear on the film. They have no parallax error .
Camera Controls
(i) Aperture
Am aperture is simply a small hole or opening into the camera which
allows light to get in. The lens aperture consist of overlapping movable
leaves which forms a diaphragm set to different ranges of diameters so
that the quality of light admitted is controlled. Apertures control the
amount of light reaching the film. The size of the aperture can be
controlled depending on the amount of light present i.e. large aperture
used for dim light and small aperture used when there is bright light.
The aperture also can increase or reduce the depth of field i.e. these is
simply the zone of sharp focus in front and behind the focussed object
NB as the aperture is reduced, the depth of field increases. The
aperture is usually adjusted by turning a narrow ring near the focus
control (in some cameras) these includes intermediate setting which
creates progressive doubles and halves the light that is admitted. Each
setting is given an f-number. Some simple cameras use weather
symbols instead of f-number
(ii) shutter
A shutter is simply a knob at the top of the camera, which is normally
pressed when taking an exposure. Shutters not only control the exact
moments when the film is exposed to light but also the duration of
exposure i.e. the amount of light that is admitted .the length of time the
shutter remains open controls the quantity of light that reaches the
film. Shutters are fitted within or just behind the lens body and use a
set of blades, which rapidly opens or shuts. They sometimes consist of
two separate blinds positioned just in front of the film.
Shutter speed (the speed in which the shutter opens and closes the
aperture to allow light) varies but the longest time it should remain
 SCIENCE LABORATORY TECHNOLOGY
open is 1 sec and the shortest is 1/250 sec although for others its
1/500,or 1/1000sec. Shutter speed affects or determines how moving
objects record on the picture, high shutter speeds eliminates the effects
of blur, it reveals details but reduces the sense of movement .to help
reduce or avoid the effect of camera shake and blurs, the shutter speed
should be high or alternatively the camera should be supported on a
tripod stand
NB shutter speed and the size of the aperture can be used
constructively to control sharpness of the image, the depth of field and
blur

The Film and The Exposure

Exposure is the amount of light that is allowed to act or fall on the film.
The aperture and the shutter settings control it. In order to control or
set the correct exposure on the camera, knowledge of light sensitivity of
the film and the brightness of the object should be known (i.e. lighting
and tone)
The right size of the film should be selected which should fit in the
camera e.g. 35mm wide that are packed in cassettes containing upto 36
exposures. The film sensitivity is given in A.S.A (American standard
association) or DIN number (for Europe) e.g. the film marked 400 ASA
is twice as sensitive as that marked 200 ASA film.
These mean doubling the ASA number doubles the speed or its
sensitivity. The faster the film, the less exposure needed to be given but
the coarser or grainy your picture will be. i.e. higher ASA films produce
coarse or grainy pictures compared to low ASA films, which give
smooth and less grainy pictures, but however low ASA films require
prolonged exposure. These is because a fast film gains its sensitivity by
using
larger grains of silver so the resulting negative film have a coarse
granular pattern that can be clearly seen on enlargement.
Therefore to decide on which film speed to use, you have to consider
the lighting conditions and the subject matter of your picture. Films of
64,32or lower ASA numbers are slow and have very fine grains. They
are the best choice is you want quality photographs with a lot of details.
But they are slow and require lots of bright light. But films of 125 ASA
still have fine grains; they are ideal for average subject surfaces
outdoors and well-lit indoor subjects. Faster films of over 400ASA
show grains and are most ideal for very dim lighting or when grainy
 SCIENCE LABORATORY TECHNOLOGY
pictures are needed or when you want to freeze motion (i.e. to make
fast moving object to appear to be stand still) while using fast shutter
speeds.
NB most cameras nowadays have exposure meters and tables on their
cameras, which help to determine the amount of exposure needed for a
particular film
There are two types of photography ;
(a) Black and white photography
(b) Colored photography
A. Black and White Photography
After taking photographs using a camera, the film has to be processed
to give the negative and the positive. Film processing is the art of
developing the exposed film to give the negative and the positive.
Photograph development involves two major steps;
(a) Film processing -developing the film to form white and black
negative (negative
image )
(b) Printing and enlargement – producing positive image onto paper
(i) Film processing
Early films were made using a light sensitive material coated on a
glass sheet made to stick using a sticky material called gelatin or
collodoic which contain the light sensitive silver halide salt . Glass
sheet films were heavy and only practicable with large cameras in
which they could fit . nowadays , plastic or cellulose is the most
versatile and useful for film sheets . They are light and flexible enough
to be wounded onto a spool for easier camera loading . They are even
cheaper than glass. The light sensitive chemical is coated on the front
surface while its back surface is coated with anti-halation dye which is
black and thus prevent light reflecting off the sheet into the light
sensitive layer on the front surface
The gelatin materials for coating the cellulose sheet where the silver
halide is coated have many advantages as a binder; it swells when
paced in a development liquid. These allow the processing chemical to
enter and react with the silver halide it
contains. But when its dried the gelatin coating returns to its normal
thickness without distorting the shape or position of the image and
therefore its most ideal for wet processing technique used in modern
photography.
 SCIENCE LABORATORY TECHNOLOGY
It takes relatively long time for light to change enough silver halide
grains into metallic silver so as to form visible image (latent image)
because only relatively few atoms of silver are affected by the initial
exposure in the camera. These can however be later amplified millions
of times through development process using development chemicals
which increase the amount of silver in those areas of the image that
have been affected by light. Three processing chemicals are needed i.e
a) Developer solution
b) Stop bath solution
c) Fixer solution
These solutions have to be diluted with water. They are usually cheaper
when bought in powder form. Most photographic chemicals deteriorate
when in contact with air and therefore they must stored in stoppered
glass bottles. Some people are allergic to some chemicals used in
development and these can cause skin rashes, its therefore necessary to
use protective clothing e.g. rubber gloves.
Films are very sensitive and therefore care must be taken not to scratch
them or to handle the picture area of the film particularly when it’s wet
or partially dry. Never expose it to dust or grits at this delicate stage.
film processing should be done in a light tight closet or black fabric
changing bag .
Items used in film processing
 Chemicals -
 Developer solution
 Fixer solution
 Stop bath solution
 Concertina storage bottles
 Tray
 Graduated cylinder
 Funnel
 Timer / clock
 Squeegee tongs
 Thermometer
 Development tank
 Rubber gloves
 Film clips
 Rubber horse
 Water filter
 SCIENCE LABORATORY TECHNOLOGY

The developing tank

These are an airtight cylinders of sizes large enough to accommodate

one roll of a film. There are two types of development tanks i.e. plastic
and stainless steel development tank
A typical development tank has four plastic parts
 The tank body that is threaded at the top to receive the light proof
lid
 The reel which loads the film from its outer edge. It have a spiral
groove designed to hold the film in the loose coil
 They have baffles in the lid which allows the solution to be poured
out of the tank without admitting any light
 Agitating rod, which is a plastic rod, which dips into the hole for
rotating the reel so as to agitate the solution.

Developing and fixing

Once the film is in the developing tank, the processing, the preceding
processes can be carried out in the open daylight or normal light. The
already diluted solution i.e. developer,stop bath and fixer solution can
now be used in systematic sequence i.e.
 Pour the solution in the tank, holding the tank at an angle to allow air
to escape. Start the timer and agitate as instructed by the
manufacturer. tap the tank to dislodge air bubbles , then close the cap
and then turn the tank over and back at half an hour interval
throughout the development time .
 At the end of the development time ,pour the developer solution out
and fill it again with the stop bath solution for 10 sec then leave the
tank to stand in warm water for 1 min .
 Pour out the stop bath, you can now use water at room temperature.
fill the tank and agitate for 10 sec empty the tank and repeat these
step again .
 Pour in the tank and agitate once every minute. Fixing time is usually
10 min. Return the fixer to the bottle. The film can now be washed.

Washing and drying.

 SCIENCE LABORATORY TECHNOLOGY
Washing is done using water to remove the processing chemicals,
which will otherwise stain the film. Wet negatives (even when dry) are
very sensitive and can easily be damaged by hairs, dust, scratches. They
must therefore be handled by their edges. Washing of films can be done
in normal light but inside the developing tank. A rubber horse is used
to deliver water into the tank but its advisable to use filters
incase the water have grits or sand particles.
After washing the film should be thoroughly clean, they should be dried
by hanging
them to dry by the clips. Remaining liquids on the surface can be dried
using squeegee tongs.
Once the film is absolutely dry, protect it as soon as possible from dust,
never fold it as these might cause scratches. A protective file is
necessary at this stage for keeping or storing these films .
Printing
Printing is the process of focusing the image on the processed negative
onto a photographic paper .it means making a positive image that is
the same size as the negative on a photographic paper, the positive
image formed have to be again enlarged to a reasonable size. Contact
printing .

Contact printing
These is a technique whereby the negative is placed on a sheet of
contact print and exposing it with a low voltage disk lamp which gives
an even lighting. An enlarger is not required at this stage. After these
period of exposure on the contact sheet, the contact sheet have to be
processed again through a series of three solutions a just like in the
film processing i.e. in developer, stop bath and fixer solutions,

Contact printing requires a normal contrast grade paper with a white

smooth glossy finish. Contrast refers to the number of grays given
between white and black (a hard contrast gives few grays, soft contrast
gives many tones of grays). Variable contrasts are also available.
Contact printing can also be done on resin – coated paper that washes
and dries faster but this type of paper is more expensive.
 SCIENCE LABORATORY TECHNOLOGY
After contact printing enlargement have to be made whereby an
enlarger is used. A good enlarger should be one that can be used for
both black and white photography and also in colored photography. (It
should have filter drawers for colored photographs. enlargement allows
several control over image, the negative can be printed to almost any
size. the choice of how light or dark the print should appear can be
determined by the choice of exposure time. there is no limit to the
number of identical prints that can b made from one negative. black
and white photographs can be made from colored negatives.
Contact printing and enlargement process requires a dark room, these
is a room specifically designed for carrying out photo printing, they are
suppose to be totally light tight, dust free, smooth plastered walls and
without any shelves and any other wall fixtures because these may trap
dust.
Its doors, frames, windows and ventilation grills should be light tight,
should instead be provided with artificial air conditionings that are
light tight, electric power and water .its floors should be made of
chemical resistant materials.
The dark room is divided into two; i.e. dry bench and wet bench
The wet bench are used for all those processes that involve use of
liquids / chemicals while the dry bench is used for those activities that
uses electric appliances e.g. enlarging and drying .
Lighting in a dark room consist of the white light, which should be
positioned within easy reach so that there is no trouble reaching for it
in the darkness. Also a well-mounted safe light should be positioned
over the developing tray so as to enable monitoring of developments.
Contact printing is done placing the negative with the emulsion-coated
side in contact with the emission-coated side of the sheet of light
sensitive photographic paper. They are then exposed to light from a
reading lamp or an empty enlarger. After these exposure process
removes the negative and start re- processing the print again
.
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER THIRTEEN

CHEMICAL SAMPLING
TECHNIQUES
A sample is a finite part of a statistical population whose properties are
studied to gain information about the whole. A population is a group
of items from which samples are taken for analysis. Samples are
representative of the population that is taken or required for
laboratory analysis.
An ideal sample is expected to mirror the population from which it
comes, however, there is no guarantee that any sample will be precisely
representative of the population from which it comes.A sample will
only be satisfactory if the properties under investigation correspond to
those of the bulk material within the limit set by nature.
Sampling is the act, process, or technique of selecting a suitable
sample, or a representative part of a population for the purpose of
 SCIENCE LABORATORY TECHNOLOGY
determining parameters or characteristics of the whole population.
Sampling is a technique used to draw conclusions about populations
from samples.Obviously, it is cheaper and more accurate to observe a
sample rather than the whole .A sample may provide you with needed
information quickly.
Sampling is a very challenging task to perform , improper sampling can
result into sampling error that can affect the entire analysis and
hence results .Sampling errors comprises the differences between the
sample and the population that are due solely to the particular units
that happen to have been selected. Most of these arrors are due to
sampling bias,Sampling bias is a tendency to favour the selection of
units that have paticular characteristics.

Sample Size
Before deciding how large a sample should be, you have to define your
study population.
Sample size can be determined by various constraints. In general,
sample size depends on;
 The nature of the analysis to be performed.
 The desired precision of the estimates one wishes to achieve.
 The kind and number of comparisons that will be made.
 The number of variables that have to be examined simultaneously .
 The heterogenicity or homogenicity of the sample .
 The available funds
Steps In Sampling
There are three major steps in sampling ;
a) Identification of the population from which the sample is to
be taken from .
b) Selection and obtaining of gross sample of the population
maneuver
c) Reduction of each gross sample to a laboratory size that is
suitable for analysis
NB The failure to obtain a proper sample makes subsequent analysis
worthless.
Factors to consider during sampling
For a sample to truly represent the batch .the following should be
done ;
a) The material to be sample should first be thoroughly mixed
 SCIENCE LABORATORY TECHNOLOGY
b) The population from which the sample was taken from should be
identified by labeling
c) The amount of the original material should be noted as the quantity
of the sample taken from it
e) The date, the owner and the place of sampling should also be noted.
Precaution observed during and after sampling
The samples should be taken in such a manner that will not undergo
chemical or physical changes .
It is necessary that the sample be kept in an air-tight container so that
the content cannot be tempered with .
The sample should be kept in cold so that decomposition and increase
in bacterial load will be minimised.The container should preserve the
physical and chemical characteristic of the sample as much as possible.
Proceedures for Sampling
The commonly used procedures for sampling or obtaining a
representative sample is that of halving and quartering.
The composite portion that have been obtained from various sections
of the bulk are first mixed as thoroughly as possible then is divided into
halve then quarter then halved again, quartered e.t.c. This process of
halving and quartering is continued until a sample that is small enough
for analysis is obtained
NB;For solid samples , they are ground first into fine sizes before
doing halving and quartering
Types Of Samples
Random samples
These are samples obtained from the bulk of the materials in which the
bulk is first divided into a number of real or imaginary segments then
the sample is there after obtained in a predetermined pattern
Systematic samples
These are samples collected so as to test changes in composition with
times, temperature, spatial location or treatment. The results obtained
from these samples are tested statistically significant differences
Representative samples
These are samples that can only be obtained from homogenous
material
Composite samples
These are samples composed of two or more samples
Gross samples
 SCIENCE LABORATORY TECHNOLOGY
It is also called bulk sample or lot sample.it is that which one or more
increment of materials is taken from a larger quantity of material for
assay or record purposes
Laboratory samples
These are a sample intended for testing or analysis. Its prepared from a
gross sample .it must therefore retain the composition of the gross
sample
Sub - samples
These is a portion taken from a sample i.e a laboratory may be a
subsample of a gross sample.
NB; samples for analysis should be large enough for all intended
determination (about 250ml)
Methods of Sampling
There are two methods of sampling
(a) Manual sampling –
in this method, the samples are taken manually and mixed thoroughly
before sampling by rotating or shaking. Mixing can also be achieved by
pouring the materials several times from one container to another
(b) Mechanical /instrumental sampling These involves use of
mechanical samplers .
Sampling Errors
They are caused by several factors i.e.
(a) Lack of randomness in sample selection which results from both
instrumental limitation and human bias e.g. powdered or granular
materials are subject to many errors which could be as a result of-
 Their particle shape i.e rounded shapes flow faster than angular
shaped of same sizes
 Surface adhesive’s especially the uncoated hygroscopic materials
tends to flow into samplers more readily than non hygroscopic
materials of similar shapes.
 Differential downward movement of samples when disturbed during
sampling
(b) Changes in composition of the samples, which occur during and
after sampling e.g. – gain or loss of water
 Loss of volatiles
 Physical inclusion of gases
 Reaction with container material
 Foreign maters in the container
 Microbial and enzymatic actions of samples
 SCIENCE LABORATORY TECHNOLOGY
NB; Most of these errors may be overcomed by fine grinding and
mixing of the sample.
The aim of sampling is to secure a portion of the material that
satisfactorily represents the whole.

Preparation of Sample
Accurate information a bout the sample to be tested can only be
obtained if samples are correctly prepared before analysis. These
require effort, care, and time.
The purpose of sample preparation is to mix thoroughly a large sample
in the lab so as to produce a homogenous sample. The sample must
then be reduced in size and the amount for subsequent analysis.
Basically there are two types of samples
(a) Wet sample
(b) dry sample
(a) Preparation of dry samples
Dry samples exist in many forms and sizes. These should be reduced
into fine sizes by grinding. The method for grinding depends. Samples
can be grinded mechanically or manually.
Several methods of grinding ranges from the simple pistil and motor to
other several other elaborate and efficient machines like the
hammermills, poshomills. But the size of the grounded particles should
be controlled by passing the particles through various screen or sieves.
Finess of the ground materials is largely affected by composition of the
sample especially its moisture content.generally samples are grounded
better after drying them.
Drying can be done in a desicator, vacuum, oven r by using hot air
(pulverization)
Grinding of hard material may lead to errors especially due to
contamination from the abrasive part of the mills which may add some
metallic elements hence cousing errors when minerals components are
supposed to be determined. Using with resistant moving parts that
cannot wear out due to friction can minimize these.
Some samples are supposed to be grounded in there frozen state. Such
samples are grounded in chilled bowl mills. Centrifuges are used to
separated solids from liquid
suspension
 SCIENCE LABORATORY TECHNOLOGY
Preparation of wet sample.
Moist materials can be disintegrated using various fine slicing devises
or bowl cutters for leafy vegetables and tubers meat cutters for fruits
roots and meat products. Such devices can be hand or power driven.
Dilute suspensions and most soft and pasty food can be grinded using
various modification of blenders that have rotating knives. (Upto
2500RPM).
Sonic and supersonic vibration have also been used for dispersion of
food.
Enzymes and Chemical Treatment of Samples

Enzymes and chemicals can be used to disintegrate samples and hence

can serve as a method of sample preparation e.g. cellulase enzyme is
useful in preparing samples of plant origin, proteases and
carbohydrases are also used to solubilise high molecular weight
components.
Also chemicals like phenol, Urea ,dimethyl-sulphate, pyridines and
detergents as well as other reducing agents are useful in preparation of
samples. NB; but sometimes presence of chemicals and enzymes in
food samples can cause change of composition and modification
following sampling or the preparation of samples for analysis. These
means that the samples must be treated in such away that potentially
troublesome enzymes and microorganisms are completely and
immediately inactivated.Several methods of enzymatic and
microbiological inactivation are used. These include use of heat, drying
or keeping the samples in extremely low temp as well as using several
other inorganic chemicals that will poison the enzymes and
microorganisms
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER FOURTEEN

SEPARATION, EXTRACTION
AND PURIFICATION
TECHNIQUES
1. SEPARATION TECHNIQUES
(i) Decantation
Decantation is a method of separating substance e.g. immiscible liquids
such as oils and water using separating funnel .it can also be used to
separate insoluble solids from liquids e.g. sand and water
(ii) Filtration
This is a method of separating a solid from a liquid using a filer paper
and a filter funnel. The filter paper have pores through which the liquid
portion of the mixture pass leaving behind the solid portion
(iii) Precipitation
These is a process of separation in which soluble substances are
converted into an insoluble substance called a precipitated
(iv) Evaporation
These a separation process in which a soluble substance is subjected to
heat so that it boils and in the process it gets separated through
evaporation i.e. the solvent evaporates leaving behind the solute.
(v) Crystallization
These is a separation technique in which the substance is dissolved
until it becomes saturated as it is heated then quickly cooled stepwise
through various temperatures ad in the process the dissolved solids
separates by forming crystals
(vi) Distillation
These is method of obtaining a solvent from a solution by heating to
boiling point then the steam produced is collected by passing it through
a condenser where it is cooled back to liquid which shall be collected.
There are two types of distillation; simple distillation, this is a process
of distillation whereby a solution is heated until to a reasonable
temperature where the solution begins to evaporate. These evaporated
solution is allowed to pass through a libel condenser where it collects
 SCIENCE LABORATORY TECHNOLOGY
as a distillate leaving behind the solid, fractional distillation these is a
process whereby two or more solutions are separated according to their
boiling point i.e. one should be having a lower boiling point than the
other .the solutions are mixed then heated together with a
thermometer attached, the distillate is allowed to pass through a liebig
condenser from where it shall be collected. Its therefore expected that
the solution with the lowest boiling point shall be collected before the
on with the highest boiling point .
(vii) Sublimation
These is the process of separation whereby one of the solids in the
mixture e.g. iodine and iron is able to change directly from solid-state
to gaseous state without passing through the liquid state.
(viii) Magnetization
These is a process of separating solids whereby one of the solid is
magnetic while the other one is not .
(ix) Sedimentation and floatation
These is a method of separation whereby a mixture is added to a liquid
and then left to settle for a while to allow it to sediment or float.
Sometimes some clarifying agents can be added which hastens
sedimentation, such clarifying agents are usually heavy metals e.g.
alum or lead salts (flocculation).
2. EXTRACTION TCHNIQUES
Extraction is a very important procedure in chemistry and biology
laboratories .its required as a clean-up procedure, as a concentration
step and also as a separation method for slightly soluble materials and
for aiding in the identification of components there are several
extraction methods;

1. CHROMATOGRAPHY
Chromatography is a method of extraction that utilizes separation of
different fractions of colors . its based on the principle that the
components of the mixture may be separated and concentrated into
zones by passage into two phase systems i.e the stationery phase and
the mobile phase . The mobile phase serves to carry the

mixture through the stationery phase

There are various chromatographic techniques these includes
 paper chromatography
 thin layer chromatography ( TLC)
 SCIENCE LABORATORY TECHNOLOGY
 column chromatography
 gas –liquid chromatography (GLC)
 high performance liquid chromatography ( HPLC)
(a) Paper Chromatography
These method involves use of a porous filter paper onto which a drop
of unknown substance is placed at one end and placed in a container
or development tank in which the atmosphere is saturated with a
solvent vapor .these technique is used to separate a group of substance
that have similar general molecular structure e.g. sugars , aminoacids
,fats etc.
These substances are separated basing on their ability to move at
different rates on a special paper whatman no1 in a given solvent .the
stationary phase in these case is water supported on a solid cellulose .
Its also called partition chromatography because the component of the
mixture are distributed or partitioned between two liquids i.e. both the
stationery and the mobile phase are liquids there are three types of
paper chromatography ;
(i) Ascending paper chromatography
These is where elution or separation is carried out such that the eluent
(mixture containing the pigment) travels upwards on a strip of paper
(ii) Descending paper chromatography
These where the elution is done in such a way that the eluent travels
down the strip of paper
(iii) Circular or radial paper chromatography
In these case ,the component of the mixture travels in the center of the
paper in form of circular zones .a circular filter paper is taken and a
spot of the eluent is put at the center of the paper ,then a small radial
strip (tail)is cut within the paper the eluent is taken in the petridish
and a paper is placed horizontally over it such that the tail dips into the
solution eluent then the setup is covered with another petridish .after
sometimes ,the different components in the mixture will travel a
different rates from the center of the paper and then forming
different color zones on the paper
(b) Thin Layer Chromatography (TLC).
These consist of a glass plate or slide that is coated with a very thin
layer of adsorbent material (a liquid that can stick on the surface) the
adsorbent is left to dry and then a spot of unknown substance is
placed on the edge of the glass in an atmosphere that is saturated
with the solvent vapour .
 SCIENCE LABORATORY TECHNOLOGY
The unknown substance will diffuse the adsorbent material depending
on their ratesof movement and therefore gets spread out .
TLC gives much more rapid results and is a more sensitive technique
.its normally made of a flat plate of sizes 10 x20cm these plates
must be thoroughly cleaned before use by washing in a good detergent
followed by hot water and finally rinsing in distilled water before
drying
The adsorbent material used as the emulsion is silica gel , alumna gel ,
or cellulose .the required quantity of the adsorbent is mixed with
distilled water to form a slurry .
After the slide have cleaned and dried , a narrow strip of adhesive tape
(elastoplast) is applied on the edge of the plate . The thickness of the
tape determines the thickness of the adsorbent coating.the prepared
slurry is then poured along the untapped edge of the plate ,then using
a uniform glass rod the slurry is drawn along the plate in one
continuos glide along the tape . Care should be taken not to roll the
glass rod .the sample is applied using a micropippete to the plate
under a stream of warm air on a line marked about 2-5 cm from the
lower edge of he plate . A plastic spotting plate can be used for correct
spacing of various samples along the starting line . The distance
between spots is about 1-3cm and the diameter of the spots should not
exceed 8mm.
After evaporation of the solvent in which the sample was applied, the
pate is placed in a chromatographic jar making sure that it does not
reach the surface of the adsorbent . To obtain rapid equilibrium , one
side of the jar should be lined with the filter paper , which extends
into the solvent at the bottom . When the jar have become saturated
with the solvent paper additional solvent is carefully poured down on
one side of the jar until about 2cm of the chromaplate is covered thus
starting development .when the solvent have reached the score line,
the plate is removed from the tank and dried
(c) Column Chromatography .
This is a method of separating solids. A glass column e.g. burettes is
used in which a pure liquid like benzene is half way filled and a slurry
of the solid adsorbent i.e. silica gel is put into the column ,these is
used as the stationery phase .
The solid stationery phase sinks to the bottom of the column and in
the solids running out of glass , a glass wool is placed at the bottom of
the tube . a disk of filter paper is placed at the top of the column so as
 SCIENCE LABORATORY TECHNOLOGY
to prevent disturbance of the solid when the solution or solvent is
added later.
The solvent (mobile phase) above the column is then run off carefully
so that the solids (stationery phase ) in the tube remains wetted so
that cracks and channels do not form or appear in it otherwise the
mixture to be separated will run evenly through it hence no separation
.
The mixture to be separated is then added at the top of the column
and the liquid emerges at the bottom which is the collected in
separate receivers e.g. beakers
After addition of the mixture solutions in different colors will emerge
in the tube. The process of emerging different colors is called elution .
Each solution is collected in different flask and the solvent is removed
by evaporation or distillation so as to leave a pure compound . If the
solid in the mixture are colored ,then they can easily be seen as they
pass down the glass , but if they are colorless, then some physical
properties e.g. fluorescent or UV light can be used to monitor how
the compounds in the mixture is moving so that the flask can be
inserted just when the solutions of the compounds is just about to
emerge .
Principle of chromatography
The principle here is that each component in the mixture has a
particular solubility in the solvent . no two compound can be held
alike and therefore those compounds be held alike and therefore
those compounds that are readily soluble and not strongly adsorbed
move quickly or rapidly down the column in a stream of solvent and
those compounds which are not soluble and are strongly adsorbed are
held on the column for longer periods
Column chromatography is used for separation of extremely delicate of
products e.g. proteins ,vitamins and hormones and other substances
which are not readily separated by other methods

(d) Gas- Liquid Chromatography (GLC)

In Gas –liquid chromatography the mobile phase is normally an inert
gas e.g. argon in which the various components in the mixture are
separated as they pass along a special column containing a stationery
phase that have been coated into a support matrix or on the inside of a
capillary column .
 SCIENCE LABORATORY TECHNOLOGY
The mixture to be separated is introduced at the start column. The gas
stream then carries the sample through the chromatographic column
which is a tube containing a relatively non- –volatile material capable
of reversibly absorbing the sample .
The adsorption process results in the sample being retarded in its
passage through the column at a degree that depends upon the
partitioning ration between the stationery and mobile phase .different
components of the sample travels through the column at different
speeds and therefore arrive at the end of the column where they pass
through the detector which gives an electric signal that’s related to the
varying composition of the gas passing through it . The signal is then
amplified in the electronic box where its converted to a suitable
response so that it can be processed by a microcomputer or on a chart
paper.
The computer shall draw a curve in a chromatogram showing the
changes in composition of the gas passing through the detector
thereby illustrating the sample .
The behavior of each component is then expressed as the time for it to
reach the detector and its known as the retention time or relative
retention time . The computer can also calculate the concentration of
each of the sample in the components .

Terminologies Used In Chromatography

Elution – these is the use of a solvent to separate components
Eluate - these is the solvent containing the components .it is
sometimes called the fraction
Origin – these is the point of application of the eluate on the
chromatogram
Loading- these is the amount of substance applied
Solvent front – these is the level at which the elution fluid have reached
Stationery phase – these is usually the solid or a liquid adsorbent e.g.
filter paper or water
Mobile phase - these is a solvent or a gas used to separate the
component .
Column – these is a cylindrical tube for holding the adsorbent or ion
exchange resin
Polarity –a polar compound is one that is held by the stationary phase
whereas a non-polar component tends to move forward in a mobile
phase .
 SCIENCE LABORATORY TECHNOLOGY
Adsorption chromatography
These where the stationary phase is a solid while the mobile phase can
either a gas or a liquid
Paper partition chromatography
These is when the stationery is the liquid e.g. water held on an inert
porous support e.g. paper and the mobile phase can either be a liquid
or gas .in these type of chromatography, more than one solvent is
usually present in mobile phase since its always saturated with the
stationery phase thus making two phases imiscible .separation occurs
between two components of a mixture when one component is
strongly retained then the other type by the stationery phase
One dimension chromatography
These when the solvent runs only in one direction
Two dimension chromatography
These is when after the solvent have run in one direction ,the paper is
dried and turned through 90o , replaced in the tank and developed in
a new direction.
Multiple development
When the development is rerun a number of times in a solvent so as
to improve resolution
Development
These the process of allowing the solvent to move along the column of
paper
Resolution
These is the degree of separation of components after development
Location
These the detection of the components after development either by
using general or specific reagents or UV light
Tank
These is an airtight container in which development takes place
Relative fraction
This is defined as the ratio of the distance the solute has traveled from
the point of origin to the distance traveled by the solvent front i.e.
Rf=distance moved by the solute from origin /distance moved by the
solvent moved from origin
NB ;Rf value is always less than 1

2. ELECTROPHORESIS
 SCIENCE LABORATORY TECHNOLOGY
Many macromolecules (colloids) are charged and their ions move in
response to an electric field .the method of separation based on
movement of ions in an electric field is called electrophoresis .Its
generally restricted to proteins .The charge on the protein depends on
the pH of the solution. Every protein have an isoelectric point (pi) at
which the net charge is zero and at which have zero mobility at pH
value below pi the protein migrate as cation. The mobility increases
with decreasing pH, but at pH value above pi, the protein migrate as
anion and in these case its mobility increases with increasing pH , such
electrophoretic movement is as a results of or depends on its net
charge ,size and shape.
Electrophoresis is a very useful technique in separation of
biomolecules from complex mixtures. Infact electrophoresis is almost
the only method available for quantitative analysis and fractionation of
biological materials (fluids) and for characterization of the purified
components . Electrophoretic methods can be divided into
(a) Paper electrophoresis
(b) zone electrophoresis
(c) Disk -Gel Electrophoresis
(d) Isatachophoresis.
(e) Capillary electrophoresis
(a) Zone Electrophoresis
These is the most widely used especially in food analysis.it is suitable
for analysis of small samples by fairly simple procedures. These is
normally done on a solid support .the sample components are
separated basing on their difference in net charge ,their shapes and
sizes . These separation takes place at a constant pH and ionic strength
in presence of a stabilizing medium like cellulose or granulated gel e.g.
in gel electrophoresis whereby, migration of ions takes place through a
gel slab .(its objective is to separate the sample according to the molar
mass of the sample) the sample is applied as a narrow zone on top of
the stabilizing gel and when electric field is applied ,the sample
component will migrate into the gel.separration in the stabilizing
media takes place because of the different mobilities of the sample
components .
Zone electrophoresis have several advantages i.e. ;
(i) Use of simple and inexpensive apparatus that permits
simultaneous analysis of several samples in routine procedure
 SCIENCE LABORATORY TECHNOLOGY
(ii) Simple procedure for visualization of zones and isolation of
fractions
(iii)improved resolution by combining electromigration with
molecular sieving
(iv)adaptability to either macroanalytical or microanaltical separation
(v) it can be used for investigation of a low molecular weight
substances that are difficult to analyze by other methods
Among the most commonly used supporting mediums are paper,
cellulose acetate and agar.

(b) Paper electrophoresis

In these case, the material to be separated into its components is
either spotted or steaked into the center of the strip of paper that is
saturated with a buffer solution.each end of the paper dips into a
container holding several millions of additional buffer solution .
An electrode is placed in each in each container i.e. one of the
electrode is connected to the positive terminal and the other electrode
to the negative terminal of a DC source .a potential of about 5-10 volts
is applied and if any charged particles are present in the sample . They
migrate towards the terminal of opposite charge.
In order for particles to migrate in an electric field , it must poses a net
electrostatic charge .this charge is an integral multiple of 4.8 x10 -10.
But many compounds that do not have a charge can be separated
electrophoreticaly provided some charging process is used e.g. the
most common charging process are ; reaction between an acid and base
, dissociation into ions by polar solvents ,hydrogen bonding , chemical
reaction ,polarization and ion pair formation.
The problem that often arises is determining the mobilities is heat
produced by the current passing through the cell.these heat. These
does not only cause conventional current which changes mobilities
but also increases rate of evaporation of the buffer solution when
paper electrophoresis is employed .The lose of buffer solution causes
change in mobility due to changes in potential and current .
The dry paper will also take up solution from the tank these causes
capillary action .water placed in a capillary tube in which a positive and
negative electrodes have been placed inside can be caused to migrate
towards the negative electrode . This indicates that water is positively
 SCIENCE LABORATORY TECHNOLOGY
charged. These migration can be altered by placing other ions in the
system .These movement of water ions in an electric field is called
electroosmosis

(c) Disk -Gel Electrophoresis

This is a modification of gel electrophoresis . It however takes long
time because the sample must be added in a broad band . Its however
easier to remove a particular fraction from the mixture without
stirring and removing the solution.
Disk electrophoresis is away of concentrating the sample in a very
narrow disk before actual separation occurs . This means that the
separation can be done faster and better . The narrow initial zone
created is infact a reason for good separation and also the gel acts as a
sieve .
The gel usually used for disk electrophoresis is polyacrylamide gel
which is lattice networked whose pores can be made of molecular size
and acts as sieves to further separate compounds based on their
differences in size and shapes
(d) Isatachophoresis.
Isatachophoresis is an electrophoretic techniques by which the sample
component are separated based on their differences in heir net
mobility .the separation is performed by a polyacrylamide gel utilizing
discontinuous buffer system
The sample component and the spacer ions are dissolved in
terminating electrolyte and the leading electrolyte is initially applied
throughout the polyacrylamide gel . When electric field is applied, the
separation of the sample takes place between the leading and
terminating electrolyte during migration .when segregation is
complete the separated zone and spacer ions migrate with the same
velocity in immediate contact with each other into a funnel shaped
elution chamber where they are flushed out by a continuos steam of
elution buffer
Isatachophoresis have high resolving power because of its
concentration effect
Isoelectric Focusing
Amino acid are amphoteric in nature i.e. they react as acids and
bases .If placed in an elrctrophoretic cell, they would migrate towards
the anode in basic system and towards the cathode in acidic system
.but there should be a pH at which the net charge is zero where amino
 SCIENCE LABORATORY TECHNOLOGY
acids would show no movements .These point is called isoelectric
point . Isoelectric point is important as it affects separation.
The principle used is that naturally occurring colloids acquire a charge
when they are dispersed in water. The overall charge usually depends
on the pH of the medium e.g. in acidic medium protons attach to the
basic group and the net charge is positive while in the basic medium ,
the net charge is negative as a result of proton loss but at isoelectric
point ,the pH is such that there is no net charge on the macromolecule.

Isoelectric focusing is the method that drift with pH e.g. a mixture of a

distinct proteins dispersed in a medium with a pH gradient along the
direction of the applied

electric field . These gradient is obtained by imposing a DC potential l

on an electrolyte system in which the pH steadily increases from anode
to cathode .Each protein in the mixture will stop moving at a position
in the gradient where the pH is equal to its isoelectric point .In these
manner the proton mixture can be separated into its components.
The two main application of isoelectric forcasing in biochemistry
are ;
(i) characterization of proteins by their isoelectric point .
(ii) the analytical separation of high molecular weight proteins
according to isoelectric point .
However isoelectric focusing also have some disadvantages i.e.;
(a) The lipoproteins are denatured at isoelectric point
(b) Some proteins are only slightly soluble at their isoelectric
point especially in salt free system.

(d) Capillary electrophoresis

Gel and isoelectric focusing are slow hence it takes several hours so as
to give good separation of a complex mixture .one way to increase
these, is to increase the electric field strength.
However these is not advisable because large electric fields can heat the
large surface of electrophoretic apparatus unevenly leading to no
uniform distribution of electrophoretic and hence poor separation .
In capillary electrophoresis, the sample is dispersed in the medium (a
buffer solution) e.g. methyl cellulose in which a potential of 300 v/cm
DC is applied through the graphite electrode and it is held on a thin
glass or plastic tube with a diameter ranging from 20-100micromters .
 SCIENCE LABORATORY TECHNOLOGY
These small size apparatus makes it easy to dissipate heat when large
electric field is applied. Usually two forces acts to cause separation i.e.
electrophoresis of the charged particles and electroosmosis. UV and
fluorescence are common means of detection .These method is fast

3. CENTRIFUGATION
Centrifugation is a technique used for separation of solids from liquids
and from immisible solvents and for resolution of emulsion that are
formed during extraction.

centrifuge
Ultracentrifugation (centrifugation at high speed) is useful for
concentrating high molecular weight materials and for estimating the
molecular weight.
Basic concept of centrifugation
The force acting on a particle is given by;

F=Ma=Mw2r
Where F =Force on a particle in dynes
M =Mass of a particle in grams
 SCIENCE LABORATORY TECHNOLOGY
W =the angular velocity of rotation in radius
Per second
R = the radial distance of a particle from the
axis of rotation in cm
Therefore the centrifugal force F1 is given by
F1 =Mw2r/g
Where g is the gravitational constant (9.80,07 cm/sec2 )
F =1.118 X10 –5(MrN2)
Where N= the speed of rotation in revolution per min (rpm)
There are three types of centrifuges
(a) solid-wall centrifuge in which separation or concentration is
by subsidence or floatation
(b) perforated –wall centrifuge (centrifuge filters ) in which the
solid phase is supported on permeable surface through which the fluid
phase is free to pass
(c) a combination of the two centrifuges in which the primary
concentration is affected by subsidence followed by drainage of the
liquid away from the solid phase
Centrifuges can be hand or power (or motor ) driven in which the
vertical spindle on which the various heads or rotors can be
mounted ,the rotors carry metal containers in which glass tubes or
bottles into glass tubes or bottles are fitted i.e. between 2-16 or even
more bottles can be fitted
Bottle centrifuges are the most preferred in analytical and small-scale
preparative separation because time, speed and temperature can easily
be controlled. They should be enclosed for safety in metal guard bowl
these tubes hang vertically at rest but as the rotor starts to run, they
gradually swing out to horizontal position where they remain as long as
the head is rotating. The advantage of the method is fractional
sedimentation across the tube length, but the long path of travel of
some particles and some particles being hindered from setting near the
bottom is the main disadvantage. These can however be overcomed by
prolonged centrifugation at high speed
All rotating part of a centrifuge are subject to stress created by
centrifugal force these stress impose limitation in the maximum
permeable speed of the centrifuge .the effect of these stress can be
minimized by ;
• Properly designed centrifuge,
• By carefully balancing before loading the sample,
 SCIENCE LABORATORY TECHNOLOGY
• By slowly increasing the speed to that desired
• By proper maintenance
• Adherence to manufacturers instruction .
ULTRACENTRIFUGATION
These is the separation and fractionation of macromolecules e.g.
proteins by subjecting them to strong centrifugal force using an
ultracentrifuge. Analytical ultracentrifuges provide a photographic
record of the migration of high molecular substances in a strong
gravimetric field .the have an automatic temperature and speed control
for the rotor, high vacuum chamber to reduce friction and optical
system for measuring the rate at which individual peaks (proteins)
move towards the bottom of the cell, automatic photographic systems
for recording changes in concentration at specific intervals and special
cells

PURIFICATION TECHNIQUES
(a) Distillation
This method involves heating the solution in the distillation flask , the
solution becomes progressively until it begins to boil. Th solvent will
escape as vapor whereas the solute is left behind in pure form in the
flask.
The vapor of the solvent passes down the side arm of the flask into the
condenser where it cools into a liquid .in this case the pure solvent is
collected as the distillate at the end of the condenser .

(b) Crystallization
Temperature increase or decrease affects the ability of the solvent to
dissolve in a given solvent in two main ways i.e.
(i) It increases the rate at which the solute dissolves e.g. solubility is
greatest at high temperature
(ii)it also affects the amount of the solute that can dissolve in a given
quantity of solvent e.g. the higher the temperature , the greater the
amount of solute , which a given quantity of a solvent can dissolve .
Similarly ,if a suitable quantity of a solution is cooled from
temperatures e.g. T1 oc-T2oc ,the solution will deposit a mass of (W-
w) g of solute .these principle of cooling can be applied on purification
in crystallization
 SCIENCE LABORATORY TECHNOLOGY
Crystallization can be used to purify solids , whereby a given quantity
of a suitable solvent and solute is heated until the solution is very
much concentrated ‘ the concentrated solution is then quickly filtered
while its still hot and the filtrate is set aside to cool in a corked conical
flask . As the solution cools , some of the solute crystallizes out and
when the solution is quite cold , the crystal of the solutes are filtered
off from the remaining solution which is known as the mother-
liquor .The crystals are then dried and a pure substance is obtained
(c) Fractional crystallization
With slight modification ,crystallization can be used to separate a
mixture of two solids .these can be done by carefully looking for a
solvent which dissolves a large amount of one of the solid e.g. solid A
but small amount of the other solid e.g. solid B.
The effect of temperature on the amount of solute that can be
dissolved by a given volume of solvent should be different for solid A
and solid B
Therefore if a concentrated solution of the mixture of A and B in a
suitable volume of the solvent in a temperature Toc and then cooled to
Tooc
Then the mass of A which would be deposited is( WA –wa)g. Similarly
the mass of B deposited from the same solution is(WA - wa) g . Now its
clear that (WA –wa) is greater than (WB-wb) i.e. more A would be
deposited than B and if the deposit are recrystalised further from the
same solvent ,the crystals of A deposited will even be greater than B
.after several recrystalisation , pure A and B shall be deposited
according to their ratios . A mixture of sugar and salt can be separated
this way using water as the solvent
(d) sublimation
These is a purification process whereby a solid on heating changes
directly into the vapour state without first becoming liquids .these
vapour also condenses directly into a solid without passing through
the liquid state . Such solids include NH4CL,iodine and nepthaline
can be sublimed under reduced pressure

METHODS OF DETERMINING PURITY

(a) Determination of melting point
 SCIENCE LABORATORY TECHNOLOGY
If a compound is pure, it will melt at a specific temperature and the
change from solid to liquid will be sharp .if the temperature is raised
gradualy,these sudden change of state allows the melting point to be
observed with accuracy .the presence impurities have two effect on the
melting point
(i) It lowers the melting point unlike that of pure compound
(ii) It prolongs the period of melting so that instead of sharp changes
in melting ,melting proceeds slowly over arrange of temperature
Sharp melting point is therefore a criterion of determing purity of a
compound
(b) Determination of the boiling point
if a compound is pure ,it will boil at a specific temperature and also
the change from liquid to vapor is always sharp
( c) Boiling point elevation
a solution have a lower vapor pressure than it pure solvent and
therefore boil at high temperature.the lowering of the vapor pressure
is accompound by a proportional of boiling point
(d) Freezing point depression
The lowering of the vapor pressure of a solution is accompanied by a
proportional depression of the freezing point NB; one mole of ionizable
solute when dissolved in o1kg of a solvent elevates the boiling point by
0.5°C i.e. the solution will normally boil at 100.5°C
Similarly addition of non ionizable solute to a solvent causes a
depression of approximately 1oc.the depression of the freezing point is
consequently proportional to the concentration of the solute in the
solution
Boiling point elevation and freezing point depression results may be
used in determing the purity of a compound by finding their RMM of
the compound on the basis that1 mole of none ionizable solute in 1kg
of water (solvent) can couse a boiling point of 0.52°C or depression of
1.8°C

(e) Determination of specific gravity

Any pure compound have a constant specific gravity impure
compounds will not have the same specific gravity
(f) Determination of absorbency
Any pure element will show specific absorbency in spectroscopy unlike
an impure compound .
 SCIENCE LABORATORY TECHNOLOGY
g) Determination of refractive index

Every pure element have specific refractive index impure compound

shall show different refractive index .

CHAPTER FIFTEEN

PH ANALYSIS
The pH of a solution is defined as the negative logarithm of the
concentration of hydrogen ion in the solution
pH =-Log (H+)
pH is measurement of acidity or basicity of a solution. Water is
polar i.e. its internal charges are evenly distributed such that one part
of the molecule is positive (+) and the other part is negative (-) .The
 SCIENCE LABORATORY TECHNOLOGY
positive part is H+ and the negative part is OH - .The H+ which is
positively charged is always attracted to the negatively charged OH -.
Water is neutral i.e. it have equal concentration of H+ and OH- acids
and bases are substances that increase the concentration of H+ and OH
– respectively, If an aqueous solution contains more H+ than OH –ions
then these solution is acidic ,likewise ,if the solution contains more
OH- ions than H+ ions ,then the solution is basic
H+ and OH- ions do not exist in water on its own, they however exist
as hydronium ionsH3O+ and OH -.t they associate with water
molecules i.e. they are hydrated (surrounded by water molecules )

H2O +H2O ----> (H++H2O) +OH -

H2O +H2O ---- > H3O+ + OH –

one useful way to express the concentration in qualitative form is by

giving the dissociation constant K

(H+)(OH-) =K
(H2O)
Where K =represent the dissociation constant while the other terms
represent the concentration of ions and molecules
Rearranging the above equation becomes
K(H2O)=(H+)(OH-)
The concentration of water in dilute solution is almost constant K.
The constant therefore k(H2o) becomes Kw. These is called the
ionization constant of water
The value of these constant at 25oc is Kw =1 x10 –14
The above equation can therefore be written as
Kw =(H+)(OH-)
1X 10-14 =(H+)(OH-)
EXAMPLE
A solution contains 0.001g of hydrogen in one liter . calculate its pH
0.001=10-3 g ions/liter
pH = -Log(H+)
pH =- Log 10 –3
pH =3
Conversely a solution whose pH =8 will have H+ concentration equal
to10 –8 g ions .
 SCIENCE LABORATORY TECHNOLOGY
In the case of neutral solution e.g. water the concentration of H+ is
equal to OH –
H+= OH-= 1X10-14
The pH of water is there fore 7,for acid solution is lower than 7 and for
basic solution is higher than 7
To obtain H+ concentration and pH the following equation is used
(H+)(OH-) = Kw= 1 X10 –14
where Kw is the dissociation constant for water
therefore (H+) = 1X10 –14
OH_
When a substance e.g. Nacl is added to water , the H+ and OH-
concentration is not changed in anyway because salt i.e. NaCL
contain neither H+ or OH- . But if HCL is added to water ,H+ from
HCL shall be added to the solution . These will increase the H+
concentration but will decrease
the OH – concentration since the ionization constant must remain
constant.
The same thing shall happen when NaCL (abase) is added in water ,
here the OH- from the NaCL shall be added to OH –ions in the water
thereby increasing the concentration of OH- while the H+ shall
decrease .
NB;Any substance that is capable of increasing the H+ concentration
in a solution is called an acid and any substance that is capable of
increasing the OH- ions concentration is called a base. Water is
regarded as neutral
Strength of acids and bases
Some acids and bases are stronger than others e.g.
HI >HBr >HCl
NaOH >CaOH >NH4OH
All acids liberates hydrogen gas .If equal volume of acids are used and
if the amount of the hydrogen gas liberated is measured ,it shall be
found that the volume of H2 liberated varies depending on the type of
acid used .
These shows that the greater the reactivity of acids must be the result
of the amount of H2 gas liberated .The strength of acid is therefore
measured in terms of H+ concentration of the solution .The scale of
acid strength ranges from very strong acid HI,HBr,Hcl, through an
intermediate range down to weak acid e.g. acetic acid . The strength of
bases is similarly accessed basing on the same reasoning for OH- .
 SCIENCE LABORATORY TECHNOLOGY
Strong acids readily dissociate in water and gives or transfer all their
H+ to water to form H3O+ ions Acids are therefore strong
electrolytes because they completely dissociate to give ions . In
contrast , weak acids do not readily dissociate or transfer its H+ ions.
Similarly strong bases are those that completely dissociate to give all
their OH- whereas weak bases do not completely dissociate.

Concentration of acids and bases

Concentrated and dilute acids and bases refer to the amount of water
added to that particular acid or base .Concentration is measured in
molarity.
Concentrated acid is that which have less amount of water dissolved in
large amount of acid while dilute acid have large amount of water is
dissolved in less amount of base.

Buffer solutions
Buffer solutions are solutions that stop sharp changes in pH .The
higher the tendency of the solution to keep or maintain the pH, the
higher its buffering power .
Buffer solutions are widespread in nature including in the living
systems .They ussualy consist of an aqueous solution of weak acids
e.g. acetic acid (CH3COOH) or bases e.g. (NH4OH) and its salt of a
strong base e.g. sodium acetate (CH3COONa ) or a salt of its strong
acid e.g.( NH4CL)
In water solution acetic acid and sodium acetate dissociate to give
CH3COOH -----> CH3COO - +H+
CH3COONa -----> CH3COO - +Na+
While a solution of ammonia and ammonium chloride dissociate to
give
NH4OH ------> NH3 + H+
NH4CL ------>NH4+ +CL

In a buffer solution , apart from H+ and OH- ions existing , there are
also CH3COOH and CH3COO- . Therefore if a small quantity of an
acid is added these buffer solution ,the H+- ions are freed by the acid
and are immediately buffered by CH3COO- ions leading to the
reaction
 SCIENCE LABORATORY TECHNOLOGY

CH3COO - + H+ ------> CH3COOH

But if a small quantity of a base is added , then the OH – ions
introduced are faced by the reaction below
CH3COOH + OH- -------> CH3COO – + H2O
Therefore adding an acid or base to a buffer solution alters the value
of the relationship CH3COO/CH3COOH
Without causing variation in the solution pH

Common Ion Effect

Buffer solutions have and give common ions i.e. these are solutions
with two dissolved solutes that contain the same ions (cation or
anion)’the presence of common ions suppresses the ionization of a
weak acid or weak base .If sodium acetate and acetic acid are
dissolved in the same solution , they both dissociate or ionize to
produce CH3COO – ions
CH3COONa(s) ------------> CH3COO + Na +(ag)
CH3OOH(ag) --------- CH3COO - + H+ (ag)
CH3COONa is a strong electrolyte, so it dissolves completely in
solution. but CH3COOH is weak acid which ionizes only slightly.
According to Le chateliers principle, the addition of CH3COO- ions
from CH3COONa to a solution of CH3COOH will suppress the
ionization of CH3COOH i.e. it will shift the equilibrium from right to
left
Thereby decreasing the H+ concentration. Thus a solution containing
both CH3COOH and CH3COONa will be less acidic than a solution
containing only CH3COOH at the same concentration . The shift in
equilibrium of acetic acid ionization is cause by the acetate ions from
salt.
CH3COO – is the common ions because it is supplied by both
CH3COOH and CH3COONa
The common ion effect is the shift in equilibrium caused by addition of
a compound having an ion in common with the dissolved solution. The
common ion effect plays an important role in determining the pH of
a solution and the solubility of a slightly soluble salt .
Consider the pH of a solution containing weak acid , HA and a soluble
salt of a weak acid such as NaA
HA(aq) +H2O -----------> H3O+(ag) + A-(ag)
Or simply
 SCIENCE LABORATORY TECHNOLOGY
HA(aq) ----------> H+(ag) + A(ag)
Ionization constant Ka is given by
Ka= (H +)(A-)
(HA)
By rearranging the above equation
(H+) =Ka (HA)
(A-)
Taking the negative logarithm of both sides
log (H+) = - Log Ka- logHA
(A-)
Log (H+) = -logKa + log (A-)
(HA)
pH = Pka+log (A-)
(HA)
Pka= -log Ka
A more general form of these expression is,
pH = pka +log (conjugate base)
(acid)
where HA is the acid and A- is the conjugate base. Thus if ka and the
concentration of the acid and salt of the acid , then the pH of the
solution can be calculated.
Example 2
(a) Calculate the pH of a solution containing 020M CH3COOH and
0.30M CH3COONa
(b) What would be the pH of 0.20M CH3COOH solution be if
CH3COONa were not present
strategy
CH3COOH is a weak acid it dissociates to give;
CH3COOH ---------->CH3COO- + H+
CH3COONa is a soluble salt which completely dissociate to give
CH3COONa ---->CH3COO- + Na+
CH COO- is the common ion,
at equilibrium , the ions in the solution shall be ;
CH3COOH , CH3COO- ,Na+,H+ and H2O
Na ion have no acidic properties therefore it is ignored
Ka for CH3COOH = 1;8 X10-5 (its always given)
Therefore we can calculate the (H+) at equilibrium hence the pH if we
know the equilibrium concentration of both CH3COOH and CH3COO-
As noted , sodium acetate dissociate completely in solution to give
 SCIENCE LABORATORY TECHNOLOGY
CH3COONa -------> Na+ +CH3CO
0.30M 0.30M
The initial concentration ,changes, and final concentration of the
species involved in equilibrium are;
CH3COOH-------->H+ + CH3COO-
Initial 0.20 0 0.30

Change M -x ---------> +x +x
Equil M 0’20-x x 0.30+ x

But Ka = (H+)(CH3COO)
(CH3COOH)
1.8X10-5 = ( x)(0.30 +x)
0.20 –x)
Assuming that 0.30 +x=0.30 and 0.20 – x =0.20 we obtain
1.8X 10-5 = (x) (0.30 + x) = (x)(0.30)
(0.20-x) ( 0.20 )
x=(H+) =1.2X10-5
PH = -log (H+)
= -Log (1.2 X 10-5)
= 4.92
(b) The pH of 0.20 M CH3OOH solution if CH3COONa were not
present

CH3COOH -----> H+ +CH3COO-

Initial M 0.20 0 0
ChangeM -x +x +
Aquilibr M 0.20-x x x
Ka = (H+)(CH3COO-
(CH3COOH)
1.8X10-5 = x 0.20-x
assuming 0.20-x = 0.20
we obtain
1.8X10-5 = x = x2
0.20 - x 0.20
x =(H+) =1.9X10-5
PH= - log (1.9 X10-5 )
= 2.72
 SCIENCE LABORATORY TECHNOLOGY

Preparation of buffer solutions

To prepare a buffer solution with a specific pH , we first choose a weak
acid whose pka is close to the desired pH , then the pKa and the pH
values are substituted to obtain the (conjugate base) / (acid ) ratio
.These ratio can then be converted to molar quantities for the
preparation of the buffer solution
pH =pka +log (conjugate base)
(acid)
Log (conjugate base) =0
(acid)
pH =pKa
Example
Describe how you would prepare a phosphate buffer with a pH of
about 7.40

Solution
For a buffer to function correctly , the concentration of the acid
component must be roughly equal to the conjugate base component .
When the desired pH is close to the pKa of the acid , i.e. when pH
=pKa

log (conjugate base) =0

(acid)
(conjugate base) =1
(acid)
Because phosphoric acid is a trprotic acid , it undergoes three stages of
ionization i.e.

H3PO4 ------> H+ +H2PO4- Ka=7.5X10-3 Pka=2.12

H2PO4- -------> H+ +HPO4-2 Ka=6.2X10-8 Pka=7.21

HPO4-2 -------> H+ +PO4-3 Ka=4.8X10-13 Pka=12.32

Therefore the most suitable of the three buffer system is HPO4-

2/H2PO4- because the pKa of the acid is close to the desired pH
pH =pka + log(conjugate base)
(acid)
 SCIENCE LABORATORY TECHNOLOGY
7.40 =7.21 +log(HPO4-2)
(H2PO4-)

log(HPO4-2) = 0.19
H2PO4
Taking the antilog we obtain
HPO4-2) =10 0.19
(H2PO4-)
=1.5
Thus one way to prepare a buffer solution with a pH of 7.40 is to
dissolve Disodium hydrogen phosphate(Na2HPO4) and Sodium
dihydrogen phosphate (NaH2PO4) in a mole ratio of 1 .5:1.0 in water

DETERMINATION OF pH
There are two common ways used to measure pH
(a) Using pH meters(potentiometric analysis)
(b) Using indicators

Indicators
Indicators are generally organic compounds which changes color
according to the pH of the solution in which they are dissolved . They
are chemicals that visually indicate the end point of chemical processes
. They achieve these by change of color, fluorescence or by formation
of precipitates . They work over some ranges normally between 2-3
unit .These therefore means a different indicator must be used
according to the range being examined .
But to avoid having to use several indicators , a standard mixture
showing gradual and continuos color changes over a wide range of
pH have been developed .These mixture is known as a Universal
indicator.
To determine the pH value of a solution of unknown hydrogen
concentration , Only a few drops of the appropriate indicator are
added to it .The resulting color is then compared to standard colors
on a pH chart which reveals the pH of the solution .alternatively ,a slip
of paper saturated with an indicator is dipped into the test solution ,
the color of the paper is then compared on the chart.
Indicators are mostly used in titration experiments to test or
determine whether neutralization ,precipitation or redox reactions
have reached their end point .
 SCIENCE LABORATORY TECHNOLOGY

Indicators can be classified into the following categories .

(a) Acid-base indicators
(b) Oxidation –reduction indicators
(c) Precipitation indicators

Acid –base indicators

They are normally used in neutralization reaction .They are generally
organic substances which any changes in H+ ion concentration or pH
causes alteration in their molecular structure which result in change
of color in the substance itself .
As a general rule , acid base indicators are generally weak acids or
bases that dissociate when immersed in water . Indicators consist of
the acid and its conjugate base i.e. Hin .
To be an effective indicator ,Hin and its conjugate base In – must
have distinct colors in acidic and basic solutions . The acid ionizes to a
small extend
Hin(aq) -------> H+ (aq) + In-(aq)

If the indicator is in a sufficient acidic medium , the equilibrium

according to Le chatelier,s principle will shift to the left and the
predominant color of the indicator is that of the unionized form (HIn)
.On the other hand ,in a basic medium , the equilibrium shifts to the
right and the color of the solution will be due to that of the conjugate
base (In-).
It then follows that the color of the indicator is directly dependent on
the pH of the surrounding solution .
The constant of these equilibrium is
K= (H+)(In-)
(HIn)

Which gives,
HIn = (H+)
(In) K

Oxidation -Reduction indicator,

 SCIENCE LABORATORY TECHNOLOGY
These are indicators whose color in oxidized states is different from
that in the reduced state .the end point is reached when the
concentration of the oxidized form is equal to that of the reduced form
Some oxidation –reduction reaction do not require use of
indicators .These is especially the case when one of the reagent itself
changes color according to whether it is reduced or oxidized e.g.
KmnO and iodine . The end point of titration in the case of iodine can
be brought out even more by adding starch which gives a dip blue
color .

Indicators of precipitation reaction

Such indicators are used to determine endpoint of precipitation
reaction .The most common ones are
(i) Adsorption indicators . These are adsorbed on the surface of
the precipitate where it colors it deeply e.g. silver nitrate in the
titration of the chlorides in presence of fluorescent . these gives a pink
complex with the fluorescent coloring the complex .
(ii) indicators that produce colored precipitates .such indicators react
with an excess titrant to produce soluble colored complex .e.g. titration
of silver ions with thiocyanate ion to produce a white precipitate of
silver thiocyanate in presence of ferric alum .Another example is
titration of a chloride with silver nitrate which can be done in
presence of potassium dichromate forming a deep red silver
chromate.

The PH Meters
pH meters are instruments used to determine precisely the pH values
of solution .Such an instrument is based on determining the H+ ion
concentration in a solution by measuring the difference in potential at
the poles of an electrochemical cell .
 SCIENCE LABORATORY TECHNOLOGY

PH meter
The glass measuring electrodes have a very high electrical resistance
in the of several hundred mega ohms. Accordingly, the potential
difference in a cell containing a glass electrode must be measured
with a special potentiometer . These electronic voltmeters forms the
measuring part of the pH meter .Before measuring the pH value of a
test solution ,the potentiometer , the reference and the glass
measurement electrode must be first calibrated for accuracy .To
calibrate the instrument , a number of solutions of known PH are used
for reference .
pH meters are very quick and easy to use , they can measure solutions
having strong oxidizing and reducing properties and also can be used
with very small quantities of solutions or liquids .

 
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER SIXTEEN

VOLUMETRIC AND
TITREMETRIC ANALYSIS
Titremetric analysis refers to the qualitative chemical analysis carried
out by determining the volume of solutions accurately and from
which the volume of the unknown concentration is determined .
Volumetric analysis is almost similar to titremetric analysis, they both
rely on methods involving accurate measurement of volumes of liquids
but it may sometimes involve weighing

General Principles
Titrimetric analysis volumetrically measures the amount of reagent
,often called a titrant, required to complete a chemical reaction with
the analyte .A generic chemical reaction for titrimetric analysis is
                       aA + tT  ------->   products
    Where a moles of analyte A contained in the sample reacts with t
moles of the titrant T in the titrant solution.
The reaction is generally carried out in a flask containing the liquid or
dissolved sample.
Titrant solution is volumetrically delivered to the reaction flask using a
burette .Delivery of the titrant is called a titration .The titration is
complete when sufficient titrant has been added to react with all the
analyte .This is called the equivalence point .
    An indicator is often added to the reaction flask to signal when all the
analyte has reacted .The titrant volume where the signal is generated is
called the end point .The equivalence and end point are rarely the
same.

Successful Titrimetric Analysis

A few rules of thumb for designing a successful titration are:
 SCIENCE LABORATORY TECHNOLOGY
(i) The titrant should either be a standard or should be standardized
(ii)    The reaction should proceed to a stable and well defined
equivalence point
(iii)   The equivalence point must be able to be detected
 (iv) The titrant’s and sample’s volume or mass must be accurately
known
(v) The reaction must proceed by a definite chemistry .There should
be complicating side reactions
 (vi) The reaction should be nearly complete at the equivalence
point .In other words, chemical equilibrium favors product
(vii)   The reaction rates should be fast enough to be practical

Types of Chemistry
Although any type of chemical reaction may be used for titrimetric
analysis, the reactions most often used fall under the categories of
(i) Acid-Base    titration             
(ii) Oxidation-Redution  titration      
(iii) Precipitation     titration         
(iv)Complex Formation titration        

Procedure
A typical titration begins with a beaker or Erlenmeyer flask containing
a very precise volume of the analyte and a small amount of indicator
placed underneath a calibrated burette or chemistry pipetting syringe
containing the titrant. Small volumes of the titrant are then added to
the analyte and indicator until the indicator changes, reflecting arrival
at the endpoint of the titration. Depending on the endpoint desired,
single drops or less than a single drop of the titrant can make the
difference between a permanent and temporary change in the
indicator. When the endpoint of the reaction is reached, the volume of
reactant consumed is measured and used to calculate the concentration
of analyte by

Ca= Ct X Vt X M
Va
where Ca is the concentration of the analyte, typically in molarity; Ct is
the concentration of the titrant, typically in molarity; Vt is the volume
of the titrant used, typically in liters; M is the mole ratio of the analyte
 SCIENCE LABORATORY TECHNOLOGY
and reactant from the balanced chemical equation; and Va is the
volume of the analyte used, typically in liters.
These process of adding one standardized solution to another solution
so as to determine the equivalent volume is called titration . A standard
solution is one in which the concentration is known .
The mole is the unit of measurement of concentration .its usually
defined as the amount of a substance which contain as many elements
as 0.012kg of carbon . using the Avogados constant , a mole can be
defined as substance having as many as 6.02X 1023 particles

EXAMPLES OF TITREMETRIC ANALYSIS

Example 1
25cm3 of HCL whose concentration was unknown was titrated with
24.5 cm3 of 0.1 M NaOH . calculate the concentration of the acid.
HCL(aq) + NaOH(aq) NaCl(aq) +H2O(l)
Let the volume of HCL be Va
Volume of NaOH be Vb
Moles of HCL be Ma
Moles of NaOH be Mb
Therefore Va = 25cm
Ma = unknown
Vb = 24.5 cm
Mb = 0.1 moles
From the equation , the ratios of reaction between HCL and NaOH is
1: 1 , to calculate the concentration
VaX Ma = Vb X Mb
But Ma is unknown , it can be worked out by substituting the other
values
25 cm X Ma = 24.5 cm X 0.1 M

Ma =24.4cm X 0.1 M
25cm

= 0.098 M
These means 1000cm3 of HCL have a molarity of 0.098
Therefore if 1000cm3 = 0.098 M
25cm3 = ? moles

To get the moles in 25 cm3

 SCIENCE LABORATORY TECHNOLOGY
25cm3 X0.098M
1000cm3
=0.00245moles

Hence therefore 25 cm3 contain 0.00245 moles of HCL

EXAMPLE 2
25 cm3 of Potassium hydroxide KOH was pipetted and transferred
into a conical flask , it was titrated against 0.02M of HCL. On titration
, the following volumes of the acid was used in three titration ;
22.5cm3 ,24.2cm3, and 23.5 cm3
KOH (ag) +HCL(ag) KCL (ag) + H2O(ag)

(a) The average volume of HCL used

22.5cm +24.2cm +23.5cm
3
=23.4 cm3

let the volume of HCL = va

KOH = Vb
Moles of HCL =Ma
Moles of KOH =Mb

Therefore
Va= 23.4
Ma =0.02M
Vb =25
Mb = ?

Va X ma =Vb X Mb
X0.02 = 25 X MbMb =23.4 X0.02
25
Mb = 0.0187M
Therefore if 1000 cm3 =0.0187M
25cm3 = ?
25 X 0.0187M =0.00046
100
 SCIENCE LABORATORY TECHNOLOGY
Therefore the concentration of KOH used = 0.000468 moles

EXAMPLE 3
You are provide with;
Solution M1 which is an alkali ,XOH containing 2.88g /l
Solution M2 which is 0.045M H2SO4 acid solution
You are required to determine the molarity for alkali and then the
relative atomic mass of X in XOH. (O=16,H=1)
PROCEDURE
Fill the burette to the mark with solution M2 pipette 15cm3 of m1 into
a clean ,dry conical flask and titrate using phenolphalein indicator
provided .Repeat the titration until two concordant reading are
obtained .Record your results in the table below.Volume of
pipette=15.0cm3.

Titration number Trial 1st 2nd 3rd

Final reading 20.1 40.1 22.95 43.05
Initial reading 0 20.1 3.0 23.0
Volume used 20.1 20.00 19.9 20.05

a). What is the average volume of M2 use

20.00 + 19/.95 +20.05 =20.00cm3.
3
(Note that you must show the values being averaged)
(b)Calculate the molarity of M1
If 1000cm3 of M2 contains o.045 moles
Then 20cm3 of it will contain
20 X 0.045
1000
=0.0009Moles.

Equation for the reaction :

2XOH (aq) +H2SO4 (aq) ------> X2SO4(aq) +2H2O (l)
From the equation for the reaction , 1 mole of M2 reacts will 2 moles
of 1.
If 1 mole of M2 reacts with 2 moles of M1,
Then 0.0009 moles of M1 will react with 0.0009 X 2 =
0.0018moles .
If 15.0 cm3 of M1 contain 0.0018 moles,
 SCIENCE LABORATORY TECHNOLOGY

Then 1000cm3 will contain

1000X0.0018 =0.12M
15.0

EXAMPLE 4 You are provided with;

• Solution A which is a mixture of a salt; XCL (RFM 54. 5g)

and an alkali ; XOH(RFM 56g).8.4g of the mixture were made up to
one liter of aqueous solution .
• Solution B which is a 0.1M solution of a monobasic acid;HA.
You are required to determine the concentration (in moles per liter ) of
salt and hence the percentage of the salt in the mixture

Procedure
Fill the burette to the mark with solution B .
pippete 20cm3 of solution A into a clean dry conical flask and titrate
it against B using phenopthalene as indicator . repeat the titration
twice to obtain consistent results . record your results in the table
below

Titration number 1st 2nd 3rd

Final reading 24.15 48.55 26.25
Initial reading 0 24.4 2.0
Volume used 24.15 24.15 24.25

(a) What is the average volume of B used

24.15 +24.25 +24.15 = 24.20cm3
3
(b) Determine the number of moles of the monobasic acid HA ,
used.
If 1000cm3 of HA contains 0.1moles,Therefore 24.20 of it will
contain
24.20 x 0.1 = 0.0024moles.
1000
(c )Write an equation for the reaction between acid HA and the acid.
HA (aq)a +XOH (aq) -----> XA(aq) +H2O (l)
(d) calculate the number of moles of the alkali that are reacting
 SCIENCE LABORATORY TECHNOLOGY
From the equation of the reaction,1 mole of the acid reacts 1mole of
the alkali,therefore 0.0024moles of the acid will react with
0.0024 x 1
= 0.0024moles of the alkali.

(e)Calculate the concentration of the alkali in ;

(i) moles per liter
If 20cm3 of the alkali contain 0.0024moles;
Therefore 1000cm3 of it will contain
{1000 x0.0024 } =0.12M
20

(ii) grams per litre

Concentration in g / l =Molarity x R . F .M.
= 0.12 X56 6.72
= 6.72 g /l.

(f)what is the concentration of the salt XCL in grams per litre ?

Concentration of XCl in g/l = 8.4 –6.72
= 1.68 g/l.

THE MOLE CONCEPT

Atomic structure

An atom is the smallest indivisible part of an element that takes part in

a chemical reaction just like the element itself, an atom is assumed to
be spherical in shape and to consist of the nucleus at the center which
is surrounded by one or many shells or orbital .
The nuclears is made of proton which are positively charged and
neutrons which have no charge .they both have mass .the shell contain
electrons which are negatively charged and have no mass .they rotate
round the nuclears in definite distances known as orbitals ,shells or
energy levels just like the sun and the planets. Each atom has specific
 SCIENCE LABORATORY TECHNOLOGY
number of protons neutrons and electrons. The number of protons,
and electrons are the same or equal in each atom e.g. sodium atom
have 11 Protons 11 Eletrons 12 Neutrons
Atomic mass and mass number
Atomic number is defined as the total number of protons in an atom
while the mass number refers to the total number of protons and
neutrons in an atom e.g. the atomic number of sodium atom is 11
because it has 11 protons while its mass number is 23 because it have 11
protons + 12 neutrons =23
Relative atomic mass (RAM)
These is the mass of an atom expressed relative to the mass assigned
to carbon 12 . e.g. the RAM of
Na =23
O =16
N =14

Relative molecular mass or relative formula mass (RMM) or

(RFM)
These is the sum of the atomic masses of all the atoms in a compound
e.g.
Nacl = 23 +35.5 =58.5
Na2CO3 =(23 X2) +12 +(16X3) =106

The mole
A mole of any element is defined as the number of atoms of that
element that is equal to the number of atoms in exactly 12.0 g of
carbon 12.
One mole of any atom contain 6.02 X1023 atoms (avogadros constant)
e.g. there are 6.02 X1023 in every mole of mole of Na, Mg , Ca , Zn etc
The molar mass
This is defined as the mass in grams of 1 mole of a substance
e.g. the molar mass of ; Na=23g
Ca= 40g
Nacl =58.5 g
Cacl =111g
The molar mass of an element can be determined from its atomic mass
or formula mass e.g.
 SCIENCE LABORATORY TECHNOLOGY
(a) calculate the number of moles in
(i) 10g NaOH
The molar mass of NaOH = 40g
1 mole NaOH =40g
? moles =10g
= 10g X 1m =0.25moles
40g
(ii) 40g Nacl
The molar mass of Nacl =58.5
1 mole of Nacl = 58.5
?moles = 40g
40g X1m =0.68mol
58.5g
(c)calculate the mass in 0.1mole NaOH
1m NaOH =40g
0.1m =
0.1m x 40g = 4g
1m
(i) how many atoms are present in 0.5moles of NaOH
1 mole of NaOH =40g =6.02X 10-23
0.5 moles = ?

0.5 X6.23 X10 23 =3.01 X10-23

1
(ii) how many atoms are in 20 g NaOH

if 40 g = 6.02 x10-23
20g = ?
20 X 6.02 X10-23
40
=3.01 X10-23 Atoms
EMPERICAL AND MOLECULAR FORMULA
The formula of a compound indicates the number and kind of each
atom present in a respective element of the compound e.g. Na2CO3
shows that there are two atoms of sodium and one atom of Carbon
and three atoms of oxygen
(a) percentage composition
These is the mass of each element in a compound relative to the
entire mass of the compound and multiplied by 100% . The
 SCIENCE LABORATORY TECHNOLOGY
percentage composition tells the percentage proportions of the
masses made up by each element in the compound .
it can be calculated by calculating the composition from a given
chemical element e.g. calculate the % of
(a) Hydrogen in water
H2O =18
H2 = 2
% composition of H = 2 X 100 =11%
18
18
(b) Oxygen in water
O =16% composition of O =16 X100 =89 %
18
Percentage composition of a compound can also be determined by
experimental analysis . In these method ,the mass of the sample is
first determined then the sample is decomposed or chemically
separated into its compound . The mass of each element is determined
and the % composition is calculated as above.

Emperical formula
These is the formula that gives the simplest whole number ratio of
the atoms of the compound or element. Such a formula simply tells
the simplest ratio of he compound e.g.
(i) Calculate the empirical formula of a compound with
80% carbon and 20% hydrogen
carbon =80%
hydrogen =20%
RAM of carbon =12
Hydrogen =1

Therefore
80% C : 20% H
12 1
= 6.7 :20

= 6.7 : 20
6.7 6.7
 SCIENCE LABORATORY TECHNOLOGY
=1 : 2.985
=1: 3
Therefore , the imperical formula will be:
= C1H3
Molecular formula

Molecular formula gives he actual number of atoms of each element in

a molecular compound . its always a whole number multiple of the
empirical formula .These is obtained by dividing the empirical formula
for the unknown compound with the molar mass of he compound .e.g.
The empirical formula of glucose is CH2O its empirical mass is 30g
but it experimentally shows that the molar mass of glucose is 180g .
Calculate the molecular formula
Molecular formula =
molar mass
Empirical mass
=180 =180 = 6
CH2O 30
Therefore (CH2O)6 = C6H12O6

MOLARITY AND NORMALITY

Molarity is defined as concentration in grams per liter i.e. g/dm3 it’s
the number of grams of a given substance dissolved in 1000cm3. It’s
abbreviated as M e.g.
1M of NaOH = 40 g
0.1M 0f NaOH = 4g

Normality is another way in which concentration may be expressed . a

normal solution of any substance is defined as a solution which
contain one gram equivalent of that substance per liter e.g.
1N H2S04 = 49g
1N Na2CO3 =53 g
IN NaOH = 40g
0.1N H2SO4 = 4.9
0.1N Na2CO3 =5.3g
 SCIENCE LABORATORY TECHNOLOGY

STOICHIOMETRIC AND IONIC EQUATION

Stoichiometric equation are chemical equations which state the
relationship between masses of the reacting chemicals and the
relationships between volumes of the reactants . e.g.
NaOH +HCL----------> NaCL +H2O
1M : 1M 1M 1M
ionic equation give an indication of the actual reactions which is
taking place in the solutions e.g.
H+ +OH- --------> H20

STANDARD ,PRIMARY AND SECONDARY SOLUTIONS

A standard solution is one whose concentration is known . It’s

prepared by dissolving an accurately weighed sample of desired
solute in an accurately measured volume of a solvent. These method
is rarely applicable (acceptable) since relatively few reagents can be
obtained in a sufficiently pure form to meet the analytical demand .
Primary standards are just few of these substances which are often
adequately used in these regard for standardizing other solution by
titration , in which the other solution to be prepared is allowed to
react with a weighed portion of a primary standard .
The reaction between a titrant and the substance to be selected as a
primary standard should fulfill or have the following characteristics .
(a) It should be readily available in a pure form or in a state of known
purity and at a reasonable cost, the total amount of impurities should
not exceed 0.001-0.02%
(b) The substance should be stable, easy to dry and should not be so
hygroscopic (takes up water during weighing ) it should also not loose
water during exposure.
( c)Should have a reasonably high equivalent weight in order to
minimize the consequence of errors during weighing e.g. Na2CO3
Secondary standards are solution e.g. acids prepared from primary
standards then are used to standardize or to obtain the concentration
of other solutions .

INSTRUMENTS USED IN VOLUMETRIC ANALYSIS

 SCIENCE LABORATORY TECHNOLOGY

(A) Weighing bottle

These consist of a cylindrical glass vessel with a ground stopper in
which the material can be weighed out of contact with the atmosphere
.its usually heated in a steam oven before use to ensure dryness and
is then allowed to cool in a dessicator
It should be protected from contamination with fingers during
handling (using a dry cloth )
A weighing bottle can be used in two ways
(i) By first weighing it when empty then when loaded. This method is
tidious ad it’s likely to expose the substance to the atmospheric
contamination during the process of weighing .
(ii) By weighing an already loaded bottle then pour the substance in a
measuring flask then letter weigh the empty bottle and finally
calculating the difference in weight

(b) Measuring flask

250cm3 capacity is usually used. Measuring flask are made to contain
a specific volume of liquid and will not deliver that volume because
part of that liquid will inevitably remain or retained as a film on the
sides of the flask .
They are usually graded to work at specific temperatures e.g. 20oc and
should be used at temperatures close to these .they should be rinsed
before use wit h distilled water or solvent to be used . the solvent
should be added from the wash bottle down the sides of the beaker so
as to avoid splashing . The flask should thereafter be filled with the
solvent to the meniscus mark a teat pipette should be used to add the
last drops the flash should then be stopered and shaken vigorously .
(c) Burette
They usually have a capacity of 50cm3. Some are glass stopered . its
first washed is first washed /rinsed with solution to be used
whereby the solution should run through the jet . its then filled to the
zero mark then read carefully to ensure it’s on the meniscus a sheet of
white paper placed behind it . The tap is opened and the required
volume is released and worked out by difference .
Rough titration is always advisable before doing the real accurate
titration ..About 1cm3 solution is released at a time into the solution
in the conical flask containing an indicator until the indicator
changes color theses at end point
 SCIENCE LABORATORY TECHNOLOGY
Burettes should always be well washed out after use . if incase an
alkaline solution have been used in the burette , it should be washed
out using about 10cm3 of dilute acid then rinsed using distilled water

(d) Pipettes
They are designed to deliver a certain volume of liquids . When filled
to the mark, it contains more than these volume .the extra volume is
always retained after delivery as a film on the sides of the pipette
and in the tips .The pipette is filled past the mark by sucking the
solution into it and allowed to slowly drain away.sunction devices
should b used for poisonous or corrosive chemicals . If the solution is
sucked by mouth it should spat out at once and the mouth rinsed .
The liquid is retained in the pipette by pressing the forefinger on the
open end of the pipette .the tip of the conical flask is then placed
inside the conical flask . No attempt should be made to expel the
remaining solution from the pipette as the pipette will have already
delivered 25 cm3

Sources of errors in volumetric analysis

(a) solution not made homogeneous

These results from the solution not being thoroughly mixed by shaking
or swirling
(b) inaccuracy in instruments used
some instruments have errors caused by manufacturing e.g. due to
poor calibration
(c) Errors in weighing
These may result as a result of carelessness or lack of concentration by
the technician.
(c) Presence of impurities in chemicals used.
These are due to contaminants or impurities on chemicals used which
may arise due to action of moisture , light , dust particles ,or oxygen .
These can be minimized by correct storage
(d) Inaccuracy in endpoint recorded.
These may occur if too much indicator is used , an extra amount of
reagent may be needed to couse color change .its helpful to perform a
blank titration so as to ascertain the volume of the reagent that is
necessary to affect the indicator which have been added to a volume
of water approximately equal to the final volume of solution likely to
 SCIENCE LABORATORY TECHNOLOGY
be obtained in the actual titration . These problem can be overcomed
by practice and experience.
NB; The result obtained in volumetric analysis is correct to three
decimal places

CHAPTER SEVENTEEN

GRAVIMETRIC ANALYSIS
Gravimetric analysis a type of chemical analysis whereby the quantity
of a substance is determined through weighing . it’s a technique based
on the determination of weight of the original substance taken for
analysis and that of the product obtained after reaction . The product
is then obtained as a compound of known definite composition.
Gravimetric analysis takes longer than volumetric analysis and it also
requires more skills of manipulation . Its however more accurate
than volumetric analysis .The aim of gravimetric analysis therefore is
to obtain a final compound of known composition from which the
mass of the element or radical which is being estimated may be
calculated .
Methods of gravimetric analysis .
There are two methods of gravimetric analysis
(a) Evolution method
(b) Precipitation method
Of the two methods , precipitation method is the most important and
commonly used , and we shall hence therefore look at it in details
Precipitation method involves the qualitative conversion of elements
or groups of elements to be determined into an insoluble compound
of known composition , which can be suitably isolated by filtration in
a state of purity and then weighed . e.g.

Ag NO3(aq)+ Nacl (ag) ----> Ag cl (s) + NaNO3(aq)

(soluble ) ( soluble) (precipitate) (soluble)

BaNO3 (aq)+ K2S04 (aq) -----> BaSO4( s ) + K2NO3( aq)

 SCIENCE LABORATORY TECHNOLOGY

General principles of gravimetric analysis

Gravimetric analysis is based on chemical reactions such as
Ar + Bq -----------> ArBq
Where ‘ r‘ are molecules of the analyte ‘A‘ react with ‘q‘ molecules of
the analyte ‘ B ‘ to produce a product ArBq
which is usually insoluble and hence it can be separated by filtration
,then dried , ignited into another compound of known composition
and thereafter its weighed or its even weighed directly after drying
e.g. calcium can be determined gravimetrically by precipitation as
calcium oxalate and the oxalate can be ignited to give calcium oxide
which can now be determined by weighing i.e.
Ca2+ + Ca2O4-2 -------> CaC2O4 +CaC2O4(s)
CaO (s) -------> + Ca2 +CO
Calcium oxalate + calcium oxide
An excess of reagent B is normally added to repress the solubility of
the precipitate
The following requirements should be met in order for a Gravimetric
analysis to be successful
(a) the separation process should be sufficiently complete so
that the quantity of the element of the analyte left unprecipitated is
less than 0.1mg in determining the major constituent of a
microsample
(b) the substance weighed should have a definite composition
and should be pure or nearly so , otherwise erroneous results may be
obtained .

Formation and properties of a precipitate

Formation of pure and filterable precipitated is of major importance
in gravimetric analysis . The process of building up into solid
aggregates that are sufficiently large enough to precipitate begins
when the solubility product is supposed (or goes beyond ) and the ions
begins clinging together forming particles called nuclei which then
grows sufficiently large enough to be pulled to the bottom of the
container by the force of gravity .
During these growth process, the size of the particles passes through
several colloidal ranges (colloids are particles that are very small and
not easily seen by eyes but not as small as the water or solvent
particles in which they are dissolved in)
 SCIENCE LABORATORY TECHNOLOGY
The size of the precipitates is determined by the relative rates of two
processes i.e

(a) the formation of the nuclei ( nucleation)

(b) the growth of the nuclei to form particles (colloids) that are
sufficiently large enough to precipitate if the rate of nucleation is small
compared to the rate of growth of the nuclei, then few particles are
produced but are relatively large in size .such precipitated is more
easily filtered and is frequently pure than precipitates whose particles
are relatively small. Hence its always good to try to adjust conditions
during precipitation so that the rate of nucleation is small compared
to the rate at which the particles grow in size .

EXPERIMENTAL TECHNIQUES OF GRAVIMETRIC

ANALYSIS
Gravimetric techniques involves the following
(a) solution making
(b) precipitation
(c) digestion
(d) filtration
(e) washing of precipitated
(f) drying and ignition
(g) weighing

a) Solution making

These is the process of dissolving the reagents which were initially in

solid form into solution form. The solid is first accurately weighed out
and then dissolved in about 50cm3 of distilled water in a clean beaker

b) Precipitation

These is the process in which the originally soluble substance to be

estimated is converted to an insoluble precipitate . the precipitated
formed should have low solubility otherwise complete precipitation
will not be possible
the precipitated must also have large particles for quick filtration and
also not to block the filter pores . it must also be stable
 SCIENCE LABORATORY TECHNOLOGY
Conditions for precipitation
for a good precipitate to form ,saturation of the precipitate must be as
slow as possible. These means that thee solution should be very dilute
because dilute solution have larger particles and are easily filtered
contamination’s of the precipitates
Sometimes the precipitated formed may not be pure due to presence of
contaminants. These contamination’s may occur due to

(i) Co –precipitation

These is a condition whereby normally soluble substance is taken up

by the precipitate formed e.g. when BaNO3 is added to K2SO4
solution .the precipitate formed is BaSO4 but these precipitate may
contain some KNO3 which is normally soluble.
(i) post- precipitation
These occur when the impurities inform of a sparingly soluble
compound is precipitated on the surface of the main precipitated
after its formation e.g. when calcium oxalate is formed in presence of
magnesium , a small amount of magnesium oxalate is formed . Post-
precipitation normally occurs after the real precipitated have formed
and it forms on the surface of these precipitated unlike in co –
precipitation where it takes place during the process of precipitated
growth .Its therefore found inside the precipitate. Therefore
Contamination due to co -precipitation is therefore difficult to remove
compared to that due to post -precipitation.

How to reduce contamination in precipitate due to co-

precipitation and post precipitation
To minimize chances of post and co- precipitation, both solutions in
use in preparation should be very dilute , these help to complete
ionization and reduces supersaturation . Precipitation should be done
when the solution is still hot as these will help to increase solubility
and to also increase the speed of crystallization . The crystalline
precipitate should be digested. The solution should also not be boiled
during precipitation because these will lead to splashing which may
 SCIENCE LABORATORY TECHNOLOGY
result in errors. The beakers used in precipitation should have their
inner surfaces free from scratches because some of the precipitates
formed may stick on the scratches and hence being lost.

(c) Digestion
Digestion is the process f keeping the precipitated formed in contact
with the liquid from which it was formed (i.e. the mother liquor) so as
to achieve complete precipitation in a form that can easily be easily
filtered . It also reduces in the amount of absorbed impurities and
makes the precipitates more regular . Digestion is carried out by
placing the covered beaker containing the precipitate in a still bath .
These process allows the precipitate to stand in contact with the
mother liquor at an elevated temperature for sometimes before
filtration . Here , small particles of crystalline substance e.g. BaS04
,being more soluble than the larger ones ,dissolves more readily
making the solution to be supper saturated with respect to larger
particles .
Consequently therefore additional materials must therefore leave the
supersaturated phase and enter the solid phase, these ions therefore
deposits on the larger particles hence causing these particles to grow
over the larger ones . These process is sometimes called ostward
ripening. The reason for the elevated temperature is to cause more
solute to dissolve causing supersaturation.
N/B The most difficult problem in precipitation is obtaining the
precipitated in higher degree of purity.these may be due to the effect of
co-precipitation and post precipitation . Sometimes crystalline
particles e.g. BaS04 may adsorb impurities especially when thee
particles are still small and as they grow in size , the adsorbed
impurities may become enclosed in the crystal. These type of
contamination is called occlusion. Occluded impurities cannot be
removed by washing the precipitated but the quantity of the
precipitated can often be removed by digestion.
To minimize co precipitation , the following should be done
(a) Washing - adsorbed impurities can be removed by washing unless
they are occluded .
(b) Digestion – these is recommended for crystalline precipitated and
not gelatinous precipitate
(c) Reprecipitation -if the salt can be readily redissolved , it can be
filtered ,redissolved and reprecipitated
 SCIENCE LABORATORY TECHNOLOGY
(d) Separation - the impurities may be separated by changing its
chemical nature through some reaction before the precipitated is
formed

( d) Filtration and washing

Filtration is done so as to separate the last traces of the mother liquor
from the precipitated . The precipitated is collected or retained on the
filter paper of filtering crucible while the mother shall pass through .
But its important to ensure that precipitation process is complete
before filtration.

How to carry out filtration

Prepare the filter paper into a cone shape and place it on a correct
and place it in a correct shape funnel . Moisten it with water and press
down so as not to leave any air space .
Place the funnel on the filter stand and place a clean beaker below
with the tip of the funnel touching the inside wall of the beaker so as
to avoid splashing
Pour the supernatant (the liquid to be filtered ) down wit the glass rod
into the filter paper . Never fill the filter paper completely.
The precipitate formed on the filter paper should not be disturbed
when filtering

Washing the precipitated

Distilled water in a wash bottle is used as the wash solution. It’s
directed along the rim of the funnel or filter paper , and then gradually
towards the apex of the corner where the precipitated is collected .
The end of washing is carried out by checking the end of qualitative
test on a small test on small sample of the filtrate from the funnel .
The precipitated should not be allowed to stand on the filter paper for
long otherwise the precipitated will cake (harden) and therefore
making it impossible to wash.
The precipitated should not be soluble in washing solution but the
impurities should be easily soluble in it . The washing liquid should
also be easily volatile at the temperature of during the precipitate
The filtrate in the beaker should be examined for particles of the
precipitated that may have run through the filter paper and if
 SCIENCE LABORATORY TECHNOLOGY
present ,the filtrate in the beaker must pass be through the filter
paper again .

Drying and ignition of precipitated

In any Gravimetric procedure , the precipitated must be converted
into a form that is suitable for weighing .its necessary for the
solution to be weighed to be pure , stable and of definite
composition for the result of analysis to be accurate
The water and any other electrolyte added to the wash water have to be
eliminated .some precipitates are weighed in the same form as that
which they are precipitated ,others undergo chemical changes during
ignition and these reaction must go to completion for correct results .
The procedure used in the final step depends on the chemical
properties of the precipitated and upon the tenacity with which water
is held by the solid .the precipitated can be dried either by
(a) air drying at ambient temperature
These can be done by washing the precipitated with alcohol or ether
and drawing air over the preoccupied for some few minutes .however
these procedure is not recommended because of the possibility of
incomplete removal of washing water
(b) Air drying at low temperature
Some precipitates loses water very easily in an oven at a temperature
of between 100 –130 °C e.g. AgCl does not adsorb water strongly and
therefore its normally dried in these manner .however not all water is
removed .

Ignition of the precipitate

Ignition is the process of burning or incineration of the substance so as

to convert it into another oxidized form of known purity . Ignition at
high temperature is required for removal of occluded water or water
that have been very strongly adsorbed and for complete conversion
of some precipitates to the desired compound .
Gelatinous precipitates e.g. hydrous oxide adsorbs water quite strongly
and must be heated very strongly at high temperature so as to
completely remove water .

Errors in ignition
 SCIENCE LABORATORY TECHNOLOGY
Errors rather than incomplete removal of water or volatile electrolytes
can occur during ignition . One of the most serious errors is the
reduction of the precipitate by carbon when a filter paper is used .
Infact substances such as Agcl that are easily reduced are never
filtered on filter paper instead crucibles are used .
Precipitates can also be over ignited leading to the decomposition of
the substances to indefinite composition .
Other errors an also result from the reabsorption of water or carbon
dioxide by an ignited precipitate upon cooling. Therefore crucibles
should be kept in dessicators as they cool
N/B Studies on ignition temperature required for different
precipitates can be made using another technique of
electrogravimetric analysis which involves a thermobalance which
allows a sample to be weighed while its in a furnace

APPARATUS USED IN GRAVIMETRIC ANALYSIS

Apparatus used in gravimetric analysis should be appropriate and
suitable for the required purpose. They should also be thoroughly
cleaned with distilled water. Glasswares and crucibles used should not
have scratches on their surfaces.

(a) Beakers
The beakers used are usually 400 –500cm3 . they are used for ;
(i) Making solutions
(ii) Carrying out precipitation
(iii) For heating solutions
(iv) for filtering solutions
Beakers are made of Pyrex or corning glass because the kind of glass
are heat tolerant ( they tolerate frequent heating and cooling )
Sprouted beakers are preferred because they allow pouring , and
steam to go out even when the beaker is covered with a cover glass or
 SCIENCE LABORATORY TECHNOLOGY
cork glass. Sprouted beakers also provide space at which glass rods
protrudes (come out) from the beaker .
Ensure that the beaker is clean and dry before use .
Beakers should be heated on an asbestos filter covered with a wire
gauze . Never heat on a naked flame.
Before placing the beaker in the beaker for heating .its outer surface
should be dried by wiping the moisture from its surface so as to
prevent uneven expansion of the beaker and hence cracking .
When boiling in a beaker , a stirring rod should not be left in the
beaker because the rod may bump up and down during the boiling
and may break the bottom of the beaker .
The content of the beaker must be covered with a clock to avoid
contamination .
The inner surface of the beaker should be smooth and not scratched
since the scratched surface may adsorb substances from the solution
and therefore leading to wrong results .

(b) Cover or clock glass

Cover glass are used for covering beakers containing solution for
gravimetric analysis . A suitable is always used . It should be largely
be larger diameter than the beaker.
Cover glass should be placed on the beaker on the beaker with
convex (bulge) downward.
Ensure that the beaker is covered with a cover glass when heating so
as to leave space for exit of steam.
When cover glass is removed from the beaker , it should be washed
with spray of water from the wash bottle .

(c) glass rod

This is a rod with a rounded tip for stirring gravimetric solutions and
for removing precipitates from the container during filtration
The length of the glass rod should be of suitable size and length of
the vessel (beaker)
if the glassrod is fitted with a short piece of rubber tubing over one
end , then the tubing is called policeman.
 SCIENCE LABORATORY TECHNOLOGY
The policemen is used to remove detached particles of precipitates
sticking on the sides of the beaker which cannot be removed by
stream of water from the wash bottle
When the glass rod is being used , the policeman should not be used
for stirring and it should remain in solution because the rubber is
attacked by chemicals andhot water .
The policeman end should not be used for directing the flow of liquid
during pouring
The rod should not be placed on the bench as it can pick dust and
other impurities but should be instead placed on an empty beaker to
rest on a sprout rim.
When the rod is taken out of the solution , it should be washed with
spray from the wash bottle.

Wash bottle
These bottle is designed in such a way as to deliver a fine jet or stream
of distilled water for transfer or washing of the precipitate .wash
bottles are either made of glass or plastic ,but plastic wash bottles are
the most commonly used because they are cheap, easy to clean and not
fragile and resistant to chemical attack .they are also flexible i.e. only a
slight application of pressure on the sides of the bottle gives a jet of
water.

Funnels
Funnels are used for directing liquids from one container to another
with a narrow neck .they are made of Pyrex or plastic The commonly
used ones are those bearing an angle of 60o to the stem and have a
diameter of 9cm .the stem diameter is 4cm
Funnels should be cleaned and washed wit distilled water.

Weighing bottle
These is a small bottle with a glass stopper .its weight is known .its
used for weighing in gravimetric analysis .
Weighing glasses that are fitted with glass stoppers outside are better
than those fitted with stoppers inside because the ones with stoppers
inside have a danger of picking up small particles from inside during
weighing hence the weight may not be accurate.
 SCIENCE LABORATORY TECHNOLOGY
Crucibles
These are small containers with loosely fitted lid for heating
substances in gravimetric analysis.
Crucibles are made up of fine quality porcelain that is glazed outside
and inside. Their capacity is usually between 25-30ml
Crucibles made of fussed silica are better than porcelain though they
are more expensive .silica crucibles are highly resistant to heat and
therefore good for heating,they do not react with acids at high
temperatures except hydrochloric acid and phosphoric acid.

Tarring process
Tarring is a process followed when cleaning crucibles and their lids.
Crucibles should be washed ,cleaned and dried and finallyIgnited or
heated before using it. the following is a systematic steps that should
be followed .
 Place the crucibles in concentrated potassium dichromate to
remove stains .
 Wash the crucibles in distilled water ,add some dilute nitric acid to
the crucibles then heat the crucible .
 Pour away the HNO3 ,wash it in distilled water ,if the stain still
persist then rub the crucible with moist sand .
 Wash it with tap water ,heat it with non-luminous flame for 15 min.
 Incase the stain still persist ,leave it alone because these means that
its now part of the crucible and therefore its harmless.
 Clean the lid using a similar process. Place the crucible and lid in the
desicator using a pair of tongs
 Always handle crucibles using pairs of tongs and not fingers because
the moisture and dust from your fingers can make the crucible
contaminated .
 Never put a wet crucible on a flame because it might break .

Crucible tongs
These are scissors like apparatus used for handling crucibles and
other hot substances .its made up of nickel steel.
Before using it ensure that its arms move freely and its tips move
freely.
 SCIENCE LABORATORY TECHNOLOGY
When using crucible tongs , the tips should be cleaned ,if its not
clean ,rub with sand paper and tap water then heat with non-
luminous flame for a while and let it cool .
When not using it , place it on the table with the tips facing upwards
so that they don’t pick dust from the table .
When lifting the crucibles ,the tips of the tongs should not touch the
content of the crucibles to avoid loss of content.

Clay-pipe triangle
These is used for supporting the crucibles on a retord stand while
heating it on a Bunsen burner or while placing it in the desicator for
drying . It’s formed by passing three pieces of iron wire through small
length of heat resistant clay tube . the wires are then twisted at the end
to form a triangle

Desicator
These is a large vessel with a tightly fitting cover in which the
atmosphere is kept free from moisture or water vapor by use of a
drying agent called a desiccant which is placed in the lower chamber or
compartment .it is usually used for holding crucibles so as to protect
them and their contents from contact with moisture.
The ground glass rims of the dessicator are coated with Vaseline or
grease to avoid air getting in .
When using a desicator;
Hold firmly when opening on the table with your left hand . hold the
knob of the cover with your right hand and pull it aside by sliding it
sideways .
Do not keep the cover on the table but hold it in your left hand upside
down in order to avoid dirt getting in contact with the grease
If the cover have to be placed on the table , the grease surface should
face upward with the knob resting on the table.
Place the hot crucible in the desicator using a pair of tongs. If the over
have to be replaced , slide it on the rim until the rims coincides . Also
remove the crucible from the desicator using a pair of tongs
Do not pace a red-hot crucible in a desicator but allow it to atleast cool
Use both hands when carrying a desicator

Filtering media
 SCIENCE LABORATORY TECHNOLOGY
These includes all categories of those devices used in filtration e.g.
filter papers and filter crucibles . different filter papers are used for
different types of precipitates depending on
(a) The nature of the precipitate
(b) The treatment given to the precipitate to convert it to the final
product for weighing
There are two types of filtering media’s
(i) Filter paper
(ii) Filter crucibles

(a) Filter paper

in quantitative work , accuracy is very important ,hence therefore
special type of filtering media’s are used i.e. ashless filter papers and
harden filter papers is important
Ashless filter papers are produced by removing the mineral
components of the paper by treating them with HCL and HF acid .
Such papers are called acid washed filter papers . they have general
diameters of 7 and 12.5cm
Filter papers come in various grades and the type of filter papers one
will choose will be determined by the fineness of the precipitate to
be filtered e.g. FeOH precipitate have large particles and therefore
requires a dense paper with fine pores . the denser the precipitate ,
the slower the filtration .
Because of the hygroscopic nature of the filter paper s , the
precipitated collected cannot be weighed in a filter paper after
washing and drying and therefore igniting the content in a crucible is
necessary and the residue is weighed . the weight of the ash formed is
negligible .

Filter crucibles
They are used for collection of precipitates to be weighed after heating
in them . the precipitate is filtered , washed and weighed in the same
vessel they are porous and the precipitated is sucked by applying
sunction pressure . There are generally three types ;
(i) Gooch crucibles .
They are made of glazed porcelain with a perforated bottom upon
which there is a glass mat or glass fiber or asbestos
(ii) Sintered glass crucibles –these are made of only sintered glass
 SCIENCE LABORATORY TECHNOLOGY
(iii) Porous porcelain filtered glass - They are made of porous
porcelain .
NB Filtering crucibles can be heated in an oven

CHAPTER EIGHTEEN

COLORIMETRIC ANALYSIS
Most test substances in water are colorless and undetectable to the
human eye. To test for their presence we must find a way to "see"
them. A colorimeter or spectrophotometer can be used to measure any
test substance that is itself colored or can be reacted to produce a color.
In fact a simple definition of colorimetry is "the measurement of color"
and a colorimetric method is "any technique used to evaluate an
unknown color in reference to known colors". In a colorimetric
chemical test the intensity of the color from the reaction must be
proportional to the concentration of the substance being tested. Some
reactions have limitations or variances inherent to them that may give
misleading results. Most limitations or variances are discussed with
each particular test instruction. In the most basic colorimetric method
the reacted test sample is visually compared to a known color standard.
However, the eyesight of the analyst, inconsistencies in the light
sources, and the fading of color standards limit accurate and
reproducible results.
To avoid these sources of error, a colorimeter or spectrophotometer
can be used to photoelectrically measure the amount of colored light
absorbed by a colored sample in reference to a colorless sample
(blank). A colorimeter is generally any tool that characterizes color
samples to provide an objective measure of color characteristics. In
chemistry, the colorimeter is an apparatus that allows the absorbance
of a solution at a particular frequency (color) of visual light to be
determined.
Colorimeters hence make it possible to ascertain the concentration of a
known solute, since it is proportional to the absorbance.
 SCIENCE LABORATORY TECHNOLOGY

colorimeter
A spectrophotometer is a photometer (a device for measuring light
intensity) that can measure intensity as a function of the color, or more
specifically, the wavelength of light. There are many kinds of
spectrophotometers. Among the most important distinctions used to
classify them are the wavelengths they work with, the measurement
techniques they use, how they acquire a spectrum, and the sources of
intensity variation they are designed to measure. Other important
features of spectrophotometers include the spectral bandwidth and
linear range. The most common application of spectrophotometers is
the measurement of light absorption.
White light is made up of many different colors or wavelengths of
light. A colored sample typically absorbs only one color or one band of
wavelengths from the white light.
Different chemical substances absorb varying frequencies of the visible
spectrum. Only a small difference would be measured between white
light before it passes through a colored sample versus after it passes
through a colored sample. The reason for this is that the one color
absorbed by the sample is only a small portion of the total amount of
light passing through the sample. However, if we could select only that
one color or band of wavelengths of light to which the test sample is
most sensitive, we would see a large difference between the light before
it passes through the sample and after it passes through the sample.
 SCIENCE LABORATORY TECHNOLOGY
Colorimeters rely on the principle that the absorbance of a substance is
proportional to its concentration i.e., a more concentrated solution
gives a higher absorbance reading.
Spectrophotometers use either a tungsten or xenon flashlamp as the
source of white light. The white light passes through an entrance slit
and is focused on a ruled grating consisting of 1200 lines/mm. The
grating causes the light to be dispersed into its various component
wavelengths. The monochromator design allows the user to select
which specific wavelength of interest will be passed through the exit slit
and into the sample. The use of mirrors and additional filters prevents
light of undesired wavelengths (diffraction of higher order, stray light)
from making it to the sample. A photodetector measures the amount
of light, which passes through the sample.
Colorimeters pass a colored light beam through an optical filter,
which transmits only one particular color or band of wavelengths of
light to the colorimeter's photodectector where it is measured. The
difference in the amount of monochromatic light transmitted through a
colorless sample (blank) and the amount of monochromatic light
transmitted through a test sample is a measurement of the amount of
monochromatic light absorbed by the sample. In most colorimetric
tests the amount of monochromatic light absorbed is directly
proportional to the concentration of the test factor producing the color
and the path length through the sample. However, for a few tests the
relationship is reversed and the amount of monochromatic light
absorbed is inversely proportional to the concentration of the test
factor.
Colorimetric methods
There are various colorimetric methods available for measuring
concentration of colored substances
(a) visual comparison
These was a technique where solutions were matched against a set of
standardized solutions using testtubes of similar diameters .the values
found between a set of standards could then be approximated
(b) Lovibond comparator
These apparatus consist of a box with compartments for tubes of the
test and the blank solution .it have a rotatable disk mounted in front
of the test tubes . The central window of the box is in front of the test
solution and the rotation of the blank solution and the rotation of the
disk permits the superimposition of the colored glass standards.
 SCIENCE LABORATORY TECHNOLOGY

(c) Visual colorimeters

These type of colorimeters are not currently in use .but they consisted
of two glass tubes with which each contained a solid plunger that
was capable of being raised and lowered . The light from an even
source of illumination at the base of the instrument was made to pass
through the two solutions and through the plunger and brought into
focus at the eyepiece . The depth of the test solution was then varied
until identical illuminations of both standard and test solutions was
achieved .
The concentration of the test solution was derived by using the
formulae below
conc of test sol = conc of std x depth of std
Dept of test solution

(d) Photoelectric absorptiometers

These measure the amount of light absorbed and not the intensity of
color. It uses photoelectric cells on which light fall on them generates
an electric current which can be made to deflect a galvanometer. The
degree of deflection is proportional to the intensity of light falling on
the cell. Since the amount of light absorbed during passage through the
colored solution is proportional to the concentration of the solution ,
then the degree of deflection is also proportion to the concentration
of the solution .
When white light is passed through a colored solution, some of the
frequencies or wavelengths of light will be absorbed while others will
be transmitted through the solution if a monochromatic light of a
frequency which is preferentially absorbed by the molecules in
solution are passed through the solution , then a proportion of the
incident light will be absorbed while the remainder is transmitted .

Absorbtiometers have five essential parts ;

(a) Light source
These can be either a tungsten filament , hydrogen or deuterium
discharge lamp
(a) Wavelength selector
Filters ,diffraction gratings or prisms are used for these purpose,.there
are many types of filters that can be used e.g. light filters .the filters
chosen are usually complementary to the color solution to be
 SCIENCE LABORATORY TECHNOLOGY
measured e.g. blue solution uses yellow filters bluish –green solution
uses red filters ,while purple solution uses red filters.
filters are made of glass or dyed gelatin between glass plates ,they have
a limited transmitance band at which they transmit maximally e.g.
bluish green solution absorbs light in he red part of he spectrum,such
solutions when illuminated by white light it absorbs red color
wavelength and transmits blue-green light together with small
amount of red ,the greater the concentration of the solution ,the
smaller the amount of light transmitted.
(c) Cells and cuvettes
Cuvettes are used to hold the colored solutions under test . Cuvettes
must be scrupulously clean, they have two parallel clear sides made of
optical glass they should not have scratches and their outside surfaces
must be wiped clean with a lens tissue and held up to the light so as to
ensure they are there are no dry finger marks or spillage fluids on
their outer surface for these may absorb light and interfere with the
measurement of color
The sets of the cells used should be optically matched before placing
the cuvettes into the light path

(b) Photoelectric cells

There are three types
(i) Barrier layer cell
(i) photoemmissive tube
(ii) photo multiplier
Light coming through the solution and falling on these photocells
generates an electric current which deflect the galvanometer needle.
This deflection is proportional to the light intensity
(c) galvanometer
Galvanometers measures the output of the photosensitive cells
Requirements of calorimetric analysis
Its essential to ensure that the color being measured is only due to the
substance under investigation and not any other reagent used .to
ensure these , its therefore important to include the following
solutions

(i) Test solution

These is that solution which contain the unknown concentration of the
substance together with the reagent used in the test
 SCIENCE LABORATORY TECHNOLOGY
(ii) Standard solution
These is a solution which is identical to the test solution except that
it contains a known concentration of a substance being determined
and is approximately equal to the concentration to that expected in
the test solution
(iii)Blank solution
These is a solution which is identical to both the test and the standard
solution and its carried through the complete test procedure and it
contain all the reagents used except the test substance its used to
correct for any error that may caused as a result of emergence of any
color that was not part or did not come from the solution under test. .
any color given by the reagent used in the analysis can then be
detected and eliminated
Its also essential to eliminate errors due to dirty glassware’s, turbidity
of the solution or air bubbles as it will interfere with light absorption .
Its especially important not to handle the cuvette by its absorptive
surface which must be clean and dry
In order to be sure that the absorbency is only due to the substance
under test, the reading given by the blank solution must be compared
with the readings obtained from the test and the standard solution
The photoelectric absorbtiometer should be first set to read zero
absorbency with distilled water ,then the blank ,test and the standard
absorbency readings are taken or recorded while rechecking the zero
absorbency between each reading . the blank reading is then
subtracted from the test and the standard readings as follows
Test sol reading- blank sol reading x conc. of the std
Std sol reading – blank sol reading

Beer –Lambert’s law

Beer’s law states that the intensity of color of a solution when viewed
through a monochromatic light is directly proportional to the
concentration of the solution .these law states that the proportion of
the incident light absorbed by the molecules in a solution is directly
proportional to the number of absorbing molecules in the light path .
in these law the characteristic of the light path remains constant
while the concentration of the solution under test is varied .
Lambert’s law states that when light passes through a transparent
medium, the rate of decrease in intensity with the thickness of the
 SCIENCE LABORATORY TECHNOLOGY
medium is proportional to the intensity of light the law can therefore
be stated as ; each successive layer of equal thickness of the same
homogenous solution will absorb the same proportion of incident
light.
Beer –Lambert’s law states that when monochromatic light passes
through a colored solution , the amount of light transmitted decreases
exponentially with the increase in the concentration of the solution
and with the increase in the thickness of the layer of the solution
through which light passes
Mathematically, these can be expressed as follows T = e –ket
Where T is the transmission , k is a constant known as the absorption
coefficient ,c is the concentration of the solution of the light path and
e is the base of the natural logarithm .
The above equation can alternatively be written as
I.e. = e –ke
Ii
Where I.e. is he intensity of the emergent light and Ii is the intensity
of the incident light
Rearranging the equation T = e –ket it becomes
Log e T= -ket
• log eT = ket Log 10 T =ket The expression -log 10 T defines
absorbance (A)
In biological sciences, k is usually a constant since the same
conditions apply for both the test and the standard solution .as the
standard and the test solutions are always compared in identical
cuvettes or tubes, the same light path or thickness isused and
therefore t is always a constant value
Therefore A =C
A test = C test
and A std = C std
A test sol X C std sol = A std sol X C test solution
By using a standard solution , the law can be applied to measure the
concentration of the substance in unknown test solution using the
formulae
Conc.test sol = A. of test sol(A) x C. of std solution x A . of standard
solution.
For Beer- Lambert’s law to hold true, the following requirements must
be met;
(a) Test solution requirements
 SCIENCE LABORATORY TECHNOLOGY
These solution requires to be homogenous, it must also not dissociate
or associate at the time when absorbency is being measured .also the
substance being measured in the solution should not react with the
solvent used. A
blank reagent should be used to correct for any absorption of color by
the solvent . a blank reagent is a reagent which contain all reagent
and chemicals used n chemical used in chemical development but
lacks the substance to be analyzed .

(b) Instrument requirement

The instrument should show satisfactory accuracy and sensitivity at
different wavelengths.the burettes and tubes (curettes) used should be
optically matched or same, free from scratches ,clean and of correct
light path distance e.g. about 10m

CHAPTER NINETEEN

FLAME PHOTOMETRY
Flame photometry is an atomic emission method for the routine
detection of metal salts, principally Na, K, Li, Ca, and Ba. In flame
photometry substances are analyzed basing on the measurements of
individual elements emission intensity produced upon ionization of
their atoms on a flame
 SCIENCE LABORATORY TECHNOLOGY

Flame photometer
Alkali metals and alkaline earth metals solutions when placed on a
Bunsen flame ,impart characteristic colors of flames whose
brightness also varies according to the concentration of these
elements in solutions i.e. the emission intensity measured from flames
correlates to the concentration of the elements dissolved in the
solution
When an element e.g. Na or K or Ca,is in its atomic state and is placed
in a flame , its atoms increases in energy ( i.e. they become excited )
and hence move to the next higher energy level .these acquired energy
makes these atoms to become less stable and they would then attempt
to fall back or come back to their original energy level by losing or
emitting these same amount of energy ,these amount of energy is
emitted in form of light . The light emitted is specific in wavelength
for each element e.g.
Na + emits 589nm
k+ emits 404 and 767nm
The intensity of light emitted is proportional to the concentration of
atoms present in the solution .The emitted wavelength is normally a
measure from which the elements can be determined
Flame photometry is usually useful for analysis of elements , which
are easily,excited aspecially the alkali and alkaline earth metals
in flame photometry, the sample is first diluted in deionised water , its
then sprayed as aerosol ,the aerosol is mixed with a gas fuel e.g.
propane. This mixture is then ignited in the Bunsen chamber . The
heat from the flame release free atoms from the molecular vapor and
 SCIENCE LABORATORY TECHNOLOGY
increase their energy state , they hence emit energy in form of light
which is then quantified in the same way as in the photoelectric
absorptiometers

Components of the flame photometers

Flame photometers consist of the following components
(i) An atomizer
These usually a special container which holds the solution and sprays it
into the flame in
form of aerosol
(ii) The burner
These could be any type of burner e.g. Bunsen or meeker burner whose
flame temperature can be controlled by a gas and air pressure control
valves . the gas commonly used are propane, acetylene-oxygen or
hydrogen oxygen
(iii) Wavelength selector
These usually a simple optical filter in simple instruments , infact
filters are quite satisfactory if the measurements are restricted to
analysis of K+ and Na+ and Ca .sometimes grating monochrometers
could be used , in these case the measurements can be extended to
include analysis of other metals such as barium , copper , manganese
and strontium
(iv) Detectors
Detectors measures light intensities falling upon a photocell
connected to a galvanometer whose deflections are related to the
concentration . in more sophisticated instruments , photomultiplier
detectors are used which are more sensitive than photocell detectors
hence more reliable .
the detection limit of an element by flame photometry depends on the
ease of excitation of metals in the flame . The most favorable element
is Na . the ease of analysis by flame photometry is in the order Na
,Li ,K , Ca ,St and
Ba. Sodium , potassium and calcium analysis are of great importance
in chemical laboratories in connection with electrolyte balance
especially in serum . but care is needed in processing of blood into
serum prior to flame photometry since the potassium ions can rapidly
be exchanged between red blood cells and serum being formed .
One of the factors which limits the sensitivity of flame photometry is
the proportion of the elements in the sample which becomes excited
 SCIENCE LABORATORY TECHNOLOGY
by the flame to emit light ..This proportion rarely exceeds 1%
therefore approximately 99% of the sample in the flame do not emit
light . but the amount of light absorbed is related to the concentration
of the particular element in the sample
Flame photometry depends on the fact that the wavelength of emitted
radiation is different for each element because each metal atom have
different nuclear charge and electrons . A means of detection of the
portion of this wavelength can therefore be used to identify which
element is present in the sample and if the intensity of this radiation
are measured ,then qualitative analysis is possible
flame photometry is designed to provide flames whose temperatures is
hot enough to;
(i) Excite as many elements as possible
(ii) Determine which wavelengths are given off
(iii) Measure the intensity of these wavelengths.
Ionization of metal atoms can however cause a decrease in intensities
of radiation , these occurs at low concentration because at low
concentration few atoms are present so they absorb all the energy , if
the concentration is increased ,the high energy is spread over atoms
and therefore few ions are produced.
To avoid this problem , add a sample of an easily ionizable alkali
metal, which shall take up excess energy and therefore less ionization .
The emission due to the flame can be measured without the sample
and the photometer reading at zero
Self absorption
Self absorption occurs when the radiation emitted from one atom is
just the right energy to be absorbed by another atom . the effect is
that several atoms may be involved but the detector may see only one
or few atoms in the final process. These results in a lower reading that
that actually present
Instrument for flame photometry
There are two types of instruments for measuring emission. I.e. the
photometer and flame spectrophotometer
(a) photometer
although its design varies from one manufacturer to another, all
instruments however have similar layout. The design comprises of ;
(i) the nebulizer or atomizer
(ii) cloud chamber with condensation vanes
(iii) a burner suitable for the fuel to be employed
 SCIENCE LABORATORY TECHNOLOGY
(iv) a wavelength selector system which consist of
monochrometers in form of filters e.g. (orange for sodium and
deep red for potassium)
(v) photocell detector which is fed to a galvanometer

Application of flame photometry

Flame photometry can be applied in the following various fields
In analysis that are difficult or impossible to perform using other
techniques or where speed is important than accuracy
For analysis of alkali metals
In biomedical research
In clinical chemistry
In food chemistry
In water and sewage treatment
SCIENCE LABORATORY TECHNOLOGY
 SCIENCE LABORATORY TECHNOLOGY
CHAPTER TWENTY

CALORIMETRIC ANALYSIS
Calorimetry is the science associated with determining the changes in
energy of a system by measuring the heat exchanged with the
surroundings. A calorimeter is a device used to measure the quantity of
heat transferred to or from an object. The calorimeter used in high
school science labs is more commonly referred to as a Styrofoam cup.
It is a coffee cup calorimeter - usually filled with water. The more
sophisticated cases include a lid on the cup with an inserted
thermometer and maybe even a stirrer.
 
Coffee Cup Calorimetry

Coffe –cup colorimeter

water will change its temperature when it gains or loses energy. And in
fact, the quantity of energy gained or lost is given by the equation
Q = mwater•Cwater•∆Twater
where Cwater is 4.18 J/g/°C. So if the mass of water and the
temperature change of the water in the coffee cup calorimeter can be
measured, the quantity of energy gained or lost by the water can be
calculated.
The assumption behind the science of calorimetry is that the energy
gained or lost by the water is equal to the energy lost or gained by the
 SCIENCE LABORATORY TECHNOLOGY
object under study. So if an attempt is being made to determine the
specific heat of fusion of ice using a coffee cup calorimeter, then the
assumption is that the energy gained by the ice when melting is equal
to the energy lost by the surrounding water. It is assumed that there is
a heat exchange between the ice and the water in the cup and that no
other objects are involved in the heat exchanged. This statement could
be placed in equation form as
Qice = - Qsurroundings = -Qcalorimeter
The value of the Styrofoam in a coffee cup calorimeter is that it reduces
the amount of heat exchange between the water in the coffee cup and
the surrounding air. The value of a lid on the coffee cup is that it also
reduces the amount of heat exchange between the water and the
surrounding air. The more that these other heat exchanges are
reduced, the more true that the above mathematical equation will be.
Any error analysis of a calorimetry experiment must take into
consideration the flow of heat from system to calorimeter to other parts
of the surroundings. And any design of a calorimeter experiment must
give attention to reducing the exchanges of heat between the
calorimeter contents and the surroundings.
 
Bomb Calorimetry
The coffee cup calorimeters used in high school science labs provides
students with a worthwhile exercise in calorimetry. But at the
professional level, a cheap Styrofoam cup and a thermometer isn't
going to assist a commercial food manufacturer in determining the
Calorie content of their products.
 SCIENCE LABORATORY TECHNOLOGY

Bomb calorimeter
For situations in which exactness and accuracy is at stake, a more
expensive calorimeter is needed. Chemists often use a device known as
a bomb calorimeter to measure the heat exchanges associated with
chemical reactions, especially combustion reactions. A bomb
calorimeter includes a reaction chamber where the reaction (usually a
combustion reaction) takes place. The reaction chamber is a strong
vessel that can withstand the intense pressure of heated gases without
exploding. The chamber is typically filled with mostly oxygen gas and
the fuel. An electrical circuit is wired into the chamber in order to
electrically ignite the contents in order to perform a study of the heat
released upon combustion. The reaction chamber is surrounded by a
jacket of water with a thermometer inserted. The heat released fromthe
chamber warms the water-filled jacket, allowing a scientist to
determine the quantity of energy released by the reaction.
 SCIENCE LABORATORY TECHNOLOGY

Solving Calorimetry Problems

Now let's look at a few examples of how a coffee cup calorimeter can be
used as a tool to answer some typical lab questions. The next three
examples are all based on laboratory experiments involving
calorimetry.

Example Problem 1:
A physics class has been assigned the task of determining an
experimental value for the heat of fusion of ice. Anna and Noah
weighed 25.8-gram of ice and place it into a coffee cup with 100.0 g of
water at 35.4°C. They place a lid on the coffee cup and insert a
thermometer. After several minutes, the ice has completely melted and
the water temperature has lowered to 18.1°C. What is their
experimental value for the specific heat of fusion of ice?
The basis for the solution to this problem is the recognition that the
quantity of energy lost by the water when cooling is equal to the
quantity of energy required to melt the ice. In equation form, this could
be stated as
Qice = -Qcalorimeter
(The negative sign indicates that the ice is gaining energy and the water
in the calorimeter is losing energy.) Here the calorimeter (as in the
Qcalorimeter term) is considered to be the water in the coffee cup.
Since the mass of this water and its temperature change are known, the
value of Qcalorimeter can be determined.

Qcalorimeter = m•C•∆T
Qcalorimeter = (100.0 g)•(4.18 J/g/°C)•(18.1°C - 35.4°C)
Qcalorimeter = -7231.4 J
The negative sign indicates that the water lost energy. The assumption
is that this energy lost by the water is equal to the quantity of energy
gained by the ice. So Qice = +7231.4 J. (The positive sign indicates an
energy gain.) This value can be used with the equation from the
previous page to determine the heat of fusion of the ice.
Qice = mice•ÄHfusion-ice
+7231.4 J = (25.8 g)•ÄHfusion-ice
∆Hfusion-ice = (+7231.4 J)/(25.8 g)
∆Hfusion-ice = 280.28 J/g
∆Hfusion-ice = 2.80x102 J/g (rounded to two significant figures)
 SCIENCE LABORATORY TECHNOLOGY
 
Example Problem 2:
A chemistry student dissolves 4.51 grams of sodium hydroxide in 100.0
mL of water at 19.5°C (in a calorimeter cup). As the sodium hydroxide
dissolves, the temperature of the surrounding water increases to
31.7°C. Determine the heat of solution of the sodium hydroxide in J/g.
Once more, the solution to this problem is based on the recognition
that the quantity of energy released when sodium hydroxide dissolves
is equal to the quantity of energy absorbed by the water in the
calorimeter. In equation form, this could be stated as
QNaOH dissolving = -Qcalorimeter
(The negative sign indicates that the NaOH is losing energy and the
water in the calorimeter is gaining energy.) Since the mass and
temperature change of the water have been measured, the energy
gained by the water (calorimeter) can be determined.
Qcalorimeter = m•C•∆T
Qcalorimeter = (100.0 g)•(4.18 J/g/°C)•(31.7°C - 19.5°C)
Qcalorimeter = 5099.6 J
The assumption is that this energy gained by the water is equal to the
quantity of energy released by the sodium hydroxide when dissolving.
So QNaOH-dissolving = -5099.6 J. (The negative sign indicates an
energy lost.) This quantity is the amount of heat released when
dissolving 4.51 grams of the sodium hydroxide. When the heat of
solution is determined on a per gram basis, this 5099.6 J of energy
must be divided by the mass of sodium hydroxide that is being
dissolved.
∆Hsolution = QNaOH-dissolving / mNaOH
∆Hsolution = (-5099.6 J) / (4.51 g)
∆Hsolution = -1130.7 J/g
∆Hsolution = -1.13x103 J/g (rounded to three significant figures)
 
Example Problem 3:
A large paraffin candle has a mass of 96.83 gram. A metal cup with
100.0 mL of water at 16.2°C absorbs the heat from the burning candle
and increases its temperature to 35.7°C. Once the burning is ceased,
the temperature of the water was 35.7°C and the paraffin had a mass of
96.14 gram. Determine the heat of combustion of paraffin in kJ/gram.
GIVEN: density of water = 1.0 g/mL.
 SCIENCE LABORATORY TECHNOLOGY
As is always the case, calorimetry is based on the assumption that all
the heat lost by the system is gained by the surroundings. It is assumed
that the surroundings is the water that undergoes the temperature
change. In equation form, it could be stated that
Qparaffin = -Qwater
Since the mass and temperature change of the water are known, the
energy gained by the water in the calorimeter can be determined.
Qcalorimeter = m•C•∆T
Qcalorimeter = (100.0 g)•(4.18 J/g/°C)•(35.7°C - 16.2°C)
Qcalorimeter = 8151 J
The paraffin released 8151 J or 8.151 kJ of energy when burned. This is
based on the burning of 0.69 gram (96.83 g - 96.14 g). To determine
the heat of combustion on a per gram basis, the Qparaffin value (-8.151
kJ) must be divided by the mass of paraffin burned:
∆Hcombustion - paraffin = (-8.151 kJ) / (0.69 g)
∆Hcombustion - paraffin = -11.813 kJ/g
∆Hcombustion - paraffin = -12 kJ/g (rounded to two significant digits)

CHAPTER TWENTY ONE

PROXIMATE ANALYSIS
Proximate analysis is an analytical technique that involves the
determination of composition of food by approximating their quantity .
the technique establishes presence of compounds contain components
such as ash , moisture ,carbohydrates , proteins , lipids ,vitamins and
other food additives
ASH
Introduction
Ash is the inorganic residue occurring from the incineration (burning)
of organic matter. The amount and composition of ash in food depend
on the nature of the food ignited.
 SCIENCE LABORATORY TECHNOLOGY
Ash is mostly composed of the minerals . the various minerals that are
in ash occur in different proportion in different food i.e.
Calcium –rich in dairy , poultry and cereals etc
Phosphorus-rich in dairy,grains, poultry and legumes
Iron –rich in grains ,dairy, legumes etc
The form in which the minerals constituents occur in ash differs
considerably from the form in which they occur in the original food.
This is because in ash , they minerals are normally converted upon
heating during ashing process.
ANALYSIS OF ASH AND MINERALS
The “ash content” is a measure of the total amount of minerals
present within a food, whereas the “mineral content” is a measure of
the amount of specific inorganic components present within a food,
such as Ca, Na, K and Cl. Determination of the ash and mineral content
of foods is important for a number of reasons:

 Nutritional labeling. The concentration and type of minerals

present must often be stipulated on the label of a food.
 Quality. The quality of many foods depends on the
concentration and type of minerals they contain, including their taste,
appearance, texture and stability.
 Microbiological stability. High mineral contents are
sometimes used to retard the growth of certain microorganisms.
 Nutrition. Some minerals are essential to a healthy diet (e.g.,
calcium, phosphorous, potassium and sodium) whereas others can be
toxic (e.g., lead, mercury, cadmium and aluminum).
 Processing. It is often important to know the mineral
content of foods during processing because this affects the
physicochemical properties of foods.

Total ash.
These is usually an index used to indicate the total composition of ash
mineral elements present in ash .Water Soluble Ash
This is the index used to refer to the proportion of the mineral
elements present in ash that can dissolve in water i.e its usually the
 SCIENCE LABORATORY TECHNOLOGY
amount of all those minerals that can be isolated from ash by simply
dissolving in water.
Acid Insoluble Ash
These is usually an index used to refer to the proportion of the mineral
elements that remain in ash after decanting using an acid e.g. HCL
,H2SO4 etc .Those minerals that don’t react with acids hence remain
intact in the ash are acid insoluble.
Salt Free Ash
These is determined as the difference between total ash and sodium
chloride in ash i.e what remains in ash after Nacl have been removed
from it

Sample Preparation
As with all food analysis procedures it is crucial to carefully select a
sample whose composition represents that of the food being analyzed
and to ensure that its composition does not change significantly prior
to analysis. Typically, samples of 1-10g are used in the analysis of ash
content. Solid foods are finely ground and then carefully mixed to
facilitate the choice of a representative sample. Before carrying out an
ash analysis, samples that are high in moisture are often dried to
prevent spattering during ashing. High fat samples are usually defatted
by solvent extraction, as this facilitates the release of the moisture and
prevents spattering. Other possible problems include contamination of
samples by minerals in grinders, glassware or crucibles which come
into contact with the sample during the analysis. For the same reason,
it is recommended to use deionized water when preparing samples.
Basically , there are two types of samples i.e wet and dry samples. Wet
samples should be first dried before ashing. For dry samples ,they
should be grounded first . The grinding materials or machines should
be clean so as to avoid introduction of contaminants to the sample .

ASHING METHODS
There are two methods of ashing
(a) Dry ashing
(b) Wet ashing
 SCIENCE LABORATORY TECHNOLOGY

a) Dry Ashing
The sample is first weighed , then burnt off without flaming for a fixed
period of time to a constant weight. Before weighing , the dish
containing the residue should be cooled in a desicator . Crucibles are
used during ashing, the type of the crucibles to be used depends on the
nature of food analyzed and on the type of analysis to be performed on
the dish . Quartz, porcelain , nickel , platinum are the most common
materials from which crucibles are made from. Crucibles should be
resistant to corrosion by halogens ,acids ,alkalis temperatures etc .they
should also retain there smooth surfaces for easy cleaning . crucibles
should be held by crucible tongs and heated on clay triangular tripods .
Ashing is normally done in ovens or furnaces but sometimes in open
Bunsen flames .Furnaces and ovens should have rheostats for
temperature control . Ashing should give a carbon free ash .Dry ashing
is the most satisfactory method . ashing methods should be relatively
fast and should prevent overall loss of minerals and should improve the
retention of the critical components . ashing can be accelerated by
addition of glycerin or alcohol .

Wet Ashing

It’s also called digestion or wet oxidation . This method is used

primarily for digestion of samples especially intended for determining
trace elements and metallic poisons . it involves use of acids e.g.
H2SO4 or HNO3 .Normally the method is slow and can be accelerated
by adding K2SO4 which raises the boiling point of the acid hence
accelerating decomposition . NB HNO3 is a good oxidant but usually
boils away before the sample is completely oxidized . Normally mixed
acids are usually used for the decomposition of organic materials. Wet
ashing is however prone to excessive foaming especially during
digestion of fats and sugar rich materials , these can be minimized by
addition of hydrogen peroxide

Comparison between Wet and Dry Ashing

Dry ashing is used in determining total ash ,water-soluble ash ,water
insoluble ash and acid insoluble and most common metals . It requires
no attention , it is simple and suitable for routine large number of
samples . no reagents is required but it takes long time and the sample
 SCIENCE LABORATORY TECHNOLOGY
normally get contaminated in the drying chamber also excess heating
makes certain mineral components insoluble .
Wet ashing requires relatively low temperature and liquid conditions
are maintained . simple apparatus are used, oxidation is rapid, but it
requires large volume of acid reagent ,its therefore suitable for small
samples, its time consuming and it requires a lot of attention.

MOISTURE
Moisture is the amount of water contained in the food
sample.determination of moisture content is most important in the
food processing industry. Moisture content in food is of direct
economic important to the food processor and consumer.
Moisture content often affect the stability and quality of food .foods
that contain more moisture are prone to rapid deterioration due to
bacterial and fungal growth and sprouting. Industrially ,moisture
content is very important and knowledge about it is very important in
evaluation of materials for optimum processing and preservation of
food.
The moisture content of food varies widely i.e –
dairy products= 87-91%
dairy milk powder= 4%
pure oil&fat = 0%
fruits= 90%
cereals = 0 –12%
No method of moisture determination is 100 % accurate and
practical.but methods that give the highest moisture content values
while minimizing the decomposition and volotization of other
compounds other than water are the most preferred. such methods
selected have high reproducibility and practicability.
water occur in foods in three i.e. –
• free water in intergranular spaces and within pores of the
material
• water absorbed on the surface of the macromolecular
colloids i.e 0n the surface of starch ,pectin ,cellulose and proteins
water in bounded form e.g. water of hydration
methods for determination of water can be divided into ;
a) drying method
b) distillation method
c) chemical assay
 SCIENCE LABORATORY TECHNOLOGY
d) physical method

DRYING METHOD
These generally involves thermal drying i.e heating under specific
condition ,whereby the water lost is taken to mean the measure of the
moisture content of the sample.
This method depends on the type of the oven used ,temperature and
the length of drying.
Drying methods are simple ,relatively rapid and is reproducible.but for
such methods to be ideal ,the weight loss should be only due to
volotization of only water and not any other organic constituent e.g.
oils and esters.
The accuracy of moisture determination is affected by ;
i. Drying temperature
ii. Relative humidity of drying chamber
iii. Depth
iv. Particle size of the sample
v. Material for oven construction
vi. Quantity and position of the samples
vii. Oven
Determination of moisture content is however also prone to several
errors, which include
(A)moisture changes during subsequent storage of sample
B)gain or loss of moisture during processing
To minimize such errors ,two stages moisture determination is
advisable since it gives higher results than one stage .also samples
should be stored in air tight containers or desicators and should be
weighed quickly after they have obtained room temperature .liquids
should be dried by spreading over a large surface ,should be evaporated
first on a water bath and completed in an oven.the other common
sources of errors is the formation of crust around the sample which Is
impervious to evaporation of moisture from the center of the dried
sample . These effect can be reduced by moistening the sample with
water and thorough mixing with sand or asbestos to increase the
exposed surface or to dry them under infra- red lamp ( Sometimes the
sample can be dried first under low temperature to low temperature)
there many types of drying ovens i.e
 SCIENCE LABORATORY TECHNOLOGY
(a) air oven method
(b )vacuum oven method
other drying methods are available i.e-
infra –red drying method -its most effective and it involves
penetration of heat into the sample being dried .its very fast.
Desiccation method – it involves use of drying dessicators containing
dehydrating or water absorbing substances e.g. H2SO4 ,Calcium
carbide . however such methods are very slow and some foods can
never dry completely.

Distilation Methods
There are two types of distillation methods i.e
(a) method where water is distilled from an immisible liquid of
high boiling point i.e the sample is suspended in a mineral oil that
have a flash point above the boiling point of water and it is heated to a
predetermined temperature in a suitable apparatus where the water
will distill off through the condenser and is collected in a suitable
measuring cylinder
(b) method where the mixture of water and the immisible
solvent e.g. xylene or toluene are distilled off and is collected in a
suitable measuring apparatus in which water separates and its volume
is measured
distillation method couses less decomposition in some foods than the
oven drying method. Many difficulties however may be encountered in
the determination of moisture by distillation method which include ;
(a) relatively low precision of the receiving measuring device
(B) difficulties in reading the meniscus
(c) adherence of the moisture to the glass wall
(d) solubility of water in the distillation solvent
(e) incomplete evaporation of water
(f) under estimation of the moisture content
(g) distillation of other water soluble components

Chemical Methods of Moisture Analysis

(a) Karl Fischer method (titration )
This method is applicable to samples that give erratic results when
heated or submitted to a vacuum especially in low moisture foods
such as dried fruit and vegetables ,candies chocolates ,oils and fats .
it’s the most appropriate method for determining the moisture in
 SCIENCE LABORATORY TECHNOLOGY
sugar –rich food e.g. sugars and honey and also with foods with high
levels of volatile oils.
The method is based on the principle of reduction of iodine by sulfur
dioxide(SO2) in presence of water
2H2O +SO2 +Iodine______H2SO4 +2H
The SO2 and iodine is first dissolved in methanol and pyridine .the
reaction takes place in two steps i.e.
C2H5 N.I +C5H5NSO2 +C5H5N + H2O 2C5H5NHI +C5H5N.SO3
And
C5H5.SO3 +CH3OH C5H5SO4CH3
These is a titration reaction where iodine and SO2 are added in
appropriate form to the water containing food..the excess of iodine
that cannot react with water is set free .
The amount required for the titration can be determined visually apon
appearance of a mahogany brown color. These can be expressed more
clearly by addition of methyl blue as an indicator, which gives a green
endpoint .
Karl Fischer reagent may be used for the determination of water in
liquids ,gases or solids .but however the titration should be carried out
in a moisture free environment so as to minimize changes in water
content .

( b) use of other dehydrating agents

apart from Karl Fischer reagents ,there are other dehydrating agents
used for determination of moisture content. They include
(i) calcium carbide
calcium carbide reacts with moisture in food to give or produce
acetylene. The quantity of acetylene can be measured by loss in
weight ,pressure or volume of gas produced in a closed system
(2)sulfuric acid
sulfuric acid is a dehydrating agent which when mixed with food it
results in heating and the increase in temperature under fixed
conditions is roughly proportional to water content in that food .

CARBOHYDRATES
Carbohydrates are the single most abundant compounds in nature .
They are manufactured by plants through a process known as
photosynthesis .The simplest carbohydrate is glucose. Glucose
molecules can be combined to form much larger molecules such as
 SCIENCE LABORATORY TECHNOLOGY
cellulose which constitutes the supporting framework of plants
,starch ,which is stored in the seed and serve as food for the new
growing plant or glycogen which is stored in animals some of the extra
glucose is converted into fats ,amino acids etc.
Carbohydrates have important nutritional and metabolic functions,
they are also the natural sweeteners, raw materials for fermentation
industry and main ingredient in cereals. Carbohydrates composition is
normally given as total carbohydrates by difference i.e 100% -(%water
+ % proteins +% fats +% ash ) = total carbohydrates .its also called
nitrogen free extract.
Carbohydrates are polyhydroxy aldehydes and polyhydroxy ketones or
compounds that can be hydrolyzed to them .a carbohydrate that cannot
be hydrolysed to simpler compounds is called monosaccharide .a
carbohydrate that can be hydrolyzed to two monosaccharide molecule
is called a disaccharide while that that can be hydrolyzed to many
monosacharides is called a polysaccharide .
Carbohydrates generally conform to te imperical formula (CH2O)n
Monosaccharides are polyhydroxyl aldehydes or ketones ,i.e. they
contain a carbonyl group [C=O].if the carbonyl is at the end of the
carbon chain ,then it is an aldehyde and therefore its is designated as
an aldose , but if it is at the interior position then it is a ketone and its
therefore refered to as ketose sugar .aldoses are more reactive than
ketoses and are thus sometimes referred to as reducing sugars .
Monosacharides are quite soluble in water .
Monosacharides are named by specifying the position of the carbonyl,
then the number of carbons and finally ending –ose . e.g. an
aldohexose is a six carbon sugar with carbonyl at the end of the
carbon chain while 2-ketopentose has its carbonyl at the second of
the five carbons
Aldoses of greater biological importance are trioses pentoses and
hexoses i.e. trioses have three carbons , pentoses have five carbons and
hexoses have six carbons
Monosaccharides usually exist as sterioisomers – two structures that
are the mirror images f each other . The two isomers are designated as
D and L
Most of the monosaccharides are optically active meaning that their
assymetric carbon cause the rotation of the plane of polarized light .
molecules that can rotate the plane of polarization to the right are
 SCIENCE LABORATORY TECHNOLOGY
called dextrorotatory and are designated as + or D while those that
rotate the plane of polarization to the left are designated as – or L
Most often , monosaccharides do occur in ring form . The ring
formation introduces a new asymmetric carbon at position one . If
the hydroxyl at position one is below the plane of the molecule , it is
the á anomer and if it is above the plane of the molecule , it’s the â
anomer . The ring form of the sacharides are referred to as furanose or
pyranose by analogy of pyran or furan which are five or six membered
ring respectively
The most abundant monossacharides is the D-glucose, which is found
in blood ,plant sap and in many fruits ,other monossacharides include
fructose and galactose. Sucrose is a disaccharide and is the most
abundant source of glucose and fructose other disaccharide’s include
lactose ,maltose and cellobiose
Oligosaccharides are two or more monosacharides joined by
condensation reaction i.e. removal of hydroxyl from one saccharine
and hydrogen from another to form water . When two
monosaccharides are joined together , they give a disaccharide , three
join to give a trisaccharide and so on
The most familiar disaccharide is an ordinary table sugar i.e. sucrose
.it consist of á-D glucose and â –D fructose . Another example is
lactose or milk sugar
Polysaccharides are polymers of glucose . They include cellulose ,
glycogen and starches . cellulose is a linear polymer of glucose ,
glycogen is a branched polymer of glucose consisting of several
thousand monomers forming a much more compact structure than
cellulose . Its found mostly in muscles and livers of animals and is
sometimes called animal starch .
The true starches however comes from plants and comes in two forms
i.e. amylose and amylopectin . Amylose is unbranched á-1,4 polymer of
glucose . Amylopectin have almost the same structure as glycogen
though it is not so highly branched as glycogen

Extraction of Carbohydrates
Before carbohydrates are analyzed , its important to firsts remove all
interfering substances e.g. lipids and chlorophyll .these are removed
by solvent extraction with petroleum ether . Clarifying agents are often
used to remove most of the interfering substances .these agents are
 SCIENCE LABORATORY TECHNOLOGY
often used to remove turbidity caused due presence of proteins and
soluble starch .
clarifying agents often work on the principle that heavy metals that
they contain will precipitate colloidal substances in the extract hence
making them to either float or sink .
clarifying agents should only remove all interfering substances
completely without absorbing or modifying the sugars
Most clarifying agents also have a decolorizing action i .e they adsorb
color of the precipiteds .clarifying agents contain various heavy
metallic salt elements e.g. lead , aluminum , barium or zinc salts .
NB ;But care must be taken not to cause changes in the composition
and properties of carbohydrates to be analyzed e.g. inversion of sugars
by chemicals should be prevented by addition CaCO3 and action of
enzymes is prevented by addition of mercury chloride .

Qualitative Determination Of Carbohydrates

Qualitative tests for carbohydrates are based on ;
(a) color reactions affected by the condensation reaction of
degraded products of sugars in mineral acids with various organic
compounds
(b) the reducing properties of the carbonyl group
(c) oxidative cleavage of the neighboring hydroxyl group
(a) methods based on color reaction in strong acids
The action of mineral acids e.g. H2SO4 ,HCL and H3PO4 on
carbohydrates leads to the formation of colored decomposition
products e.g. hydroxymethyl furfural which in acid further
decomposes to levulinic acid . Such coloration often becomes distinct
especially after the addition of some other organic compounds e.g.
phenol ,aromatic amines thio compounds ,urea etc
Methods commonly adopted in these type of analysis include ; molish
reactions
salivanof test
bial procedure’s
tollens test
tauber test
some of these test or reagents are selective for certain sugars while
others show wide application and still some give various color reaction
depending on the sugar present. phenol-sulfuric acid method for
example is simple ,rapid , sensitive, accurate , specific and widely
 SCIENCE LABORATORY TECHNOLOGY
applicable for carbohydrates and almost all classes of sugars can be
determined .these reagent is not expensive ,readily available and
stable, a stable color is produced and the results are reproducible .
(b) methods based on the reducing properties of carbonyl
group
These reactions involve use of cupric (CU2+) or ferrous (Fe2+) salts
which are reduced to cupric oxides and ferricyanides etc . This reaction
normally gives different colors and their intensities can be amplified
upon addition of acetone.
In alkali solutions, reducing sugars which contain aldehydes and keto –
groups can reduce copper, silver, bismuth and mercury salts to
compounds of lower valency or metallic states.fehling solution is the
best known example and is prepared by mixingtwo solutions i.e cupric
sulphate and sodium potassium tartarate and sodium hydroxide .These
solution normally gives a yellowish orange precipitated upon heating
depending on the concentration of sugars in the solution .
Tollens reagent is based on the oxidative effect of the complex ions
(Ag(NH3)2)+ and it is most sensitive of all reagents. It utilizes the
reduction of silver metal by sugar
These method is however non specific and can only be used after the
removal of other reducing organic compounds .but nevertheless ,the
reactions are very sensitive and suitable for routine assays .

DETERMINATION OF MONO ,DI AND OLIGOSSACHARIDES

The available assay methods include ;
(A)chemical methods
(B)colorimetric method
(A) chromatographic method
(B) electrophoretic method
(C) optical method
(D) biochemical method

(A) Chemical Methods

(i) cupric method
These is the most utilized method for the reducing sugars .it involves
the oxidation of copper ions to copper oxides. When a reducing sugar is
treated with an alkali at elevated temperature,the sugar is degraded
and some of the degradation products reduces cupric ions to copper
oxide precipitated
 SCIENCE LABORATORY TECHNOLOGY
In most of these methods ,excess of alkaline tartarate-cupric sulphate
is added to a sugar solution and boiled under specific conditions and
the amount of the precipitated formed is determined by weighing
gravimetricaly, by titration or by measuring the unreacted cupric ions
in the complex.
(ii) alkaline ferricyanide method
This method is based on the reduction of alkaline ferricyanide to
ferricyanide in the presence of a reducing sugar. Normally the amount
of the reduction is taken as the measure of the amount of sugar the
sample contains and its normally determined as the difference between
the ferricyanide added and that remaining after reduction .these
changes normally occur in the presence of potassium iodide
2K3Fe(CN)6 +2KI ___ 2K4Fe(CN)6 + I
The liberated iodine is titrated with thiosulphat
(iii) iodometric method
Iodine in an alkali media is converted rapidly to hypoiodide, which can
oxidize aldose, but ketoses are oxidized the least. Therefore these
method is applicable to aldoses alone.
The dissolved sample is treated with iodine and sodium hydroxide is
added and mixed rapidly ‘the solution is then acidified with HCL or
H2SO4 and left to stand for a few minutes. The excess of the
standardized iodine is titrated with a standardized thiosulphate
RCHO +I +3NaOH ---- RCOONa + NaI +2H2O

(B) Chromatographic and Electrophoretic Method

Chromatographic methods are always used to fractionate
,isolate,identify and determine carbohydrates in complex mixtures
.chromatographic methods includes ;
Paper chromatography
Thin layer chromatography (T.I.C)
Gas –liquid chromatography
Column chromatography
Ion –exchange chromatography
electrophoresis
Paper and thin layer chromatography is the simplest method
commonly used to distinguish btw various forms of sugars present in
food .but does not serve well in mixed or complex sugars .in such cases
the paper chromatography may be sprayed with various reagents e.g.
aniline pthylate which gives a color reaction with reducing sugars only.
 SCIENCE LABORATORY TECHNOLOGY
(C) Optical Methods
These includes ;
(i) Refactometry
these involves use of instruments e.g. refractometers. the method is
commonly used in sugar industries to measure the content of the
dissolved solids in sugar solutions.
(ii) Polarimetry
carbohydrates are opticaly active and therefore they can be assayed
polarimetricallly. Polarimetry involves measuring the angle of rotation
using a monochromatic light on a scale .polarimetric assays are non
destructive and rapid they are also accurate provided ;
• The solution is clear and colorless
• The concentration of the test solution is within the limit of
the instrument
• The solution contain no interfering optically active
compounds .

(D) Biochemical Methods

This method can be classified into ;
(i) microbiological method
(ii) enzymatic method
(i) Microbiological method
These involve use of various strains of microorganisms e.g. yeast for
various purposes that are useful in differentiating between various
aldoses and ketoses.

(ii) Enzymatic methods

These involves use of prepared enzymes to offer selective cleavage of
various sugars
NB; microorganisms and enzymes can be used in the pretreatment of
substrate prior to assay by chemical or physical methods

DETERMINATION OF POLYSSACHARIDES
(a) Starch
Starch is the second most abundant Substance in the vegetable matter
after cellulose. it occurs in form of granules as food reserves in various
parts of plants e.g. seeds. infact the shape and sizes of starch grains
are characteristics for each plants
 SCIENCE LABORATORY TECHNOLOGY
Starch is a polymer of D-glucose units ,there are two forms of starch i.e
amylose and amylopectin .starch can be fractionated into amylose and
amylopectin by geletinization in water at an elevated temperature and
pressure. Amylose gives intense blue color with iodine ,but
amylopectin gives a red or violet color with iodine .
Several methods are available for determining starch content in food .it
is first normally extracted and dispersed into colloidal solution in
which extraneous matter can be separated
Starch can be extracted in fat and proteins rich food by reacting with
an alkali to form an alcohol –insoluble complex that can be readily be
separated from other complex .but however theses method is not
suitable for extraction of starch in plant materials that contain a
mixture of other polysaccharides.
Starch can be solubilized in boiling water but however its complete
extraction from plants tissues is hard to achieve due to its high
molecular weight and colloidal properties .
Perchloric acid is an efficient extractant but it first require to remove
soluble sugars using 80 % ethanol .the sugar free residue is then
treated with perchloric acid solution and the extracted starch is
precipitated with iodine .the starch iodine complex formed is
decomposed with an alkali.
Starch can also be extracted with hot concentrated solution of
calcium chloride .starch must be separated from other Interfering
substances .these can be achieved by precipitating starch dissolved in
calcium chloride with iodine to form starch iodine complex then it is
determined gravimetricaly ,titremetricaly, colorimetricaly or
polarimetricaly
Acid hydrolysis of starch yield glucose which can be determined by the
usual chemical or physiochemical method .
Dextrins
these are mixtures of carbohydrates produced by partial enzymatic
hydrolysis of starch .they include glucose polymers above hexose and
below those colored blue with iodine.they dissolve in water to give
milky suspension upon the addition of ethanal,but starch and proteins
are copreciptated along side these precipitation.

These can simply be removed by precipitating them with lead acetate.

Dextrin can be separated through T.L.C method
 SCIENCE LABORATORY TECHNOLOGY
Dextrin can be determined chemicaly,but it first requires determining
glucose by barfoed method then assaying the reducing sugars by
fehling procedure and finally dextrins and oligosacharides are
hydrolyzed by acid then determined as glucose by fehling method
Glycogen
Glycogen is a polysaccharide of animal origin found in muscles and
liver .it resembles plant amylopectin except that it is highly
branched,it is water soluble and it colors brown-red after addition of
iodine .

STRUCTURAL POLYSSACHARIDES
They consist of cellulose, hemicelulose and pectins
(i) Cellulose and hemicellulose
they are partly soluble in alkali. They are most abundant carbohydrates
in plants.There content increases with maturity they are generally
determined in defated food following extraction of soluble
carbohydrates.defating is done using ether and alcohol .
(ii) pectin
pectin is a product found in plant cell and it cement cellwalls together.

Crude Fiber
crude fiber is the material which remains after food have been
subjected to rigorous treatment with acids and alkalis .they include all
indigestible materials in humans and animals .
it is determined from an already defated food and boiled under reflux
for exactly 30 minutes with 200mls of a solution containing 1.25 ml
H2SO4 per 100ml of solution. The solution is filtered through linen
and washing with boiling water until the washings are nolonger acidic
the residue is transferred to a beaker and again boiled for 30min with
200ml of a solution containing 1.25ml NaOH per 100mls solution .the
final residue is filtered through a thin cloth pad of washed and ignited
asbestos in a gooch crucibles ,dried in oven ,weighed ,
incenerated,cooled and weighed again .the lose in weight in
incineration is taken as crude fiber .

Dietary Fiber
These is that part of carbohydrates that is available for digestion by
secretions of human digestive systems .dietary fibers can be classified
into three major groups according to there structure and properties
 SCIENCE LABORATORY TECHNOLOGY
(a)cellulose –these is the chief constituent of the frame work of plant,
its not digested by humans due to lack of enzymes .it however
facilitate the movement of bowel down the alimentary canal and also
peristalsis
(b)non- cellulose - these includes hemicelullose . pectins ,gums
.mucilage and alga substance .they absorb water and therefore slow
down the gastric emptying time . they help in binding cholesterol and
controlling its absorption
(c) lignin – these is the only the non-carbohydrates dietary fiber. It
forms the woody parts of plants . in the intestines ,it combines with
bile acid to form insoluble compound thus prevents its absorption .

LIPIDS
Lipids is a general name used to denote fats and oils. They are
compounds that are insoluble in water but soluble in organic solvents
like ether ,petroleum ether,acetone , benzene ,chloroform ,alcohol e.t.c
.they are oily or greasy in nature. Lipids serve as structural
components and source of energy in living organisms
Foods vary widely in their lipids content and composition e.g.;-
vegetables and animal cooking fats contain almost 100% lipids while
most nuts contain between 47-70% lipids,cerialls contain 40% ,fruits
contain 10-35%
ANALYSIS OF LIPIDS
Analysis of lipids involves ;
(a)the extraction of lipids
(b)determination of lipid content and composition
(c)the assay of the extracted lipids basing on their physical and
chemical characteristics

Extraction Of Lipids
Being sparingly soluble in water ,lipids are extracted using non-polar
solvents .the extraction methods used depends on the samples to
analyzed and the nature of the subsequent analytical problem e.g.
extraction of lipids in milk Is easier than in grains because grains will
first require to be grounded its necessary to keep the chemicals,
physical and enzymatic degradation action as minimum as possible
during lipid extraction process .these is usually achieved by controlling
of ; (i) temperature
(ii) the chemical environment
 SCIENCE LABORATORY TECHNOLOGY
(iii)time of exposure of the sample to the solvent
The following requirements must therefore be met ;
 all procedures must be carried out under the atmosphere of nitrogen
 the solvent used should be first purified and should be of the right
solute solvent proportion
 the sample should be sliced as soon as possible
 heating should be minimized
 -the extract should be purified to remove non –lipids component
 the purified lipids should be stored In proper conditions to minimize
alteration
the moisture content in the atmosphere is important in the extraction
of lipids because these affects the amount of lipids extracted using the
solvent since it prevents penetration of solvent into the tissue and the
extractant becomes saturated with water hence becoming inefficient
for lipid extraction .
also drying at an elevated temperature is undesirable because some
lipids get bonded to proteins and carbohydrates hence becoming
impossible to extract.
Selection of which solvent to use depends on ;
 The nature of food sample
 The cost of the solvent
 Availability the solvent
 Explosion hazards associated with the solvent
 Easiness of evaporating the solvent after extraction
 The speed of extraction
 The tendency of the solvent to pick moisture from the environment
Ether and petroleum ether are the most commonly used solvents .but
no single solvent is 100 % effective and therefore combination of
solvents are normally used in extraction.
Purification of extracts
Lipids extraction is sometimes accompanied with extraction of other
water soluble –non lipids extracts from tissues along with lipids .the
non lipids must b removed prior determination so as to avoid
contamination during subsequent analysis .the removal of
contaminants may be accomplished by evaporation of the extract to
dryness in a vacuum under nitrogen followed by re-extraction with
another non-polar solvent .
Soxhlet Apparatus is the most used apparatus in the extraction of
lipids
 SCIENCE LABORATORY TECHNOLOGY

Fractionation of Extracted Lipids

Extracted lipids consist of heterogeneous mixture which are often hard
to analyze and therefore have to be divided into various subgroups
Two methods are used for fractionation ;
(a) countercurrent distribution
(b) chromatography
(c) countercurrent distribution
This is the separation of solutes on the basis of their differential
solubilities in two immiscible solvents depending on the partition
coefficient of compounds in two immiscible solvents. The differential
solubility is described as the partition coefficient K and is given by
K =C1
C

Where C1 and C2 are the concentration in the two solvents layers.

(B) Chromatography
(i) column chromatography
These is achieved by eluting the lipid through an adsorbent soaked in a
solvent placed inside a long glass column
(ii) thin layer chromatography (T.L.C)
These is the most commonly used method of fractionating lipids .
infact almost all types of lipids are can be separated from other lipids
by T.L.C Each fraction separated consist of a family of related
compounds differing only on chain length and degree of unsaturation.

METHODS OF LIPIDS ASSAY

(A) Physical method of lipid assay
Physical methods of lipids assay are useful for identification, for
checking purity and for control of certain aspects of processing. They
include;

(i) color
These involves identification of fats basing on color by comparing them
against standard color solution
(ii)melting point ,solidification and consistency
lipids being a complex mixture don’t have a definite melting and
therefore they pass through a gradual softening before becoming
 SCIENCE LABORATORY TECHNOLOGY
liquids. But nevertheless the melting point must be defined by specific
conditions of the method. There are two methods of determining the
melting point
(a) capillary method
in these method , 1mm thin walled capillary tube is fitted to the height
of 10mm with melted fat .one end of it is sealed and the fat is allowed
to stand at 4-10 oc for 16hrs . the tube is then attached to a
thermometer and placed in a water bath at 8 – 10 oc below the
expected melting point .the water is then heated at a rate of 0.5oc per
min. The melting point is taken as the temperature at which the fat
becomes clear

(b) Wiley melting point

This is a much more reproducible and reliable method it uses a disk of
fat 3/8 inch in diameter and 1/8 inch in thickness . these is solidified
and chilled in a metal form
for two or three hours and suspended in alcohol-water bath maintained
at15 –20 oc below titre . the sample is stirred with a rotating
thermometer. The melting point is taken as the temp at which the fat
becomes completely spherical
(c) Titre test
This method determines solidification of fatty acids . a titre tube filled
with fatty acid is suspended in air bath surrounded by water bath
that’s maintained at 15-20oc below titre.the sample is stirred until the
temperature begins to rise and remain constant for 30 sec, after which
the stirring is stoped and the maximum temperature that the fatty acid
attain as a result of heat of crystallization is determined.
(d) Dilametry
it involves measurements of changes in specific volume that occurs
with change in temperature .it is useful in the field of fats and oils
especially in the analysis of phase transformation because fats expands
when they melt and contract when they undergo polymorphic change
to a more stable form
(e) infra red spectroscopy
This method is useful for identification of lipid source , for detecting
alteration ,in studies of autoxidation, rancidity and drying properties of
lipids . in following the effect of food processing and in nutritional
investigation on the effect of food composition and interaction with
lipids.
 SCIENCE LABORATORY TECHNOLOGY
(f) UV spectrophotometry
These involves the study of absorption of a particular wavelength in the
UV region
(g) Refractive index
this involves determination of lipids basing on their refractive index
.the refractive index is related to unsaturation

(B) Chemical Method of Lipid Assay

(i)Determination of impurities
Impurities in lipids are mainly moisture ,volatile compounds insoluble
matter, unsaponifiable mater ,trace metals and there soaps i.e (MIU-
Moisture, Insoluble and Unsaponifiables) . MIU is used to designate
the amount of non-fatty constituents of crude oils and other fatty acid
products.
Insoluble matter found in oils d fats and oils include dirt , meals and
any other substance that is insoluble in petroleum ether . While
unsaponifiable matter are those substances that cannot be saponified
by KOH they include sterols , higher alcohol’s and some hydrocarbons
Presence of metallic elements in food is detrimental to quality and
stability of fats
(ii) Determination of unsaturation
Determination of unsaturation is important both in classification of
fats and oils for use and for control of manufacturing
process.unsaturation is normally expressed in terms of iodine value i.e
its defined as the grams of iodine that add to 100g of sample. There are
several methods of determining the iodine value eg. Wijs method
which is most widely used . it involves dissolving the sample in 15ml
carbon tetrachloride (CCL4) in which 25ml of wijs reagent( that’s
composed of 9g of iodine and 700mls of glacial acetic acid and 300mls
of CCL4) is added in a stoppered conical flask and allowed to stand
for about 1hr in dark at 20oc.then 20mls of 10% KI solution and about
150ml of water is added. The unreacted iodine is titrated with
accurately standardized thiosulphate solution in presence of starch
towards the end of titration. Mercury acetate is sometimes added to
shorten the reaction time the iodine value is calculated from he
difference in titration of a blank and test sample according to the
equation below
 SCIENCE LABORATORY TECHNOLOGY
iodine value =(B-S) N X 12.692
weight of sample
where B- Titration of blank sample
S-Titration of test sample
N- Normality of Na2S2O3
Determination of iodine value gives a measure of unsaturation

Saponification Value
These the measure of the amount of alkali that is required to saponify a
definite weight of fats .it is expressed in mg required to saponify 1g of
fat .
Saponification equivalent is the amount of oil or fats saponified. by 1g
equivalent of KOH . it is equivalent to
56,108
Saponification value
saponification procedure involves saponifying 4g of filtered oil in with
50mls of 0.5N KOH sol in 96% ethanol under reflux for 30 min
,excess KOH is determined by back titration with aqueous
standardized 0.5 N HCL in presence of phenopthaline
saponification value is en indicator of he average molecular weight of
fats

ANALYSIS OF PROTEINS
Protein analysis involves use of those techniques for determining
specific elements common in proteins e.g. carbon and nitrogen .but of
all the elements present in proteins , nitrogen is the most reliable one
and therefore the most commonly used in proteins assay .it is assumed
that a mixture of pure protein will contain 16 % nitrogen, thus the
protein content of the sample is obtained by multiplying the
determined nitrogen by the factor

100 = 6.2
16
Kjeldahl Method
these the most used method of protein analysis in which the sample is
first heated (digested) in sulfuric acid until carbon and hydrogen are
oxidized and the nitrogen is reduced and transformed to ammonium
 SCIENCE LABORATORY TECHNOLOGY
sulfate (NH4SO4) . Concentrated NaOH is then added and the digest
heated to drive the ammonia gas into a known volume of a
standardized acid solution solution. The unreacted acid is determined
by calculation into a percentage of protein in the organic sample.

Procedure
A sample of known weight is put in a kjeldahl flask containing
concentrated H2SO4 and left to digest for several hours. This is
normally accelerated by adding a catalyst i.e mercury. K2SO4 is added
to raise B.p to 370- 410 degrees of digestion mixture so as to shorten
the reaction time.
Mercury combines with the NH3 to form a complex i.e mercury-
ammonia complex during digestion and this complex is set free by
addition of sodium thiosulphate
NB; the use of mercury as a catalyst have been subject to criticism due
to being highly toxic and have nowadays being replaced by selenium.
The ammonium released from mercury ammonium complex combines
with So4 to form NH4SO4 in the digest. This is alkalized by addition
NaOH.
(An alkali) so as to liberateNH3 gas.
The NH3 gas liberated is passed into a known volume of standardized
acid where it is determined titremetrically or colorimetrically
The NH3 gas liberated is passed into known volume of standardized
acid where it is determined titremtrically or colorimetrically
Colorimetric determination involves reacting the solution containing
NH4 with alkaline phenol and hypochlorate. This solution gives intense
blue color upon heating.
Titremetric determination involves reacting the NH3 gas with a known
volume of standard acid and the unreacted acid back titrated with a
known volume of a standardized alkali e.g. NaOH to determine the
concentration of Hcl

.Dumas Method
In these method nitrogen is set free by pyrolysis (subjecting the
proteins into high temperature by heating).and the freed nitrogen is
determined volumetrically . Pyrolysis is done in presence of a metal i.e
copper
The organic compound is passed through a tube containing first a hot
copper oxide and next ,hot copper metal gauze .
 SCIENCE LABORATORY TECHNOLOGY
The copper oxide oxidizes the compound converting the combined
nitrogen into molecular nitrogen . the copper gauze reduces any
nitrogen oxide that may be formed also to molecular nitrogen .the
nitrogen gas is collected and its volume is measured.

Buret Method

It’s a method based on the principle that substances possessing peptide

bonds forms a purple complex with the copper salts in alkali solution .
These procedure is simple ,rapid and inexpensive.however these
method is not an absolute method , therefore the color must be
standardized against a known protein or against another method
.however results are affected by turbidity and other interfering
substances

Lowry Method (Phenol Reagent)

These is based on the interaction of proteins with the phenol reagent

and copper under alkaline conditions. The color reaction involves a
copper catalyzed oxidation of aromatic aminoacid and other group by
aheteropolyphosphate reagent
Many of the functional groups found in proteins are responsible for the
final blue color concentration. These procedure is very sensitive
,relatively specific since few substances found in the proteins causes
interference’s , the results are affected little by turbidity of the original
protein solution. However these method is more time consuming , it is
destructive and requires multiple operation on each sample and
incubation between the addition of the reagents also the color
intensity vary with amino acid composition of the protein and the
analytical condition hence the color is not strictly proportional protein
concentration

Direct Spectrometric Methods

These involves measurement of UV absoptions .most proteins exhibit
distinctive UV absorption (max at 280nm) these is due to presence of
specific amino acids e.g. tyrosine ,tryptophan phenylamine etc whose
content varies from sources with a reasonable range .This method is
rapid ,simple and generally non-destructive method for the assay of
 SCIENCE LABORATORY TECHNOLOGY
protein content of biological fluids and solution from fractional
procedures .the results however must be interpreted with care .

Nephelometric or Turbidometric Method

These involves measurements of turbidity produced when the protein
is mixed with low concentration of the common precipitant e.g.
trichloroacetic acid , potassium ferricyanide and salfosalicilic acid

Dye Binding Method .

Proteins bind quantitatively under specified conditions with certain

organic dyes .these dye binding can be used to determine total acidic
and basic groups of proteins.
When a food sample is treated excess dye ,the dye and protein in the
food react quantitatively to form an insoluble compound that can be
separated either by centrifugation of filtration . from the concentration
of the unbound dye , the binding capacity can be calculated.

CHAPTER TWENTY TWO

MUSEUM TECHNIQUES
A museum is a place where objects or materials that represent nature
are assembled and conserved . there are different types of museums
depending on what specimen they preserve .
Function of Museum
(a) Social role – museums serves to preserve the cultural
values .
(b) It helps to display some of the communities diverse
exhibition
 SCIENCE LABORATORY TECHNOLOGY
(c) They also serve educational role ie they serve as learning
centers for students who will learn how to care , collect and maintain
these materials
(d) They help in the reconstruction or restoration of
materials back to look as original as they were
(e) They offer or produce scientific publication
Types of Museums
(a) Aesthetic museums
These is where materials are collected and preserved for their beauty
or aesthetic value . these may include things like paintings ,
sculptures , decorative arts , antiques or things of the old age.
(b)Historical museums
They are those that collect and preserve materials of
historicalperspective . they present materials showing events in
chronological order e.g. political history and archeological history

(c) science museums

These are museums of natural scienc , applied science and technical
museums . Its aims is to communicate scientific information in three
diamensional perspective ie scientifically , spiritually and mentally .
these help in giving informatiom for research workers and progress .
they also help in arousing scientific interest . it also helps people to
understand and appreciate science of conservation especially of our
environment .the earliest museum was set in paries in 1700 -1800.
Types of science museums
Depends on the type of collection e.g.
Ornithology – deals with birds
Herpetology – deals with reptiles
Mammology – deals with mammals
Entomology – deals with insects
Oestology – deals with bones
Herbarium – deals with plants
Methods of collecting museum specimens
Museums speciments are collected from several areas e.g. soil , forest ,
water ,rocks , air etc .Methods of collecting includes ;
(a) Hand picking
These method is suitable for large animals or insects . however these
method is tedious and slow .
(b) Netting
 SCIENCE LABORATORY TECHNOLOGY
Involves use of nets to collect insects (anthropods ) and aquatic
animals . There are various types of nets ie
(i) Sweep nets made of long sticks tied to a net . it is used to collect
small animals especially
insects
(ii)Butterfly net –they are made of mosquito nets . are used for
catching butterflies in flight ,
(iii)P ond nets ,
(iv)Drag nets ,
(v) Dip nets .
(c) Beating trays – Used to collect insects which rest on branches . it
is held under a branch which is then beated with a stick .for collecting
land animals ,the following methods are used .
(d)Trapping – Baits are used which attract animals towards traps
where they will be trapped. Traps normally work well at night and in
the absence of the collector .

There are many types of traps .

 Sticky traps –For collecting insects that are active at day and night .
they consist of a cylinder covered with a sticky substance.
 Light trap – These consist of a bright bulb placed inside a standard
special container . it is normally used for nocturnal insects.
 Water traps – Consists of shallow trays of water . the inside of such
trays is painted white or yellow to make it attractive . few drops of a
detergent are added to water to break the surface tension so that the
insect may sink.
 Pitfall traps – Used for large insects and small animals . these may
consist of a wide mouth container or a dark hole which is either left
open or covered with a weak / delicate cover. The top of the body is
leveled with the ground . as the animal moves across it , the fall into
thecontainer where they are collected .
 Break-back trap or break jaws – They have strings that tightly grip
the animal and breaks the animal jaws or backs therefore killing it
(n) Small animals inside soil can be dug out using jembe ,then sieved
and filtered out after mixing thesoil with water
Preservation of insects
After collection , specimens are to be kept in a killing jar to protect
them against struggling out . The standard killing jar should contain
 SCIENCE LABORATORY TECHNOLOGY
potassium cyanide ; the jar should also have a covering because these
chemical is poisonous to handle . also ether or chloroform can be used
in a killing jar . large winged insects should be wrapped in an envelop
paper to prevent damage of wings then place in jars .
These insects should be labeled with the following special information
 location of collection
 date of collection
 The common name
 Scientific name
 Vernacular name
 Name of thecollector
 Preservative used
Mounting of insects
Mount insects immediately after collection
(i) keep large insects in large envelops (ii) Insects becomes brittle ,
the antennae and the legs breaks off . if they are kept for long . tese
brittleness can be solved by placing such insects in a relaxing jar to
soften them . place them on a spreading board with their wings
stretched , its advisable to pin insects on board with one
of their wings left hanging .
- Relaxing jars are of different sizes and are repaired to accommodate
any insects of various sizes. Inside each jar , insert a wet cotton wool
to with a few drops of carbolic acid have been added to inhibit growth
of moulds . cover the cotton woolwith a layer of bloating papers and
place the insect on top and cover the jar.
After 24hrs , the insects are soft enough to handle for mounting and
for speading such insects should be handled with care because they
are very weak . a spreader may be used to spread insects wings , such
spreaders can be purchased or made locally . they consist of two side
sections made of soft wood with a channel along the center that is
wide enough to accommodate the insects body .
Pin the insect via the thorax by inserting a mounting pin into the
groove of the spreader using a forcept .
Arrange the legs of the insects as they are when alive . use a strip of
paper to hold the wings into position . the duration for drying for
insects depends on the insects body and may vary from days to 2
weeks
Mounting insects for display
Set insects on pins which can be inserted into a soft box .
 SCIENCE LABORATORY TECHNOLOGY
Pin insects by thorax , but small insects e.g. weavils,fastened them on
a triangle of light weight stiff paper and pins are inserted via a broad
based triangle . small fragile insects e.g. mosquito are first pinned to a
piece of cork
Care of collected insects
Collected and preserved /mounted insects can be destroyed by other
living insects which attack and feed on them e.g. termites ,black
ants ,weevils etc . To keep these insects away, chemicals are used to
protect them e.g. paradichlorobenzene

Preservation of other animals speciments

(a) Chemical method – preservatives are used in which the animal
specimen can be stored . These method is used especially for large
animals which cannot be easily dried qualities of a good preservative
(a) should not change the chemical composition of the
organism store or preserved
(b) should have a fair pleasant smell
(c) Should be colourless
(d) Must not be reactive to the user
Examples of common preservatives
(a) Formalin /formaldehyde – it is
usually sold at 40% from which different solutions can be made . the
recommended dilutions for common animals are
Fish – 10%
Amphibians- 3.5%
Reptiles -5-10 %
Birds -15%
Mammals 10%
Advantages of formalin
o Cheap and easily available
o it hardens the organs
Disadvantages of formalin
o It has a nasty smell
o some people are allergic to it
o It is slightly dangerous to animal skin
o It is acidic and decomposes bones

(b) Buffered alcohols

 SCIENCE LABORATORY TECHNOLOGY
Any of the alcohol that exist as liquid at room temperature can be
used as a preservatives e.g. methanol , pentanol , hexanol , propanol ,
heptanol. The most common diluton of alcohol is 70%
Advantages of alcohol
o Have no abnoxious smell
o Some are odourless
o Not associated with any allergy
Disadvantages of alcohols
o It couses shrinkage of tissues due to dehydration
o Some are expensive
o Does not harden the organism
Procedure followed in chemical preservation
(a) Put the organism in a suitable container . the container should be;
o Of appropriate size
o Have wide mouth
o Be transparent
(b) chose the preservative required and make necessary dilution
. the preservative should completely cover the specimen
(c) close tightly the lead and lable
NB/ alternatively the organism may be injected with the preservative .
these process of injecting the preservative into the organismis called
embalming
(d) fixation procedure should be
followed

Labeling of museum specimens

Labeling is important for easy identification. The information
necessary during labeling include

 Date of collection
 location of collection (habitat)
 Scientific name
 Common name
 Vernacular name
 Study purpose
 Name of collector

Preservation of bones
 SCIENCE LABORATORY TECHNOLOGY
Bones may be preserved and stored separately or in a reconstituted
form into a skeleton
Materials require for preservation of bone
- Knife
- Cotton bag
- Concentrated ammonia
- Household ammonia
- Sodium hypochloride (full strength )
- Commercial hydrogen peroxide
- Carbon tetrachloride
Proceedure
- Add hydrogen peroxide to whittenthe bones .
- Keep the bones in cool dry place to dry slowly
preparation of animal skull
 Remove the outer skin from the skull
 Allow the skull to remain in boiling water for half an hour
 Cool the boiled skull by running cold water in the boiled hot water .
these will facilitate the removal of brain . it should be constantly
shaken
 thoroughly
 Place it in a wide jar filled with ammonia and allow it to remain in it
for 2-7 days (it should be covered )
 Rinse with cold water and scrap it off with a brush soaked in
sodium hypochloride to remove the remaining flesh ( wear gloves and
work out in a
 well ventilated room
 Pour a solution of hydrogen peroxide into a container and allow the
skull to remain in these solution
 for 24hrs . rinse the skull again in old water
 degrease the skull by immersing it in a suitable solvent (methylene
solvent)

Reconstructing and mounting skeleton

for further scientific studies
Avariety of items can be used to reconstruct an animal skeleton by
piecing together the appropriate bones . Such items include – pieces of
wire , wood , scissors ,
pliers , forceps , straight and hooked pins .
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER TWENTY THREE

HERBARIUM TECHNIQUES
A herbarium is a storehouse of plants specimens collected from
various parts of the world and mounted in appropriate sheet and
arranged according to some known system of classification and kept
in pigeon –holes of steel or wooden cupboards specially prepared for
that purpose . Herbariums can also be defined as a great filling
systems for information about plants both in their primary form i.e.
inform of actual specimens and in their secondary form i.e. inform of
published information , pictures and record notes. Herbariums are
always generally associated with botanical gardens , educational and
research institutions where they serve as a conservatory of materials
and data ,they also plays a major role in teaching and research .
There are many types of herbariums and they vary according to the
interest of the organization or institutions e.g. those institutions that
only specialize in teaching will only those herbariums specimens only
of interest for teaching and research while medical and drug
industries will keep herbarium consisting of plants of medicinal value ,
similarly agricultural organizations will maintain herbarium of plants
such as weed and field crops . the first herbarium of the world was
established in the university of padua in Italy in 1545 and until
currently , several thousands of herbariums have been established .
among the many important world herbarium include , Royal
botanical garden , Kew in London , V.L Komarov in Russia , New
York botanic garden etc
Botanical gardens are scientifically planed collection of shrubs ,
herbs and climbers and other living plants from different parts of the
world botanical gardens are different from public parks and gardens
in the sense that public parks and gardens are only meant to provide
for aesthetic beauty and recreation whereas botanical gardens are
primarily centered on scientific purpose i.e. for education and
research e.g. to study taxonomy , morphology, breeding and
provision of shelter to most endangered plant species of the world
 SCIENCE LABORATORY TECHNOLOGY
.They also help in simplifying the task of acquisition of plant materials
that may not be available in those parts of the world .

Functions of herbariums
(a) Herbarium is a store of reference materials hence adequate
arrangement for preservation of specimens and simple form of
indexing should be put in place so as to enable them to be retrived
easily
(b) Herbarium serves as a means of identification , these is done
by matching unnamed plant with the named speciment in the
collection or by conforming to identification arrived at by using
botanical keys . for these exercise to be more effective , alphabetical
arrangements of species shoud be followed .
(c) Identification of the plant may be needed because of
o curiosity
o in order to write about the plant
o for researching more about the plant
(d) It is a collection in a herbarium which fully represents the
diversity and distribution of various regions of a country `s vegetation
. these calls for a geographical arrangement
(e) It is a laboratory for plants taxonomy and sometimes other
botanical fields like ecology of biogeography
(f) It gives the scientist an opportunity to study about plants
(g) It makes comparative studies convenient and economical

Types of herbariums
(i) International herbarium – they are large herbariums with a
million or more specimens and global representative of comprehensive
range of taxonomy e.g. Kew in england
(ii) national /regional herbarium – specimen here are from
phylogeographicaly similar e.g. east africa herbarium . it covers the
areas in details . and some neighbouring countries and some
neighbouring countries with representative collections
(j) local herbariums – These deals with a region , a country ,
province , district , forest and these includes different national parks
e.g. herbarium covering marsabit
district in kenya
(k) Special herbariums – these contain a collection of specific
plants for special purposes e.g. research (medicinal plants)
 SCIENCE LABORATORY TECHNOLOGY
(l) Historical herbariums – the contain / caters for historical
plant specimen
(m) Teaching herbariums – these are mostly found in the
institution of learning e.g. in colleges and universities for teaching
purposes .

Collection of plants
Plants specimens are collected from; Ponds , arboterioum ,Botanical
gardens , Forest .
Whenever possible , the whole plant parts including the underground
roots should be collected . The branches should also have the flowering
structures . if the plant is too small, try as much as possible to show its
size and incase the plant is large , it is best to collect as much as it can
be mounted so as to show as many characteristics as possible
It is ideal that both flowering and fruiting parts for each plants be
included.
The best way to collect the plant specimen is to first take time to study
all the stages of growth of the plant and taking a few of each stage
structures and characteristics at a time .
The best important part of plant collection is making of field notes .
each specimen collected should be numbered and notes made
concerning it should be put in a note book while in the field .
There are three main ways of collecting plant specimens
(a) Uprooting and digging
(b) cuttings done for shrubs and woods
(c) Net picking done fo aquatic forms e.g. algae

Equipments used for collecting plants speciments includes

;panga , jembe , secatuers , axe .plant specimens should be put in bags
called vasculum.. specimens should be collected at day time , when dry
and not rained on .

Preservation of plant specimens

Plant speciments should be preserved as quickly as possible soon after
collection in order to maintain their characteristics of the plant as
natural as possible . There are two main methods of plant preservation
ie chemical and drying method
(a) chemical method
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The plant specimen is preserved under a chemical preservative . these
method is useful for preserving freshly succulent plants e.g. aloe vera .
the plant after collection is put in jar in as natural as possible where
chemical preservatives are added and tightly closed to avoid
evaporation of the preservative . it should be completely filled with
preservative to avoid oxidation of the plant .

In order to ensure the penetration of the preservative ,the plant should

be pricked to allow penetration of the preservative .most preservatives
used are formalin and ethanol . The plant specimens have to be put in a
fixative before being put in a preservative

(b) Drying method

Most plants stored in a herbarium are dry specimens . drying is
hastened by pressing the plant between two sheets of bloating papers
(adsorbent papers). Plant pressing serves two objectives
- To flatten the plants to avoid shriveling
- To fasten drying ie to reduce drying time .
A plant press consist of a pair of frame 80x 45 cm. each made from
wood . currently , plant press are made from wire mesh . in between
the wire mesh or frames , is placed a bloating paper where the plants
are placed on very thin sheets of papers .
The press is held with straps or strings or rubber bands tightly over
it . the bloating papers should be replaced every time (old newspapers
can be used as bloating papers )The plant should be arranged on the
bloating papers so as to look as much raw as possible with leaves
flattened and flowers displayed to show different views of florescent .
suplus leaves can be removed . succulent plants must also be killed by
being presses since if dried in a normal way , they often continue to
grow . killing is done by emmersing them in methylated spirit or
alcohol for some hours or put in paraffin or boiling water for 5-10
min though the later method adds water to the plant . Plants should
be pressed as soon as possible
Thorny plants e.g. accasia should have its thorns broken or bend . the
drying method should be conducted as soon as possible . the drying
paper used should be changed on daily basis .

How to dry
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 The paper changing can be done by keeping the press in warn dry
place
 The press may also be arranged over a source of heat so that warm
air can circulate over the press . these reduces the drying time
 An oven wit its dial indicating warm terms can be used as a drier
 Too much heat should be avoided since it results in the plant being
baked and becoming extreamely brittle and break when mounted
 A small jiko can be used for drying specimen in the field

Labeling
Each specimen collected should be labeled and the labesls should
contain the following information
o The flora
o The scientific name
o Vernacular name
o Local name
o Locality
o Longitude and altitudes
o Habitat
o Description of the plant characteristic
o Economic importance of the plant
o Frequency – the seasons when is the plant
common
o The collectors name

Pests in the herbarium

Insects are the chief agents of herbarium destruction .they include
beetles , book lice , silver fish , ants .
Fungi is also a pest but does not destroy dry specimens
Insects and fungi can be kept out of herbarium by keeping the
speciments dry on a well fitting cupboard
Control of pest in herbarium
Paradichlorobenzene (pcb) should be scattered among the
speciments . PCB sublimes slowly producing vapour , these affects
noxious insects and are repellant to insects but harmless to
humans .
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PCB eventually disappears and leaves residues to the specimen which
gives protection to the specimen for as long as 6 months . it is
sometimes reffered to as moth balls .
Insects can also be killed by dip freezing , fumigation using methylene
bromine can also be used but also several conditions should be
considered before using fumigation because ;
(a) Gas used is deadly /poisonous
(b) Pest control firms should be consulted first
Mercuric chloride can also be used as a preservative especially for
specimens to be mounted ( they provide a long lasting solution) . it is
prepared as a solution in alcohol . the composition of these solution is
as follows
Mercury chloride -500g
Phenol – 2505
95%ethanol -20l
Mercury chloride dissolves easily . phenol crystals are melted with
gentle heat before storing them in tightly stoppered bottles .
These solution is applied with a a soft brush to the mounted specimen
which are then left to dry
Care must be taken so that the fluid must not reach the labels . These
treatment kills all insect and their eggs present in the specimen and
leaves residue solution for severally days . however it sublimes slowly
and specimens may become attscked , therefore repeated treatment is
necessary
NB LPCP can be used as a substitute for mercuric chloride . it is less
poisonus to man but evidence shows that they accumulate in mans
tissues
- silver fish is another pest that attack labels on herbarium
specimens . it can be controlled by use of sodium fluorosilicate bait
which is applied on labels . it is however dangerous to human health
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER TWENTY FOUR

 SCIENCE LABORATORY TECHNOLOGY

AQUARIUM AND VIVARIUM

TECHNIQUES

An aquarium is an artificial aquatic environment made using shallow

bowels. it is used to keep fish and other marine animals . Aquariums
are important in learning and research institutions for teaching and
studying aquatic biological systems like food webs, courting behavior ,
breeding patterns and for studying their morphological and
anatomical structures.

An aquarium should be as wide as it is deep to allow free circulation of

oxygen from the air . Aeration of the tank is done using diffuser
blocks and pumps The most appropriate standard for school aquarium
is 60x30 x30 cm .

They should be sited near a power supply so that electric equipment

can be used without trailing the cables . it must be away from the sun
because sun will encourage growth of green algae. Artificial lighting is
the most preferred since it can be regulated. The aquarium frame and
hood should be earthed if it is made of metal to avoid electrocution .
Heating or warming of the tanks should be by means of heaters and
thermostats

When the tank is in position , a layer of well washed aquarium gravel is

spread at the bottom, the depth of the gravel should be about 8cm at
the back of the tank but slopping to 2cm at the front. To fill the tank,
place a layer of paper over the gravel with some stones or saucers on
top then run water gently without disturbing the slope of the gravel.
The paper is then slowly and carefully removed when the water in the
tank is upped the required level. Never use chlorinated tap water, but if
this is the only available water ,it may first be allowed to stand for
24hrs before introducing the plants and aquatic animals e.g. fish
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Plants are planted at the sides and the back of the tank so as to allow
free swimming area for fish. When fish are introduced into the
aquarium , the container in which they arrived in should be floated in
the aquarium tank until it have assumed the temperature of the
surrounding and then its gently sunk into the water so as to allow the
fish to swim out .

Fish is fed on dried food and feeding should be done once in a day but
however fish can still be safely left without feeding at weekends and
even holidays provided that the tank is well established with plenty of
food including life food .

In a nice set aquarium, little cleaning should be necessary ,

Fish diseases
(a) Fungal disease
This is often seen as opaque white patches on the fish . They are
normally non-pathogenic but may attack fish when the mucus
membrane on the surface of the skin is damaged especially when
injured by fishing nets during fishing . Therefore never always handle
fish using nets as they may damage the skin . Affected fish should be
immediately transferred to separate tanks containing 40cm3 of 1%
phenoxetal per gallon and left inside until cured
(b) Swim bladder disease
In these case , the fish experience difficulty in maintaining their
balance . Some will even swim while upside down others vertically .
Some fish will however recover naturally while others are incurable .
The disease is not infectious but its good to kill the infected fish
(c) Dropsy
In these case , fluids accumulates in the fish tissues and the fish swells
with its scales standing out of the body . The disease is believed to be
caused by virus followed by bacterial attack. No cure is available at
present, the affected fish should be humanely killed .
(d) White spot
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These is a common infectious disease of tropical fish , the disease is
curable , its symptoms are white bubbles on the fish bodies , these are
mainly due to protozoan parasites with cysts. The cyst will shortly
burst releasing many parasites. Various drugs are however available to
cure the disease in the tanks

VIVARIUMS
A vivarium is an artificial ecosystem within the laboratory where
reptiles and amphibians are kept . The size of the vivariums should be
related to the habits of the animals kept inside i.e. jumping and
climbing animals e.g. frogs and toads and sometimes reptiles need
deep vivariums , while crawling animals e.g. snails need more floor
space . They are usually made from old aquarium tanks which have
developed leaks , they should have slopping glass fronts .Small reptiles
can escape hence needs well fitting lids made of perforated zinc
The ecosystem I the vivarium should be a true reflection of the animals
natural habitat i.e. amphibians need cool moist environment because
they physiologically depend on moist animal skin for respiration .
Covering the aquarium with damp peats and providing a dish of water
at one end can provide for these environment. Shady hiding places
should also be provided at one end using flat stones and fern plants ,
stiff branches should also be provided for the climbing species , if
reptiles are to be kept ,then the floor should be covered with sand or
gravel and with a dish of water at one end and a piece of branch and
some stones for basking and hiding, The temperature for the vivarium
should not exceed 20oc . The vivaruims should be sited in shady parts
of the laboratory.
Young frogs should be fed on filamentous algae but afterwards ,
tadpoles become carnivorous and will start feeding on small flies or
small pieces of lean meat or liver which should be suspended on
string . Lizards and chameleons should be handle

gently and not by their tail, they should be fed on small insects ,
earthworms and slugs . Snakes can be kept in vivariums but not for
long because they will soon refuse to eat, their food mainly comprises
of newts ,lizards , mice and small birds . They are normally dangerous
if provoked . Tortoise are herbivorous and can be kept outdoors , they
however require small deep water in which to immerse their heads .
They are fed on carbages and lettuce and should also be given
 SCIENCE LABORATORY TECHNOLOGY
opportunity to grace on grass. They hibernate in autumns and should
hence be provided with peats and dried leaves placed in a cool dry
place for hibernation and should never be disrupted .

CHAPTER TWENTY FIVE

MICROSCOPY
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The word microscope comes from the fusion of the Greekwords micros
which means small and skopien, to see or examine. A microscope is
one of the most useful tools in any Microbiological laboratory .
Therefore proper microscope use is one of the most important skills
that every microbiological laboratory technician should have .
Depending on the contrast system, microscopes are given diff erent
names.
Among the most common are the following:
 Clear field optical microscope
 Dark field optical microscope
 Fluorescence optical microscope
 Phase contrast optical microscope
 Interference optical microscope
 Polarized light optical microscope
 Inverted optical microscope
 Stereoscopic microscope

THE LIGHT MICROSCOPE

These was invented in the 17th century.the light rays from a light
source beneath the stage is transmitted through the glass lens in
series i.e. eye piece lens and objective lens .
Depending on their strength , these two lens can routinely provide a
magnification of over X400.
The light microscope have had profound influence in microbiology and
cell biology but its however have a limit in the amount of details it can
show . These limit is set by its resolving power. The resolving power is
the minimum distance by which two points must be separate for them
to be perceived as two separate points rather than a single fused
point .For light microscope these image should be 2 ìm apart .
The limited resolution of light microscope is imposed by the
wavelength of the visible light and it means that more can be gained
by magnifying the object more than 1500 times .
These puts a limit I the amount of structural details that can be
detected within the cell . higher magnification can be achieved by
using a special objective lens with a fluid situated between the lens (oil
emersion ) but even then it is not possible to achieve magnification of
2000
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Types of light microscopes

(a). Monocular microscopes

Monocular microscopes are those microscopes having one eye piece
lens fixed to the microscope.

Monocular microscope

They include
(i) The standard microscope
These is the ordinary common light microscope. Its stage is not
permanently fixed and it can be inclined or and its movable
(ii) fixed inclined limb microscope The stage is maintained in a
horizontal plane and the body tube is inclined towardsthe user , in
some models , the rotation of the body tube in a horizontal plane is
possible . These may be useful in teaching as the teacher can share
the instrument with students without moving or exchanging places
with students .
(iii) Inverted microscopes
These type of microscope have its source of light on the upper side and
the specimen on the slide is placed on the stage and a serrated wheel is
turned to move the single objective lens up and down so as to achieve
focus .
Different degrees of magnification are achieved by inserting one , two
or three magnifying tubes between
the objective and the eyepiece
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Inverted microscopes operate at lower magnification than the other
microscopes e.g. X 60- X200. They are therefore suitable for junior
work.

(b) Binocular microscopes

Monocular microscopes leads to eyes straining, these can be reduced
by using binocular microscopes which have two eyepiece lens mounted
to microscope . They can have either single or double objective lens

Binocular microscope

( c ) stereo/ dissecting microsope

Dissecting microscopes are commonly used for the observation of

larger objects and generally have magnifications of less than 100x. The
light source used with the dissecting microscope can be located above
the microscope stage, or the light may be transmitted through the base
of the stage.Dissecting microscopes do not have a fine adjustment
knob. The light source (illuminator) may also be separate and it may be
mounted on the arm, below the stage plate, or at the level of the stage.
Many dissecting microscopes do not have separate objectives to
increase the magnification but they have a magnification knob that can
be turned to increase or decrease magnification. Samples are placed on
the stage plate and are moved by hand. Light can be transmitted
 SCIENCE LABORATORY TECHNOLOGY
through the slide by adjusting the mirror below the sample or the
sample may be viewed with the light coming from above

Stereo microscope

Parts of light Microscopes

Base- Metal or plastic part on which microscope rests
Arm- Somewhat C shaped pillar arising from base that supports the
stage and ocular components
Stage- Flat platform attached to lower portion of arm on which slides
or samples areplaced
Condenser or Iris Diaphragm lever - Lever beneath opening in the
stage, consists of a shutter-like group of metal leaves which regulate
the amount of light coming through the slide on the stage
Condenser - Not present on all microscopes - collects illuminator light
rays and focuses them - increases the resolution, enhances contrast of
sample
Condenser adjustment knob- Raises and lowers the condenser
Tube/Barrel- Cylindrical/vertical part attached at the top of arm for
support of optical system
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Turret- Revolving plate which bears the objective lens, attached at
lower end of tube, can be turned to change the objective lens
Coarse Focus- Larger knob which moves the tube up or down rapidly to
get the sample into coarse focus
Fine Focus- Smaller knob which moves the tube through short
distances slowly and is used to get the sample into sharp focus
Transformer or illuminator- Controls the amount of light transferred to
the sample
Eyepiece (ocular lens)- Removable, short metal tube that contains lens
that fit into the top of the tube - generally 10-15x magnification
Eyepiece Focusing Ring- Adjustment used to compensate for
differences between eyes
Objective Lens- Small metal tubes screwed into the turret which
increase the magnification of the sample. (often

referred to as just objective)

Mirror- Used to reflect light through a sample
Mirror Axle- Used to adjust mirror to reflect light through a sample
Auxiliary Lens- Also referred to as supplemental lens, may be found on
dissecting microscopes at the base of the objective cover or tube

How to Move and Transport Microscopes Properly

Microscopes are often stored in cabinets when they are not in use and
must be removed in order to use them. To remove microscopes from
the storage area, place one hand completely around the arm, and then
place your other hand underneath the microscope base. Always carry
the microscope in an upright position. Carrying in any other way may
allow parts to fall from the microscope. Use care to ensure that
electrical cords are not entangled with those of other microscopes.
Place microscope on a clean area of the desk or laboratory bench.
Light sources are required for most microscopic observations. The cord
for the light should be plugged into a suitable outlet. Dissecting
microscope lights are often a unit separate from the microscope. Such
lights may be attached to the arm to illuminate the stage from above
the sample. Alternatively, light sources may be placed next to the stage
at the level of the sample, or below the stage

Determination of Magnification
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Magnification is a measure of the ability of the microscope to enlarge
an image. Resolution is a measure of the ability of the microscope to
separate different points of the image. Determining magnification is
vital when comparing the sizes of different objects being viewed with a
microscope. Note the magnification of the eyepieces and the different
objectives on your microscope. The magnification is printed or etched
on the side of the objective and on the side or top of the eyepiece. This
magnification is shown as a number followed by an x (e.g. 15x).
Dissecting microscopes may havean additional lens (referred to as an
auxiliary or supplemental lens) on the base of the objective cover in
order to increase magnification. Note if your dissecting microscope has
such an auxiliary lens. Dissecting microscopes also may have a
magnification knob which changes the magnification when turned.
Note the markings on or near this knob that are used to determine the
magnification.
Compound microscopes often have objectives which are designed
strictly for use with immersion oil. These objectives are identified by
having the word "oil" engraved on the side, near the number stating the
magnification of the objective. Oil objectives cannot be used without
immersion oil. Other objectives cannot be used with oil and can be
damaged if inadvertently immersed in oil. The use of an oil immersion
lens is essential when viewing structures less than 10 µm in size. For
example, magnification requiring an oil immersion lens is necessary to
determine the shape of an individual bacterium. Oil immersion does
not increase the magnification of the lens, but it improves the
resolution or sharpness of the image produced by the objective. When
light passes through any material to another, such as from glass to air,
the light is refracted or bent. Light of different wavelengths bend at
different angles. Such refraction results in distortion which can be
significant at higher magnifications. By putting a drop of immersion
oil, which has the same refractive index as glass, between the 100x
objective and your slide you significantly reduce the light scattering
that would otherwise occur, thereby increasing the resolution of the
image. To determine the magnification of any image, you will need to
multiply the eyepiece magnification by the objective magnification. If
an auxiliary lens is present on a dissecting microscope, its
magnification must be multiplied times the objective and eyepiece
magnifications. It is important to note the magnification on all
drawings so that you can compare the relative sizes of images that you
 SCIENCE LABORATORY TECHNOLOGY
observe at various times. In many cases you will need to use two or
more different magnifications to view all the details of a sample.

Adjustment of Eyepieces
Several adjustments are necessary before looking at specimens. On
binocular microscopes (microscopes with two eyepieces) the distance
between eyepieces should be adjusted so that you can look through
both eyepieces easily at the same time and see a single image. There are
several different methods of adjusting this distance depending on the
microscope being used. In some microscopes there is a knob at the top
of the microscope between the eyepieces that adjusts the eyepiece
distance . Other microscopes are adjusted by pushing on the eyepieces .
This adjustment is specific to each person so minor adjustments may
be required when one person looks through a microscope set up by
another person. Make this adjustment before continuing. Adjusting for
differences between a person's eyes using the focusing sleeve on left or
both eyepieces is another important adjustment to prevent discomfort
and fatigue of the microscope user. The procedure for making this
adjustment follows the initial focusing since a specimen has to be
observable for making the adjustments.

Resolving power of alight microscopes

Magnifying power is always linked to the resolving power . The higher

the magnification , the higher the resolving power and hence the closer
the details of the specimen can be seen .The resolving power of an
objective lens depends on the numerical aperture (NA) of that
objective lens . The following are the usual NA values of the commonly
used objective lens

X10 objective lens = NA O. 25

X20 objective lens = NA O.45
X40 objective lens = NA O.65
X100 objective lens = NA 1.25

Immersion oil
When a beam of light passes from air to glass to air and back to
glass again through the microscope lenses, it’s normally bent due to
refraction property of light . These bending have little effect on low
 SCIENCE LABORATORY TECHNOLOGY
power objective lens i.e. X10,X20,X40 objectives , but it have
significant limitation to the amount of light which enter through the
high power objective lens i.e. X100 objective lens and consequently its
resolving power .
Such bedding can be avoided by replacing the air between the
specimen and the lens with an oil Immersion which have the same
optical property as that of glass . These
makes light to pass in a straight line as though it was passing through
the same media i.e. glass all the way . These therefore provides better
resolving power

Chromatic and spherical aberration

Chromatic aberration occurs when a biconcave lens splits whit light

into its component colors and in the process blue light is magnified
more than red light so that blue comes into focus near to the lens.
While spherical aberration is due to the edges of the lens giving lightly
higher magnification than its center

Illumination system of the light microscopes

Good microscopy requires an adequate well-ventilated and
controllable illumination system. These can be achieved by using a
microscope with an inbuilt illumination . Daylight illumination should
be discouraged because its variable , difficult to use and rarely
adequate for oil emulsion work
Glare
Glare in microscopes is simply that inconvenience caused as a result
of light reaching the eye which does not go to making up the perfect
image but instead only interferes with the image and the ability of the
objective to distinguish details in a specimen . To test for glare,
remove the eyepiece and check to see if the inside of the tube is
illuminated . These indicate presence of glare.
Glare can be reduced in the following ways
(a) positioning a microscope with an inbuilt illumination in a
subdued light i.e. not in front of a window
(b) avoid using a large source of illumination than its necessary
(c) reducing the condenser glare by reducing the condenser
aperture i.e. by adjusting the iris diaphragm when using low power
objective
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Microscope filters
Filters are special devises in microscopes are used to
(i) reduce the intensity of light when it is required
(ii) to increase contrast and resolution
(iii) to transmit light of selected wavelength
(iv) to protect the eyes from injuries caused by UV light

Procedure for using light microscope

 Place the microscope on a firm bench

 Turn or adjust or switch on the in-built bulb of the microscope to
direct light into the microscope
 Switch or rock the lowest objective (x10) into the optical train
 Place the slide containing the specimen on the stag
 Rock down the objective using coarse adjustment until it comes to
stop. Do so while your eyes are looking at the objective , not into the
microscope
 Now look into the microscope and using the coarse adjustment
knob, rock up the objective until you see the image . then rock the fine
adjustment knob to focus properly
 If you need higher magnification , switch the x40 objective into train
and
 then use the adjustment to focus the image and if you need still
higher magnification , apply oil immersion on the specimen placed on
the stage and switch the x100 objective
 Rock down the objective to touch the slide while the eyes are out of
the microscope
 Rock up the condenser to the maximum
 Focus clearly using fine adjustment

Care and maintenance of light microscopes

 carry microscopes using both hands by the limb and the base and
should be in upright position so as to avoid dropping loose parts e.g.
mirror and eye- pieces
 Do not touch the microscopes lens using fingers
 clean microscope lens using lens cleansing tissues microscopes
should be kept clean and covered to avoid dust
 The optical parts should be cleaned using a little xylene on a lens
cleaning tissue or soft cloth. do not use sleeves or labcoats
 SCIENCE LABORATORY TECHNOLOGY
 keep microscopes away from direct sunlight and dust i.e. always
cover microscopes and keep then in locked cupboards
 never place microscopes at the edge of benches
 avoid using a high power objective when a low power objective is
satisfactory
 It is dangerous to rock down the objective while you are looking into
the microscope. You risk breaking the slide, the objective lens or the
condenser
 repair should be left only to a trained expert
 do not place wet preparation on the stage without wiping the
undersurface of the slide

Use of Dissecting Microscope

There are several differences between using a compound microscope
and using a dissecting microscope (sometimes referred to as a stereo-
microscope because it is like two microscopes set to focus on one point
providing a 3-dimensional or stereoscopic view of the specimen).
Generally most dissecting microscopes do not have a fine adjustment
knob. The light source (illuminator) may also be separate and it may be
mounted on the arm, below the stage plate, or at the level of the stage.
Your instructor will give you specific instructions related to your
microscope. Many dissecting microscopes do not have separate
objectives to increase the magnification but they have a magnification
knob that can be turned to increase or decrease magnification. Note:
Some dissecting microscopes also have an auxiliary or supplemental
lens on the bottom of the objective cover or body. If this lens is present,
it must be included in the determination of magnification.
Samples are placed on the stage plate and are moved by hand. Light
can be transmitted through the slide by adjusting the mirror below the
sample or the sample may be viewed with the light coming from above.
When viewing the sample with the light coming from below you may
need to adjust the mirror so that the light is properly adjusted for
viewing. Use the steps as outlined below, ignoring those that do not
apply to your microscope.
After you have finished using your microscope for the day, care should
be taken in properly storing it. Make sure that objectives are clean and
slides are removed. Turn off all lights. Unplug electrical cords and
loosely wrap around the base. Remove lights that are associated with
dissecting microscopes. Place all microscopes and lights carefully back
 SCIENCE LABORATORY TECHNOLOGY
into their proper storage area. Instructors may provide further
instructions as appropriate.

THE ELECTRON MICROSCOPE

The development of electron microscope has revolutionized
microscopic studies since 1950s. These microscope uses electron
beams instead of light and
electromagnet instead of glass lens . The electrons are recorded in
efflorescent screen which then forms a viewable image on the
screen(photomicrograph).
Electrons travel in cathode ray tube which is a vacuum chamber . The
specimens are mounted in total vacuum in order to enable the
electrons to travel with high velocity without colliding with air or
any atoms in space within . These electrons pass through an
electromagnet which acts as lenses where they produce electric fields .
Also the specimens are prepared by sectioning using an extremely
delicate macrodome which produce extremely thin slices then are
fixed using osmic acid or glutaraldehyde for cytoplasm components .
Electro microscope have very high resolving power that is 1000 times
more than an optical microscope , thus specimens can be magnified
much more without lose of clarity . With these microscope , materials
which were initially described as structureless have been shown to have
elaborate internal organization and the so called homogeneous fluids
have now shown to contain a variety of complex structures . These
microscope have had a greater
impact in biology and have helped in opening up a new world whose
existence was barely realized in the 1950s .
The electron microscope however have some disadvantages e.g.
materials for examination have to be mounted in a vacuum and
therefore only dead specimens can be viewed . also the materials have
to be fixed and stained – these preliminary treatment may distort the
delicate structures inside the cells and create images that are not real
(artifacts). These problem can be overcomed by using electron
microscopes together with other types of microscopes.
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Types of electron microscopes

(a) Transmission electron microscope
The electrons pass through the specimens
(b) Scanning electron microscope
Solid specimens are bombarded with a beam of electrons which causes
secondary electrons to be emitted from the surface ofspecimen .
These electrons are recorded on a photoelectric plate as in
transmission electron microscope . The scanning electron microscope
enables some details on the surface to be seen more clearly . images
seen on these microscope are three dimensional
PHASE CONTRAST MICROSCOPE
These microscope was developed in 1940s. it enables transparent
objects to be seen and it is ideal for studying unstained living cells .in
appreciation of living cells , there may be sudden changes of refractive
index between the inside and the outside of the cell or between the
nucleus and the cytoplasm . An annulus is used to give a halo cone of
rays since full cone illumination will cause direct or diffracted rays to
be superimposed
The region on the center have a low refractive index and the outside
have a high refractive index. The transmitted rays are defracted and
will not enter the annulus . The region will appear dark . The defracted
and the undefracted rays strike different parts of the phase plate
where the phase contrast as well as the amplitude can be
altered .
The diffraction partterns are produced by interfering and
reinforcement of light waves . variation in brightness occur at
boundaries of different difractve indexes in the specimen
which gives the characteristic of a halo appearance .

THE UV MICROSCOPE

The visible region of the electromagnetic spectrum extends from 650

Ao(deep red ) –4500Ao . Wavelengths shorter than 4000Ao are called
UV . In these region , some cellular compounds e.g. nucleic acid and
proteins absorb a particular wavelength . If the UV microscope is used
in conjunction with visible light microscopy , much useful information
 SCIENCE LABORATORY TECHNOLOGY
may be obtained. These microscopes uses quartz lenses to transmit the
UV light. These microscopes however have limited use in living cells
because the UV rays rapidly kill the living cell. They also require the
use of filters to protect the eyes from the harmful effect of the UV
radiation’s.

FLUORESCENT MICROSCOPE
When certain chemical substances are irradiated by UV light ,they
absorb the radiation and emits visible light .Objects which emits such
chemicals within living cells can therefore be absorbed as as
fluorescent areas when illuminated with UV light . The chemicals
absorbed are known as fluorochromes .the method is extremely
sensitive and can detect minute quantities of materials .
It is particularly useful for studying how proteins and other molecules
enter or are adsorbed into cells . The proteins are labeled by coupling
with the molecules of fluorescent dyes . The fluorescent substances
may be naturally present within the cell or e.g. norepinephrine within
certain neurons or it may be artificially introduced into the cell as a
marker .e.g. administration of antibodies tagged with fluorescent
substances . The fluorescent substances will be visible wherever there
are antigens ,

POLARIZING MICROSCOPE

Polarized light is produced when the light waves lies in one plane .
Light of these kind is produced by calcite prism which splits the light
into beams or by selective absorption of the polarized component in
a plastic material in which the molecules are oriented parallel to each
other .
Polarizing microscopy uses the fact that the speed of polarized light
passing some objects varies with the direction of polarization . These is
especially used for studying filamentous structures such as mitotic
apparatus . It differentiate between different types of materials
embedded in another substance.
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DARK FIELD ( ILLUMINATION ) MICROSCOPE

It is a method of lighting microorganisms suspended in fluid .These

method enables clear viewing of microorganisms structures and
motility,it makes some living organisms visible which cannot be seen
by ordinary transmitted light .
In these type of microscope , light enter special condensers which has
a central ‘blacked out ‘area so that the light cannot pass out directly
through it to enter the objective instead the light is reflected to pass
through the outer edge of the condenser edge at wide angle so that
the only light entering the eyes come from the specimen themselves
with no light entering directly from the light source .
In these way ,specimens are seen brightly illuminated against a black
background like stars in a night sky or dust in a shaft of sunlight
across a darkened room.
. Compound microscopes are used for the observation of smaller
specimens which are placed on microscope slides and topped with a
cover slip. Such specimens should be transparent or translucent
because light must be transmitted through the specimen to reach the
lens of the microscope. Magnifications commonly found on compound
microscopes are between 10x and 1000x.
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER TWENTY SIX

THE CELL
Cell is a basic fundamental unit of life that is capable of its own
independent existence and can reproduce itself. The discovery of a cell
as a structural unit of life was made possible with the development of
the microscope
Cell theory states that cells originate from already existing cells
and that each cell is capable of maintaining its own vitality
independent of the rest cells i.e. they have the ability to continue living
in the absence of other cells . Cells can exist as unicellular
multicultural .all animals and plants are actually aggregates of cells of
various kinds and growth results from an increase in size and number
of cells or both.. cell range in size from the smallest e.g. bacteria to
the largest e.g. the birds egg .
Cells are divided into two broad groups i.e. the prokaryotes and
eukaryotes according to whether or not their genes or chromosomes
are enclosed in a nuclear membrane .
Prokaryotic cells include all those cells whose chromosomes are not
enclosed in a nuclear membrane e.g. bacteria and few species of blue-
green algae while the eukaryotic cells include all those advanced cells
whose chromosomes are enclosed in the nuclear membrane e.g. plants
and animal cells
Irrespective of their diversity , most cells share many common
characteristics. Eukaryotic cells and prokaryotic cells share many
common structures or organelles such as the cell membrane , the
endoplasmic reticulum , lysosomes , mitochondria etc
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They have developed many mechanisms and other metabolic

machineries that makes them capable of obtaining energy from
the environment and the ability to utilize these energy to
support their essential life processes. Cells are separated from
the rest of the world by a cell membrane

Difference between prokaryotes and eukaryotes

structure prokaryote eukaryote

organism Monera, eucabacteria, Plants, animals, prostists
archaebacteria, cynbacteria and fungi
Nuclear membrane absent present
Chromosome Single Double
DNA Cellular molecule naked Long DNA chain bound
(nucleotide) by histones
Cell division Binary fission Mitosis or meiosis
organells Non except ribosome. Mitochondria,
Plasmalemma is perhaps chloroplast(in plants),
infolded as mesosome in green
golgi bodies, lysosme,
bacteria and concentrically endoplasmic reticulam,
infolded in cyanobacteria e.t.c

Cell wall Peptidoglycans, absent in Cellulose(in plants)

mycoplasma absent in animals
Exocytosis and Endocytosis
Absent present
cytoskeleleton Absent present
Cellular organisation Mainly organized Mainly multicellular with
differentiation
metabolism Anaerobic or aerobic Aerobic
Cell size 1-10um 10-100um linearly
Compatments in cells Absent Present due to membrane sys

Similarities between prokaryote and eukaryote cells

1. capable of cell growth and development
2. contain hereditary DNA molecule
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3. carry out cellular metabolism
4. They are highly organized

Difference between plant and animal cell

Animal cell Plant cell

Cell wall Absent Present(cellulose)
Cell membrane present present
Flagella May be present Absent except in sperms of a few
species
Centrioles present absent
Endoplasmic Usually present Ussualy present
reticulum
Golgi bodies present present
Nucleurs present present
mitochondria Present present
chloroplast Absent present
vacuole absent present
Ribosomes present present
chromosomes Multiple units , DNAMultiple units , DNA associated
associated with with ribosomes
ribosomes
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Plant cell

Animal cell
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(a) The plasma membrane
They are found in both prokaryotes and eukaryotes . they serve as the
boundary to the cell and permits selective movement or passage of
materials in and out of the cell
(c)Nucleolus
these organelles is found within the nucleus and infact its
composition resembles that of chromosomes except that it has large
numbers of granules rich in RNA which are the precursors of
ribosome . infact the size of the nucleolus varies with the
requirements of the ribosome and the amount of protein synthesis.
The nucleolus has no membrane of its own .
(b) The endoplasmic reticulum these is a complicated
network of cytoplasm membranes that often appear to be continuous
with the nucleus membranes it constitute more than a half of the total
membrane of the cell .its highly folded and convoluted structures and
forms a single continuous sheet enclosing one closing one continuous
sac .it creates what looks like the tubules or cisternae or channels
which function in intracellular storage and transport and sometimes
in protein synthesis carried out by the ribosome’s attached to them .
The endoplasmic reticulum can be can be classified into two
depending on the presence or absence of m ribosome’s on the
endoplasmic reticulum , i.e. rough endoplasmic reticulum (RER) and
the smooth endoplasmic reticulum (SER). The RER has its outer
surface studded with ribosome whereas the SER don’t have ribosomes.
Ribosomes are the site of protein synthesis. Therefore RER are more
dominant in cells which are actively involved in synthesis and export of
proteins e.g. in pancreatic acinar cells and plasma cells. SER is
found in those cells which are specialized in lipid metabolism and
which secretes steroids such as cells of adrenal cortex, the testes and
ovary. Also SER is also present in liver cells where it helps in
detoxification of drugs and poisons.
(c) golgi apparatusThey were discovered by Camillo Golgi in
1898. They are located near the nucleus. Golgi apparatus are similar in
both plants and animals except that they are more distinct in plants
and are called dictyosomes in plants. they consist of stack-like or
plate like bodies and small rounded transport vesicles together with
large vacuoles filled with amorphous or granular materials . They
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perform many functions in the cell such as processing of molecules
secreted by ER and packaging of molecules called secretory granules
according to their final destination be and disposing hem off by fussing
with the plasma membrane and opening itself to the exterior in a
process called exocytose
(d) Lysosomes and peroxisomes
(e) lysosomes are also called suicide bags because they cause lysis to
other cells. by secreting digestive enzymes that are capable of
destroying a wide variety of substances. the digested products are
removed out of the cell by exocytosis .lysosomes are belived to be
derived from maturing golgi apparatus.
(f) peroxides are also found in almost every cell. It’s similar to
lysosomes in size and appearance and was infact discovered at almost
the same with lysosomes. they have enzymes which are responsible for
producing and degrading peroxides e.g. catalase whose function is to
degrade hydrogen peroxide to water and oxygen
(g) chloroplast and mitochondria
chloroplast and mitochondria share almost the same function and
characteristics. they are all enclosed in a double membrane. they are
the main energy transducers in plants and animals respectively
mitochondria are sausage shaped they are commonly known as the
power house of the cell because they are the site for ATP synthesis.
mitochondria are considered to be capable of movement and they can
change both their shapes and position within the cell. they consist of
two membranes i.e. the outer smooth membranes and the inner
highly folded membrane called cristae , the number of cristae or
foldings depends on the metabolic activities of the cell i.e. the
greater the number of foldings , the larger the surface area for ATP
synthesis . mitochondrias are often found in high concentration in
region of high metabolic activities such as in the cardiac muscles, flight
muscles of insects and birds. the fluid filled space between the outer
and the inner membrane is called intermembrenal space . the space
sorounded by the inner membrane is called mitochondrial matrix ,
its dense and its made of protenaceous materials . the matrix is
generally homogeneous but it may sometimes contain small dense
granules which are the site for binding of Mg2+ and Ca2+. The matrix
also contains ribosomes , RNA , and sometimes DNA .its infact because
of the presence of RNA , DNA and ribosomes that makes mitochondria
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self replicating and can synthesis their own proteins

mitochondria

chloroplast is plastids found in plants . they are also bounded by

double membrane . they also self replicate as they contain their own
DNA, RNA and ribosomes ..
apart from chloroplast , there are also many types of plastids such as
chloroplast , leucoplast etc .
chloroplast is mainly found in green plants . its outer membrane is also
smooth and thin while the inner membrane is again highly folded and
these inner foldings gives rise to internal parallel membranes sheets
called lamellae . the lamellae are suspended in a fluid like matrix
called stroma which contains about 50% of soluble proteins
,ribosomes , DNA and the machinery for protein synthesis .
most of the lamellae in the chloroplast are organized to form a sac –
like structure called thylakoids . thylakoids may be stacked like a pile
of coins hence forming grana .(when thylakoids are unstacked , they
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are called stroma ). Pigments such as chlorophyll, carotenoids and
plastiquinone are present in thylakoid membranes and are involved in
photosynthesis . they trap light energy required for splitting of H2O
during the light stage .. chlorophyl is green in color , carotenoid is
yellow while leucoplasts are colorless
NB both mitochondria and chloroplast originates and develop in the
same way i.e. they are formed by fission of pre-existing organelles .
they are both semiautonomous i.e. they contain DNA, RNA and
ribosomes . hence they can survive independently of other cell
organelles

chloroplast

(a) cilia and flagella

these are locomotor organelles that help to propel a cell towards or
away from the material surrounding the cell . cilia are generally
shorter and many in number than the flagella. Flagella usually occurs
single ( monoflagelate ) , two (biflagellate ) three (triflagelate ) or many
flagella(multiflagelate)

(b) centioles and the basal bodies

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centrioles are usually found near the cell nucleus and they occur in
pairs , they play an important role during cell division .basal bodies
are structurally similar to centrioles but they produce cilia and
flagella
The cell membrane
Cell membranes are biological membranes formed by assembly of
lipids , proteins and carbohydrates molecules .plasma membrane also
called the cell membrane encloses all living cells and is selectively .its
highly organized dynamic structure and the site of innumerable
physiological and biochemical processes and reactions . the basic
framework of all membranes is a continuous double layer of lipids
molecules in which proteins are dispersed .
Plasma membrane separates the internal contents of the ell from its
surrounding . it is selectively permeable and acts as a barrier by
maintaining the difference in concentration of various ions and
molecules between the inside and he outside of the cell . it also
exchanges material with the extracellualr environment by exocytosis
and endocytosis
The lipids and proteins in the plasma membrane are held together by
a non covalent bond while carbohydrates occurring in form of
oligosaccharides are covalently bounded to some of the proteins and
lipids . various protein molecules are distributed on the surfaces of the
membrane as well as embedded in the lipid bilayer forming a mosaic
arrangement (mosaic theory). There is considerable variations in lipid
, protein and carbohydrates ratio among different cell membranes
and also between the two layers of the membrane
Cell membranes are asymmetrical , dynamic and fluid in nature . this
membrane fluidity is crucial to many of its functions . most of the
proteins and the lipid molecules are able to move about freely within
the membrane . the major lipid components of the cell membrane are
phospholipids, cholesterol; and glycolipids . cholesterol is ubundant in
the plasma membrane of mammalian cells and is absent in most
prokaryotic cells . plant cells have little or no cholesterol but large
quantities of related sterols . all membranes regardless of the source
have phospholipids .
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The reason why cell membranes are bilayer is that the phospholipids
it contains is amphapathic in nature i.e. they have both a hydrophilic
heads (water loving ) and hydrophobic tails (water hating ) . these
amphiphatic molecules in aqeous solution spontaneously associate in
a way that their hydrophilic heads are in water while their
hydrophobic tails remains away from water i.e. in other hydrophobic
material such as oil . these means that phospholipids can fit in the
bilayer by fitting their forked hydrophilic and hydrophobic ends in
the bilayers forming a contious hydrophobic interior
In addition to the lipid bilayer , the cell membrane also contain
protein molecules which is normally concerned with carrying out the
specific functions of the membranes such as transport of molecules
across the membrane , receiving signals from hormones and
chemicals , stabilization of cell shape etc . these protein molecules are
mobile and diffuse rapidly across the membranes due to the fluid
property of the lipid bilayer . they form channels for the movement of
ions and small molecules and serve as carriers for large molecules .
just like phospholipids , the protein molecules are also amphipathic .
they also have their hydrophobic heads deeping into the interior of the
bilayer where they interact with the hydrophobic tails of the lipid
molecules . the hydrophilic tails may be exposed to water on one or
both sides of sides of the membrane
Carbohydrates in plasma membranes occur as glycoproteins and
glycolipids . most of the membrane carbohydrates are bound to
protein molecules . glycoprotein’s are absent from all prokaryotic
membranes . carbohydrate chains are found exclusively on the outer
surface of the cell membrane , they seem to be involved in cell
communication and recognition process e.g. the human ABO blood
group are characterized by different glycolipids on the surface of the
red blood cells
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The cell membrane

Membrane transport process

All process which is characteristic of living organism such as growth
, respiration ,excretion etc is carried out with he help of the cell
membrane transport at some stage and many process such as
digestion absorption ,gaseous exchange formation of ATP in
mitochondria and chloroplast etc involve membrane transport.
The internal environmement of the cell differs from the external
enviroment these difference is brought about due to presence of he cell
membrane which acts as a selective barrier between the cell and the
medium in which its suspended in . the rate of movement of molecules
across the membranes is called flux and its controlled by a number of
riving forces such as differences in pressure , electrical potential and
concentration . the transport of these substances may be passive
transport i.e. requires no energy expenditure e.g. diffusion , or active
i.e. requires energy expenditure e.g. active transport .the rate of
flux is proportional to both the surface area and the concentration
gradient ( first law of diffusion - by fick) .the movement of water in
response to solute concentration across the cell membrane is called
osmosis
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A substance can pass through a membrane by two routes i.e. by
simple diffusion ( especially the lipophilic substances i.e. those that
can easily dissolve in lipids ) and facilitated diffusion or active
transport (especially the lipophobic substances i.e. those that do not
dissolve easily in lipids )

(a) Diffusion
Diffusion is the spontaneous process that leads to the net movement
of a substance from a region of higher concentration to a region of
lower concentration through a medium eg water . incases where there
is a barrier , diffusion takes place relatively slowly . the greater the
thickness of the barrier , the lower will be the rate of diffusion . Ficks
first law state that the rate of flux is directly proportional to both
the surface area and the concentration gradient acting
perpendicularly to that area

(b) Osmosis
Osmosis takes place when two solutions of different concentration
are separated by a semipermiable membrane . water will move across
the membrane in the direction of the solution of higher concentration
. for osmosis to take place , the membrane should be permeable to
water but not permeable to the solute .
As water crosses through the semipermiable membrane in to either the
interior or exterior of the cell .Assuming that water gets into the cell ,
the cell will increase in size . this increase in size will cause pressure to
build up against the cell membrane . this pressure is called osmotic
pressure . animal cells usually succumb to this pressure increase
and finally the cell can burst . plant cells can however withstand this
pressure and therefore cannot burst . these is because plant cells have
rigid walls which protect the cell from extreme swelling and bursting
when the cells gain water i.e. in dilute solution . In dilute medium , the
cell absorbs water by osmosis .these builds up the hydrostatic
pressure within the cell . these are called turgor pressure .
 SCIENCE LABORATORY TECHNOLOGY
Turgor pressure is responsible for maintaing the crispness or rigidity
of most soft tissues such as leaves , petioles , young stems etc . when
turgor pressure is lost , these plant organs become limp and flabby
and the plants are said to wilt .
How a particular cell will behave when put in a given solution will
depend on its tonicity tonicity refers to the relative concentration of
the external solution in which the cell is place in with respect to the
internal concentration of solute of the cell itself . The terms iso-
osmotic , hypo-osmotic and hyper-osmotic are used to refer to
tonicities of different cells in respect to the solutions they have placed
in .two solutions having the same osmotic pressure are said to be iso-
osmotic while a solution having lower osmotic pressure than the
other is said to be hypo-osmotic whereas a solution having higher
osmotic pressure is said to be hyper-osmotic.
Tonicity of a solution is defined in terms of the response of a cell to
that solution . if the cell swells in that solution then that solution is
hypotonic and if the cell shrinks in a solution , then it’s a
hypertonic medium , but if the cell is not affected in that medium ,
then the medium is isotonic . animal cells placed in hypotonic
solutions normally burst i.e. they undergo lysis. Lysis of red blood
cells is called hemolytic .While cells placed in hypertonic solutions
normally shrink i.e. they become flaccid

Simple or passive diffusion

Passive is a form of diffusion in which molecules are transported
across the membrane from the region with higher concentration to
the region of lower concentration . in this case the driving force is only
the concentration gradient . it requires no transport molecules . these
form of diffusion obeys Fick`s first law

Facilitated diffusion
Facilitated diffusion is almost similar to passive except that it
requires transport molecules . without the transport molecules , the
membrane is practically impermeable and hence no diffusion can take
place . i.e. the transport molecules mediates or facilitates of the
 SCIENCE LABORATORY TECHNOLOGY
movement of specific molecules across a membrane from a higher
to a lower concentration gradient .
Many animal cells poses facilitated diffusion systems for molecules
which they receive from blood plasma e.g. sugars , amino acids etc .
also red blood cells have facilitated diffusion systems to absorb
glucose from plasma . in general , certain highly soluble metabolites
e.g. sugar amino acids and glycerols are transported by facilitated
diffusion . Facilitated diffusion is a mediated permeability i.e. it
requires special transport molecules to aid in the movement of
molecules across the membrane .

Active transport
Active transport is another form of mediated permeability in which
the molecules move against the concentration gradient i.e. form
region of low concentration to a region of high concentration.
Transport molecules and energy expenditure are also involved in
active transport. Because of active transport, the cells ca accumulate
substances from the environment where the concentration of the
substance are extremely low .its infact through these process that
kidneys reabsorbs most of the salts and essentially all amino acids
and sugars in the extra cellular fluid . active transport also provides for
the creation and mainainance of a concentration gradient which is
very essential for the proper functioning of the cells . active transport
is also involved in many other cellular functions e.g. in muscle
contraction , , propagation of nerve impulses , detoxification of blood
in the kidneys , absorption of metabolites in the intestinal mucosa ,
energy production in mitochondria and chloroplast , flagella
movement in bacteria etc
Active transport is commonly known as pump g Na+ pump , H+ pump
(also called proton pump) etc. energy for active transport comes from
hydrolysis of ATP
Sodium ions for example are actively transported outwards across the
cell membrane at the same rate as they leak in . this process is
attributed to sodium pump –an energy requiring enzyme system that
operates in the cell membrane
 SCIENCE LABORATORY TECHNOLOGY
several basic features of active transport can be recognized .
(a) transport can take place against substantial concentration
gradient
(b) the active transport systems generally exhibits high degree of
selectivity
(c) ATP or other sources of chemical energy are required .
(d) Certain membrane pumps exchange one kind of molecules or
ion from one side of the membrane for another kind of molecule or
ion from the other side
(e) some pumps can perform electrical work by producing net flux
of charges
(f) active transport can be selectively inhibited by certain
membrane blocking agent e.g. cardiac glycosides
energy for active transport is released by hydrolysis of ATP by enzyme
(ATPases) present in the membrane

PINOCYTOSIS AND EXOCYTOSIS

Membranes sometimes transport substances by transfer of small bulk
quantities of materials into and out of the cell . the membrane can
take up bulk quantities into the cell by first tracing the material in a
vesicle formed fro a tiny invagination .the vesicle then pinches off ,
isolating the material on the cytoplasm side of membrane .digestion
or rapture of the vesicle liberates the content in the cymosely . these
process is termed as pinocytosis if fluid is ingested and
phagocytosis if solid is ingested .
A related process called exocytosis plays an important role in
endocrine and nervous system . In these case the vesicles coalesces
with the surface membranes and releases their content to the cell
exterior

CELL DIVISION
MITOSIS AND MEIOSIS
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Mitosis is the process by which the cell nucleus divides to produce
two daughter nucleur containing identical set of chromosomes like
those present in the parent cell . Mitosis is immediately followed by
another process caled cytokinesis which involes the division of the
cytoplasm
Meiosis is the process by which a cell nucleus devides to produce 4
daughter cells each containing half the number of chromosomes of the
original nucleus . Meiosis is sometimes called a reduction division.
Cytokinesi is also followed by cytokinesis
THE CELL CYCLE
The sequence of events which occur between one cell division and the
next is called the cell cycle. The cell cycle have three major stages
(a) Interphase These is a period of synthesis and growth .
The cell produces many materials required for its growth and carry
out their functions .
The DNA replicates during these stage
(b) Mitosis -These is a process of nucleur division
(c) Cytokinesis - These is where the cytoplasm divides to form
two cells

MITOSIS
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Stage Events
Interphase DNA replicates
Chromosomes exist as a pair of chromatids
joined at centriomere
Chromosomes look like thin threads called
chromatids
Centrioles replicates and they lie close to
the nuclear membranes
Nucleolus and nuclear membrane are
present
These is ussually the longest stage
The chromosomes shortens and thickens
and become very vissible . They are seen to
consist of two chromatids joined at
centriomere
centrioles move to the opposite poles of
the cell
Prophase spindles may be seen radiating from the
centriomere
Nucleolus disapear as their DNA gets
distributed to certain chroosomes
The nucleur membrane breaks up and
disapears towards the end of these stage

Chromosomes line up around the equator

of the spindle
The spindle fibers are attached on the
chroosomes at the centriomere
The chromosomes arrange themseves end
to end at the equator

Mataphase
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These is a very rapid stage

The centromeres splits into two and the
spindle fibers pull the daugghter
centriomeres to opposite poles
separate chromatids are pulled along
behind the centriomer forming a `V`
shaped

Anaphase

Telophase The chromatids reach the poles of the cell

where they uncoli to form the chromatin
threads that are not vissible
The spindle fiber diaspears
The nuclear membrane reforms around the
chromosomes at each poles
The nucleolus reapears
cytokinesis begins and splits the cellinto
two
The centrioles replicates

Cytokinesis the cell organels becomes evenly

didtributed towards the two poles of the
telophase cell
the cell surface membrane begins to
invaginate along the equator
the cell surface membrane eventually
joins up and splits the cell into two cells

Signficance of mitosis
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(a) genetic stability - mitosis produces two nucleus

which have the same number of chromosomes , they have
exact copy of genes and DNA from the mother cell .
therefore the daughter cells are genetically identical
(b) growth – the number of cells within the organism
increases by mitosis . These is the basis of growth in
multiccellular oganism
(c) cell replacement – the cells are constantly dying
and being replaced by new ones . these replacement
involes mitosis
(d) regeneration - some animals e.g. lizards, crustacean
etc have the ability to regenarate their lost parts or organs
of the body like tails , arms etc . also some internal cells
and organs e.g. liver can be regenerated back to their
normal status . these process of regenration involves
mitosis
(e) asexual reproduction – mitosis is the basis of
reproduction in cellular organisms .

MEIOSIS

Meiosis is another process of cell division in which the diploid status of

the cells are reduced to haploid status . These process normally takes
place in gametes cells . The process is important in the sense that the
chromosome number in gametes is reduced to half so as to maintain
the diploid status in the offspring that will form after fertilizatin of the
gametes cells to give the new offsprinng

Meiosis involve two stages i.e. 1st meiotic division and second meiotic
division

1ST MEIOTIC DIVISION ( MEIOSIS 1)

Stages in prophase 1
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Leptonema

In pre –leptonema, chromosome are very thin and difficult to observe.

In leptonema chromosome are more apparent and nuclear increases in
size. Chromosome show bead like structure called chromomere.
Chromomeres are formed due to folding of chromosomes.

Chromosomes show a definite combinations. The telomere attach

themselves to the nuclear membrane near the centriomere. This
peculiar arrangememt is called Boaguent

Zygonema

The homologous chromosomes becomes aligned and undergoes pairing

called synapsis. Pairing is highly specific and involves the formation of
synaptomal complex. Due to this complex, pairings become specific for
the corresponding pair of genes.

Due to this formation, the two homologous chromosomes don”t fuse

with each other but remain separated by a space/ gap of 0.15- 0.2um
The functions of the synaptomal complex are
1. It stabilizes the pairing of the homologous chromosomes.
2. Facilitates recombination

Pachynema
Pairing reaches conclusion when chromosome becomes contact
longitudinally resulting in short , thick chromosome. Nucleus appear to
contain half the original number of chromosome. At this stage, each
paired unit of chromosome is called Bivalents, because they contain
two chromosome or called tetrads because they contain 4 chromatids.
In the late pachynema, alline of separation perpendicular to the plane
of pairing appears.
They separate from each other but don’t separate compleletely. They
remain attached to each other forming CHIASMATA. Out of the two
chromatids, only one will take part in the formation chiasmata. Where
they attach themselves, they interchange their segments. The
enzmes endonuclease helps in the breaking the segments while the
enzymes ligases helps in unifying the broken segments
 SCIENCE LABORATORY TECHNOLOGY
Its here where the difference of the offspring from the parent occurs
Diplonema
Repulsion of the homologous chromosomes in pieces
They remain attached at chiasmata
Synaptomal complex disappears
Diakinesis
Contraction of the homologous chromosomes
Tetrads are more evenly distributed in the nucleus.
nucleolus disappears
Homologous chromosome appear to attach with each other at terminal
ends. This is terminalization separation

1ST MEOTIC DIVISION

Meiotic phase Events

interphase 1 These stage is just ike in mitosis
Early Prophase 1 These is the longest phase
The chromosome shortens and become
vissible
Homologous chromosomes pairs up .
These process is called synapsis and
each pair is reffered to as bivalent . each
of these pair is the same in length , same
centriomere position and same number
of genes arranged in the same order
 SCIENCE LABORATORY TECHNOLOGY

The bivalent shortens and thickens

further and
each chromosome in each pair is seen
to be composed of two chromatids
two chromosomes are seen to be found
at several points along their length. The
points of contact are called chiasmata
Each chiasmata is the site of an
exchange of genetic material between
chromatids . The exchange is produced by
Late
breakage and reunion between any two of
prophase 1 the 4 chromatids
as a result , genes from one
chromosome may swap with genes from
the other chromosome leading to new
genes combination in the resulting
chromatids . the process is called
crossing over
By the end of prophasse1, all
chromosomes are fuly contracted and
deeply stained , centrioles have migrated
to the poles of the cell , the nucleoli and
crossing over the nuclear membrane have disapeared
and the spindle fiber are beginning to
form

Here the bevalents become arranged at

Metaphase 1
the equator
The spindle fibers are attached to the
bivalent at the centriomeres
the arrangement of the bivalents at the
equator is in ( i.e. one on top of the other
)
 SCIENCE LABORATORY TECHNOLOGY

The spindle fiber pulls the homologous

chromosomes towards the poles. These
process separates the homologous
chromosomes

Anaphse 1
The homologous chromosomes arrive at
the poles
Telophase 1
Halving of the chromosomes have
ocured but the chromosomes are still
made up of two chromatids
NB if crossig over had occurred , then
homologous chromosome shall not be
genetically identicle
The spindle fiber disapears and the cell
starts undergoing cytokinesis . At each
pole the chromosomes becomes
sorounded by a nuclear membrane . NB
most plant cells do not have telophase 1
but instead the cell just passes straight
from anaphase 1 to prohase 11 of the 2
meiotic division

2ND MEIOTIC DIVISION

Phase Events
Interphase 11 These stage is only present in animal cells . the DNA d
again
Prophas 11 Nucleoli and the nuclear membrane disapear
 SCIENCE LABORATORY TECHNOLOGY

Centrioles moves to the opposite poles of the cell

ussually the centrioles moves to the poles in such a
perpendicular to the originalspindle ussually at right a
of meiosis 1

Metaphase 11 Chromosomes line up separately(but not in pairs as i

the equator of the spindle

Anaphase 11 The centriomere devides and the spindle fiber pull t

the opposite pole. NB here , it’s the chromatids that are

Telophase 11 -just like in mitosis, each cell shall undergo cytokines

these case , 4 daughter cells wil be formed
the chromosomes uncoils , lengthens and becomes v
the centrioles replicates in eachcelland the nuclear m
around the chromosomes , the nucleolus reeapears in e
four cells formed are haploid
 SCIENCE LABORATORY TECHNOLOGY

Summery of the comparison of Mitosis and Meiosis

MITOSIS MEI
1Occurs in somatic cells 1 Occurs in reproductive
formation of gametes or
2Number of chromosomes remain constant 2 The number of chromos
after every division after division
3Has a single division of the chromosomes 3 A single division of the c
and nuclear double division of the n
4 Same number of chromosomes remain 4 Half the number of chro
5Homologous chromosomes do not associate 5 Homologous chromosom
6Chiasmata is never formed 6 Chiasmata may be form
7Crossing over never occurs 7 Crossing over may occu
8Two douter cells are formed 8 Four daughter cells form
one is usually functiona
9Daughtercells are identical to the parents(in 9 Daughter cells are gene
absence of mutations) parental ones
10Prophase is shorter 10 Prolonged and divided i
11Chromosomes forms a single row at the 11 Forms a double row at t
equator of the spindle fibre spindle fibre

Similarities between mitosis and meiosis

1.Both process involves the division of the parent cell
2. In both process the chromosome migrates to the central plane and
arrange themselves at the equator of the spindle during metaphase
 SCIENCE LABORATORY TECHNOLOGY
3. Cells cycling through interphase of both processes have an
accumulation of energy to be used for other stages of division. During
this stage there is also duplication of genetic materials and other
cytoplasmic organelles
4. prophase marks the disappearance ofnucleolus and centrioles move
to the opposite poles
5. Both start at the interphase stage
Differences between nuclear division and cell division
1 Nuclear division is the division of the nuclears alone while cell
division involves nuclear division and other cellular processes
2. cell division is proceeded by nuclear division
3. nuclear division is similar in both plants and animals cells while cell
division is differen

GENETICS
THE NUCLEUS AND GENES
Nucleus is the most dominant organelle in the cell and it controls all
activities of the cell Most cells have one nucleus. Some cells lack
nucleus at maturity stage e.g. in red blood cells and the sieve tubes
cells. The nucleus in eukaryotic cell is enclosed in a nuclear membrane
which is composed of two membranes i.e. the outer and the inner
membrane.The inner and the outer membrane are separated from each
 SCIENCE LABORATORY TECHNOLOGY
other by a narrow space called perinucleur space . at certain places ,
the nuclear envelop is interrupted by the presence of some small pores
i.e. the nuclear pores . These pores helps in selective exchange or
movement of materials in and out of the nucleus and the cytoplasm .
The nuclear envelops usually disappears but it will reappear during
nuclear reorganization
The nucleus contain the chromosomes which carries the genetical
material
Chromosomes are nucleoprotein bodies which are dark staining with
basic dyes and are microscopically observable in the cell during cell
division. They carry genes which are arranged in linear order . Each
species have a characteristic number of chromosomes e.g.
humans beings – 46
Cats - 38
Dogs – 78
fruit fly - 8.
chromosomes transmits hereditary information from one generation
to the next .
Chromosomes of prokaryotic cells (in bacteria and cynobacteria )
differ from eukaryotic cells (algae , fungi , protozoa , higher plants and
animals . the chromosomes in prokaryotic cells are circular while
eukaryotic cells are not .
Chromosomes in eukaryotic cells contain DNA ( Deoxyribonucleic
acid ) and protein coat , but prokaryotic cells only have chromosomes
made up of DNA only but no protein coat
In eukaryotic cells , the DNA , have negative charges along its length
and the positive charges I the protein that bind it . These proteins are
called histones .
The DNA and protein complex is called chromatid. Experiments
have shown that the genetic information responsible for inheritance is
in the DNA molecule , but however , a few viruses like tuberculosis
 SCIENCE LABORATORY TECHNOLOGY
In eukaryotic cells , the chromosomes , just before cell division, are
seen to be made of two parallel strands called chromatids held
together at a point called centriomere.
Genes for different characteristic are located at a particular position
or locus on a particular chromosome . Genes can be therefore defined
as a particular hereditary determinant or a unit of inheritance or a
unit

STRUCTURE OF DNA / RNA

In eukaryotic cells , chromosomes at interphase stage are usually
seen as thin – thread like structures with some swelling at intervals . it
have been shown that the DNA molecules combine with groups of 8
histones protein molecules to form these swelligs called nucleosomes .
they appear like beads on a string . This nucleosomes and the DNA
strands linking them are packed closely together to produce a
structure called solenoid

DNA or RNA are composed of repeating sub-units called

nucleotides ,therefore a DNA molecule is actually polynucleotide . a
nucleotide is made up of
(a) Phosphate group ,
(b) five carbon sugar (Pentose sugar ).a pentose sugar is called ribose
hence the origin of the name ribonucleic acid (RNA). There also exist
another type of pentose sugar in which the carbon number 2 is
missing one oxygen atom hence the name deoxyribonucleic acid
(DNA)
 SCIENCE LABORATORY TECHNOLOGY

(c) nitrogen bases present in DNA are

 adenine
 guanine
 thymine
 cytosine
the nitrogen bases found in RNA include
 adenine
 guanine
 cytosine
 uracil
NB RNA molecules does not have thymine but instead it has
uracil as the nitrogen base
Adenine and guanine are double ring bases and are called purines
 SCIENCE LABORATORY TECHNOLOGY
whereas cytosine , thymine and uracil are single ring bases called
pyrimidines

The nucleotide are linked

The Polymerization together by phosphodiester bond
RNA usually exist as a singe
Reaction strand that is composed of a long
sequence of nucleotide as shown
below .
DNA Usually exist as a double
strand . the two strands exist as a
double helix in which the two
chains or strands are in spiral
form and are coiled about one
another in a spiral .the two
polynucleotide strands are held
together by hydrogen bonds
between base in the two strands .
These therefore means that the
bases must pair up . The pairing
is specific i.e. adenine will pair
with thymine and guanine will
pair with cytosine . in RNA which
do not have thymine , uracil pairs
with adenine .
The base pairing is very specific ,
and due to these specificity, once

Figure 11.10
 SCIENCE LABORATORY TECHNOLOGY
the sequence of bases on one strand of DNA is known , then the
sequence of the other base in he second strand shall be known
automatically . The two strands of DNA are therefore said to be
complementary. Its these property of complementarity that makes the
DNA molecule to store and transmit genetic information . DNA is a
very stable molecule due to ;
(a) the large number of hydrogen bonds between the base pairs
(b) the hydrophobic bonding between the base pairs with each
other when present in aqueous solution
(c) covalent bonds between base and a sugar molecule or between
the sugar molecule and phosphate group
(d) the DNA strands are antiparallel and these contribute a lot to
their stability . they have opposite chemical polarity . the
phosphodiester bond in one strand is in the direction of
51 3 1 while on the other strand is
31 51

DNA REPLICATION
DNA is replicated in order to create a second DNA molecule that is
identical to the original one. DNA replication is generally bidirectional.
And it always starts from a distinct point along the strand. Replication
of double stranded DNA is semi conservative. I.e. each of the two DNA
molecules generated contains one of its original strand and a newly
synthesized strand. the process of replication requires the coordinated
action of many different set of complexes called replisome which
contain enzymes and other proteins e.g. DNA polymerase which is
responsible for synthesizing DNA using one strand as a template to
generate the complimentary strand.
 SCIENCE LABORATORY TECHNOLOGY

This enzyme can only add nucleotides onto the preexisting fragments
of nucleic acid called Okazaki fragments. These fragments serve as a
primer from which synthesis can continue. DNA polymerase always
elongates the chain in the 51 to 3I direction.
Replication process is very accurate. The accuracy is assured because
of its proof reading ability of some DNA polymerases which always
plays an important role in editing the chain formed

During replication , the template strand continue to unzip due to the

enzyme called helicase . synthesis of one new strand proceed
continously in the 51 to the 31 direction as thefresh DNA strand called
the leading strand is exposed from which the oposing strand called
the lagging strand is synthesized . The enzyme called DNA ligase
seals the gaps between fragments by catalyzing the formation of
covalent bonds between the adjacent nucleotides
 SCIENCE LABORATORY TECHNOLOGY

Mechanics of DNA Synthesis

RNA

RNA exist in three types ;

- messenger m RNA
- ribosomal RNA
- transfer t RNA
all these three types of RNA are synthesized directly from the DNA
molecule . They are all involved in protein synthesis in the cell
meaning that the amount of RNA in the cell depends on how much
protein that cell makes . DNA makes RNA and RNA makes proteins
 SCIENCE LABORATORY TECHNOLOGY
mesenger RNA
They make upto 3-5% of the total RNA in a cell .it’s a single stranded
molecule formed from a single strand of DNA in a process called
transcription . One strand of DNA is used for copping during the
formation of messenger RNA . the base sequence of the messenger
RNA is a complementary copy of the DNA being copied . Its length
usually varies i.e. some may be short or long depending on the length
of the protein that will be made .Most messenger RNA exist within the
cell for a very short time . They are synthesized in the nucleus and
they move out through the nuclear membrane pore into the cytoplasm
where they are used for protein synthesis
synthesis of m RNA
During the synthesis of m RNA ,the DNA of the double helix
unwinds by breaking of the weak hydrogen bond between the bases .
These is done using an enzyme called helicase .Only one strand of DNA
acts as a template . The messenger RNA is formed by linking of the
free nucleotide under the influence of enzyme called RNA
polymerase . The rules of the base pairing are as follows:
T-A
C-G
A-U
C-G
The base sequence of a section of a DNA representing a gene is
converted into the complementary base sequence of m RNA . When
the m RNA have been made , they leave the nucleus via the nuclear
pore and carry that genetic information to the ribosomes . After the
transcription , the DNA double helix unzips up again until when
another similar mRNA would be required
Ribosomal RNA (r RNA)
r RNA makes upto 80% of the total RNA in the cell . They are
synthesized by genes present I on the DNA of several chromosomes
found within a region of the nucleolus known as organizer . The base
sequence of rRNA is similar in all organisms including bacteria ,
higher plants and animals,
 SCIENCE LABORATORY TECHNOLOGY
rRNA is found in the cytoplasm where its linked with protein
molecule which together forms an organelle called ribosome , which
is the site for protein synthesis
Transfer RNA (t RNA )
They make upto 15 % of the total RNA in the cell . They are very mall
in size i.e. about 80 nucleotide . They have the same basic structure as
shown bellow. The 5 1 end of the t RNA always end in the base
guanine while 3 1 end always end in the base cytosine . At one end of
the t RNA are three bases reffered to as the anticodon which vary
from one tRNA to another .The anticodon base sequence is directly
related to the amino acid carried by t RNA . ( The base sequence
determines which amino acid by that tRNA ). tRNA transfers amino
acids present in the cytoplasm to the ribosomes , they act as an
intermediate between a set of 3 bases on mRNA and the amino acid
sequence of the protein . Each amino acid has its own specific family of
tRNA
 SCIENCE LABORATORY TECHNOLOGY

The structure of t RNA

DIFFERENCES BETWEEN RNA AND DNA

RNA DNA
Smaller molecular mass Larger molecular mass
May have a single or double helix Double helix
 SCIENCE LABORATORY TECHNOLOGY

pentose sugar is ribose Pentose sugar is deoxyribose

Organic bases present are guanine, Organic bases present are

cytosine, adenine, uracil Adenine, guanine, cytosine,
thymine

The ratio of adenine and uracil to cytosine

The ratio of adenine and thymine
and guanine varies to cytosine and guanine is one

Made in nucleus but found throughoutFound entirely in the cell nucleus

the cell cytoplasm

Amount of RNA varies from cell to cellAmount of DNA is constant for all
and within a cell according to metabolic
cells of a species except gametes
activity and spores

May be temporary Chemically stable

-Exist in three basic forms mRNA, tRNA
- Are permanent
rRNA
-in one basic form but with an
almost infinite variety within that
form

KARYOTYPES
Each chromosome is made up of two chromatids held together at a
point called centriomere . Chromosomes differ in sizes and location of
their centriomeres . If the 46 human chromosomes are prepared
and photographed just before cell division, enlarged , then cut out
from the photograph and lined out according to their sizes , It will be
realised that the chromosomes will be in 23 pairs . each pair will be
made up of two chromatids of the same size , same centriomere
 SCIENCE LABORATORY TECHNOLOGY
position and same gene position . such a pair is called homologous
chromosome .
A photograph of such an arrangement of human chromosomes is
called karyogram and the set of chromosomes is called karyotype .
The human karyogram shows 23 pairs of chromosomes . These is
because one set of chromosomes comes from the male and the other
set comes from the female partner during fertilization which later
forms 2 Sets of chromosomes
Out of the 23 pairs of chromosomes in the human cell, 22 pairs are
called autosomal and the remaining one pair is called sex
chromosomes . Autosomal chromosomes contain genes for general
body characteristics while sex chromosomes contain genes that will
determine sex and a few other sex linked characteristics . the human
male sex chromosomes i.e. the 23rd pairs are XY while the human
female chromosome are XX. the Y chromosome is more smaller
than X chromosome meaning that there are some genes that are
located in X that are not located in Y due to limited space .
DIPLOID AND HAPLOID CELLS
Diploid cells have two sets of chromosomes (2n) per nucleus or cell .
Majority of animal species and a half of plant species are diploid
cells . Haploid cells have one set of chromosomes (n) . A few simple
organisms are haploid but in higher animals , only the gametes cells
are haploid . polyploid cells have three or more sets of chromosomes
i.e. (3n , 4n ,5n etc) polyploidy is common in plant cells
There are many advantages of possessing 2 sets of chromosomes i.e. ;
(a) The genetic variation is increased – each individual cells will
have a mixture of characteristics from both parents . genetic
variation is as a result of different combination of genes
(b) If a gene of one set of the chromosome of a pair is faulty , the
second set of chromosome may provide a backup

PROTEIN SYNTHESIS
The sequence of bases in the DNA is a code for the sequence of amino
acid which will form a particular protein molecule. The relationship
between the bases on the DNA and the amino acid that will make a
 SCIENCE LABORATORY TECHNOLOGY
protein is known as the genetic code . a genetic code is a triplet code ,
these means that every three base on the DNA determines the position
of one amino acid in the protein .
There are four different types of base on the DNA i.e. G ,C,T and A
.each base is part of the nucleotide and the many nucleotides make up
a polynucleotide . a polynucleotide represent a strand of DNA .
There are about 20 amino acid which are used to make proteins ,
these means that the base on the DNA stand must be able to code for
these 20 Amino acids .if one base determines the position of a single
amino acid in the protein structure ,then the protein could only be
made of only four amino acids . these could mean that the 16 different
amino acids could not be used .therefore the genetic code is not based
on one base for one amino acid .
If two bases on the DNA strand determines the position of the amino
acid i.e. 2 bases coding for one amino acid , then only 16 amino acids
would be used to make the protein , these means that 4different
amino acids would not be used out of 20. Thereore the genetic code
could not be consisting of two bases coding for one amino acid

A G T C
A AA AG AT AC
G GA GG GT GC
T TA TG TT TC
C CA CG CT CC

If three bases determine or codefor one amino acid , all the 20

aminoacids would be incoperated since there would be 64
combination of bases which would be more than enough i.e.
SCIENCE LABORATORY TECHNOLOGY
 SCIENCE LABORATORY TECHNOLOGY
From the above observation , Watson and Crick predicted that the
genetic code could be made of triplet code . But having established that
the genetic code was indeed a triplet code , the task remaining was to
find which three bases coded for which amino acid . this process was
refered to as the breaking code .
To break the code , an outline of protein synthesis is required ( i.e.
knowing the process involved in protein synthesis ) protein synthesis
involve DNA and RNA
 There are three types of RNA involved i.e. the m RNA ,t RNA and r
RNA
 DNA is transcribed or ( copied ) on to m RNA ,
 The m RNA leaves the nucleus and moves to the cytoplasm through
the nuclear pore where it becomes attached to the ribosomes .
 After attachment to the ribosomes , the base sequence becomes
translated onto the amino acid sequence (translation)
 The t RNA becomes attached or linked up with the
complementary triplet bases on the m RNA
 THE adjacent amino acids link to form a bond between them
making a polypeptide
 These process requires DNA molecules , m RNA , ribosomes ,
t RNA , amino acids , ATP , enzymes , co- factors , co enzymes ,
initiation factors (if) and termination factors
Nirrenberg used the above information to break the genetic code .He
used a known base sequence of m RNA to analyze the amino acid
sequence of the polypeptide that would be produced . He started his
experiment by synthesizing an m RNA that consisted of the same
triplets UUU ( such a triplet is called poly U or poly uridylic acid ) ,
He then prepared a seriesof 20 test tubes containing ribosomes , t
RNA , ATP , enzymes , co -factors , etc and he also incoperated a
specific radioactive labled amino acid in each test tube . poly U m
RNA was added to each test tube and time was allowed for synthesis
of polypeptide . analysis was then made for each test tube and it was
shown that the polypeptide formed only in the test tube containing
the amino acid phenylanine . these indicated that the triplet UUU was
coding for phenylanine in the polypeptide . the whole procedure was
repeated for the other 64 combination and by the year 1964 , the
triplet code for the 20 amino acids was done
Features of the genetic codes
 SCIENCE LABORATORY TECHNOLOGY
 All genetic codes are triplets
 They are universal i.e. the same triplets codes for the same amino
acid in all organisms
 They are degenerate i.e. a given amino acid can be coded for by
more than one triplet base sequence e.g. ACA and ACG encode the
amino acid threonine
 They are non –overlapping i.e. no base of a given triplet contribute
to part of the next adjacent triplet
 genetic codes are punctuated i.e. there are triplet codes reffered to
as non –sense triplet codes which do not code for any amino acid .
Existance of such a point on the m RNA act as a break and its
probably a signal to stop making a polypeptide during translation
genetic information is transferred from DNA to DNA during DNA
replication and from DNA to RNA during transcription . These
information can also be translated from RNA to polypeptides in
ribosomes . Therefore proteins synthesis involve transfer of genetic
information from DNA to RNA to proteins
The m RNA is a single strand formed from a single strand of DNA
during transcription
r RNA is synthesized by genes present in a certain region of
nucleoulus . These genes are known as nucleolar organizer. rRNA is
found in the cytoplasm where it is associated or combined with
protein molecules to form ribosomes . Ribosomes are the site for
protein synthesis where
m RNA attaches during translation. The t RNA transfers amino
acids present in the cytoplasm to the ribosomes. Each amino acid
have its own group of t RNA that carry it . such t RNA are known as
a family of t RNA specialized to carry a particular amino acid . they
act as intermediate molecules between the triplets codes of mRNA
and the amino acid sequence . There are more than 20 tRNA in a given
cell carrying a specific amino acid .
structure of t RNA
An anticodon represent three bases that are complementary to the
three bases on the m RNA (codon) . the 5 I end of the t RNA ends in
the base guanine while the 3 1 end ends in the base sequence CCA .
 SCIENCE LABORATORY TECHNOLOGY
The anticodon is directly related to the amino acid that is going to be
carried by that t RNA . These explains why t RNA is specific .each
amino acid is attached to the t RNA by an enzyme called amino –
acyl t RNA synthetase . The complex of the amino acid and t RNA
is reffered to as amino acyl – t RNA complex
Transcription is the process of synthesis of mRNA from DNA . It’s
the first step of protein synthesis. Transcription begins when RNA
polymerase enzyme and binds to the promoter region on the double
stranded DNA molecule . the binding melts a short stretch of DNA
creating a region of exposed nucleotides that serve as templates for
RNA synthesis. A particular sub unit of RNA polymerase i.e. sigma
(σ) factor (which is only a loose component of the enzyme )
recognizes the promoter region prior to initiation of transcription .
After transcription is initiated , the sigma factor dissociates from the
enzyme leaving the remaining portion of the RNA polymerase called
core enzyme to complete the transcription . A cell may have different
types of sigma factors that represent different promoters .
The RNA polymerase moves along the template strand of DNA
synthesizing the complementary single stranded RNA molecule in the
51 to 31 direction as the enzyme adds nucleotides to 31OH group at the
end of the growing chain. The core RNA polymerase enzyme advances
along the DNA melting a new stretch and allowing the previous
stretch to close . these exposes a new region of the DNA strand h thus
permiting the elongation process to continue . A single gene can be
transcribed multiple times in a short time interval.
When RNA polymerase encounters a terminator , it falls off the DNA
template and releases the newly synthesized RNA . The terminator is a
sequence of nucleotide in the DNA that when transcribed permits
the complimentary regions of the resulting RNA to base –pair
forming a hair pin loop structure . These cause the RNA polymerase to
stall or stop resulting in its dissociation from DNA template to release
off the RNA
Translation is the process of decoding the information carried on
the m RNA to synthesize the specific proteins . it’s a process by which
the sequence of the bases on m RNA are converted into a sequence of
amino acids in a polypeptide chain . Proteins are synthesized by
adding amino acids sub units sequentially to the ribosomes . Several
 SCIENCE LABORATORY TECHNOLOGY
ribosomes marked become attached to one mRNA forming a
polyribosome or polysome . These arrangement is advantageous
because several polypeptides can be synthesized at the same time .
Each ribosome is composed of small and large subunits represented as
50 S and 30 S respectively .
The process of translation begins as the m RNA is still being
synthesized . The 30 S subunit of the ribosome binds to the sequence
in m RNA called the ribosome- binding site . The 1st time the
codon for methionine AUG appears after the site , translation
generally starts . infact the UAG function as a start codon especially
when preceded by by ribosome – binding site .The position of the 1st
AUG is very critical as it determines the reading frame used for
translation of the remainder of that protein . AHt these UAG ,
ribosomes begin to assemble and several other initiation complexes
forms here which shall cause the elongation of the protein molecule to
take
place . The elongation of the polypeptide terminates when the
ribosomes reaches a stop codon . These is a codon that do not code for
an amino acid and is not recognized by t RNA . At these point , an
enzyme called release factor free the newly synthetized polypeptide
by breaking the covalent bond that joins it to t RNA
The proteins must be modified after they are synthesized e.g. some
proteins require the assistance of another protein called chaperon
to fold into the final functional shape
Mendelian inheritance
Mendelian inheritance or Mendelism is a set of primary tenets
relating to the transmission of hereditary characteristics from parent
organisms to their offspring; it underlies much of genetics. They were
initially derived from the work of Gregor Johann Mendel published in
1865 and 1866 which was "re-discovered" in 1900, and were initially
very controversial. When they were integrated with the chromosome
theory of inheritance by Thomas Hunt Morgan in 1915, they became
the core of classical genetics.
The laws of inheritance were derived by Gregor Mendel,a 19th century
Austrian Priest/monk conducting hybridization experiments in garden
peas (Pisum sativum). Between 1856 and 1863, he cultivated and
 SCIENCE LABORATORY TECHNOLOGY
tested some 29,000 pea plants. From these experiments he deduced
two generalizations which later became known as Mendel's Principles
of Heredity or Mendelian inheritance. Mendel's conclusions were
largely ignored. Although they were not completely unknown to
biologists of the time, they were not seen as generally applicable, even
by Mendel himself, who thought they only applied to certain categories
of species or traits.

Mendel's Laws
Mendel discovered that when crossing white flower and purple flower
plants, the result is not a blend. Rather than being a mix of the two, the
offspring was purple flowered. He then conceived the idea of heredity
units, which he called "factors", one of which is a recessive
characteristic and the other dominant. Mendel said that factors, later
called genes, normally occur in pairs in ordinary body cells, yet
segregate during the formation of sex cells. Each member of the pair
becomes part of the separate sex cell. The dominant gene, such as the
purple flower in Mendel's plants, will hide the recessive gene, the white
flower. After Mendel self-fertilized the F1 generation and obtained the
3:1 ratio, he correctly theorized that genes can be paired in three
different ways for each trait: AA, aa, and Aa. The capital "A" represents
the dominant factor and lowercase "a" represents the recessive. (The
last combination listed above, Aa, will occur roughly twice as often as
each of the other two, as it can be made in two different ways, Aa or
aA.)
Mendel stated that each individual has two factors for each trait, one
from each parent. The two factors may or may not contain the same
information. If the two factors are identical, the individual is called
homozygous for the trait. If the two factors have different information,
the individual is called heterozygous. The alternative forms of a factor
are called alleles. The genotype of an individual is made up of the many
alleles it possesses. An individual's physical appearance, or phenotype,
is determined by its alleles as well as by its environment. An individual
possesses two alleles for each trait; one allele is given by the female
parent and the other by the male parent. They are passed on when an
individual matures and produces gametes: egg and sperm. When
gametes form, the paired alleles separate randomly so that each gamete
receives a copy of one of the two alleles. The presence of an allele
 SCIENCE LABORATORY TECHNOLOGY
doesn't promise that the trait will be expressed in the individual that
possesses it. In heterozygous individuals the only allele that is
expressed is the dominant. The recessive allele is present but its
expression is hidden.
Mendel summarized his findings in two laws; the Law of
Segregation and the Law of Independent Assortment.

Law of Segregation (The "First Law")

The Law of Segregation states that when any individual produces
gametes, the copies of a gene separate so that each gamete receives
only one copy. A gamete will receive one allele or the other. The direct
proof of this was later found following the observation of meiosis by
two independent scientists, the German botanist, Oscar Hertwig in
1876, and the Belgian zoologist, Edouard Van Beneden in 1883. In
meiosis, the paternal and maternal chromosomes get separated and the
alleles with the traits of a character are segregated into two different
gametes.
OR

The two coexisting alleles of an individual for each trait segregate

(separate) during gamete formation so that each gamete gets only one
of the two alleles. Alleles again unite at random fertilization of gametes.
 SCIENCE LABORATORY TECHNOLOGY

Law of Independent Assortment (The "Second Law")

The Law of Independent Assortment, also known as "Inheritance Law"
states that alleles of different genes assort independently of one
another during gamete formation. While Mendel's experiments with
mixing one trait always resulted in a 3:1 ratio (Fig. 1) between
dominant and recessive phenotypes, his experiments with mixing two
 SCIENCE LABORATORY TECHNOLOGY
traits (dihybrid cross) showed 9:3:3:1 ratios.
 SCIENCE LABORATORY TECHNOLOGY

But the 9:3:3:1 table shows that each of the two genes are
independently inherited with a 3:1 phenotypic ratio. Mendel concluded
that different traits are inherited independently of each other, so that
there is no relation, for example, between a cat's color and tail length.
This is actually only true for genes that are not linked to each other.
Independent assortment occurs during meiosis I in eukaryotic
organisms, specifically metaphase I of meiosis, to produce a gamete
with a mixture of the organism's maternal and paternal chromosomes.
Along with chromosomal crossover, this process aids in increasing
genetic diversity by producing novel genetic combinations.
Of the 46 chromosomes in a normal diploid human cell, half are
maternally-derived (from the mother's egg) and half are paternally-
derived (from the father's sperm). This occurs as sexual reproduction
involves the fusion of two haploid gametes (the egg and sperm) to
produce a new organism having the full complement of chromosomes.
During gametogenesis—the production of new gametes by an adult—
the normal complement of 46 chromosomes needs to be halved to 23
to ensure that the resulting haploid gamete can join with another
gamete to produce a diploid organism. An error in the number of
chromosomes, such as those caused by a diploid gamete joining with a
haploid gamete, is termed aneuploidy.
In independent assortment the chromosomes that end up in a newly-
formed gamete are randomly sorted from all possible combinations of
maternal and paternal chromosomes. Because gametes end up with a
random mix instead of a pre-defined "set" from either parent, gametes
are therefore considered assorted independently. As such, the gamete
can end up with any combination of paternal or maternal
chromosomes. Any of the possible combinations of gametes formed
from maternal and paternal chromosomes will occur with equal
frequency. For human gametes, with 23 pairs of chromosomes, the
number of possibilities is 223 or 8,388,608 possible combinations. The
gametes will normally end up with 23 chromosomes, but the origin of
 SCIENCE LABORATORY TECHNOLOGY
any particular one will be randomly selected from paternal or maternal
chromosomes. This contributes to the genetic variability of progeny.
The reason for these laws is found in the nature of the cell nucleus. It is
made up of several chromosomes carrying the genetic traits. In a
normal cell, each of these chromosomes has two parts, the chromatids.
A reproductive cell, which is created in a process called meiosis, usually
contains only one of those chromatids of each chromosome. By
merging two of these cells (usually one male and one female), the full
set is restored and the genes are mixed. The resulting cell becomes a
new embryo. The fact that this new life has half the genes of each
parent (23 from mother, 23 from father for total of 46 in the case of
humans) is one reason for the Mendelian laws. The second most
important reason is the varying dominance of different genes, causing
some traits to appear unevenly instead of averaging out (whereby
dominant doesn't mean more likely to reproduce—recessive genes can
become the most common, too).
There are several advantages of this method (sexual reproduction) over
reproduction without genetic exchange:

1. Instead of nearly identical copies of an organism, a broad

range of offspring develops, allowing more different abilities and
evolutionary strategies.
2. There are usually some errors in every cell nucleus. Copying
the genes usually adds more of them. By distributing them randomly
over different chromosomes and mixing the genes, such errors will be
distributed unevenly over the different children. Some of them will
therefore have only very few such problems. This helps reduce
problems with copying errors somewhat.
3. Genes can spread faster from one part of a population to
another. This is for instance useful if there's a temporary isolation of
two groups. New genes developing in each of the populations don't get
reduced to half when one side replaces the other, they mix and form a
population with the advantages of both sides.
4. Sometimes, a mutation can have positive side effects. For
example, sickle cell anemia is a mutation that can causethe benefit of
malaria resistance. The mechanism behind the Mendelian laws can
make it possible for some offspring to carry the advantages without the
disadvantages until further mutations solve the problems.
 SCIENCE LABORATORY TECHNOLOGY
Mendelian trait
A Mendelian trait is one that is controlled by a single locus and
shows a simple Mendelian inheritance pattern. In such cases, a
mutation in a single gene can cause a disease that is inherited
according to Mendel's laws. Examples include sickle-cell anemia, Tay-
Sachs disease, cystic fibrosis and xeroderma pigmentosa. A disease
controlled by a single gene contrasts with a multi-factorial disease, like
arthritis, which is affected by several loci (and the environment) as well
as those diseases inherited in a non-Mendelian fashion. The Mendelian
Inheritance in Man database is a catalog of, among other things, genes
in which Mendelian traits cause disease.

Genetic linkage
Genetic linkage occurs when particular genetic loci or alleles for
genes are inherited jointly. Genetic loci on the same chromosome are
physically connected and tend to stay together during meiosis, and are
thus genetically linked. Alleles for genes on different chromosomes are
usually not linked, due to independent assortment of chromosomes
during meiosis.
 SCIENCE LABORATORY TECHNOLOGY

Linkage Prevents
Independent Assortment

Figure 5.1

Because there is some crossing over of DNA when the chromosomes

segregate, alleles on the same chromosome can be separated and go to
different daughter cells. There is a greater probability of this happening
if the alleles are far apart on the chromosome, as it is more likely that a
 SCIENCE LABORATORY TECHNOLOGY
cross-over will occur between them.

The relative distance between two genes can be calculated using the
offspring of an organism showing two linked genetic traits, and finding
the percentage of the offspring where the two traits do not run
together. The higher the percentage of descendants that does not show
both traits, the further apart on the chromosome they are.
Among individuals of an experimental population or species, some
phenotypes or traits occur randomly with respect to one another in a
manner known as independent assortment. Today scientists
understand that independent assortment occurs when the genes
affecting the phenotypes are found on different chromosomes or
separated by a great enough distance on the same chromosome that
recombination occurs at least half of the time.
An exception to independent assortment develops when genes appear
near one another on the same chromosome. When genes occur on the
same chromosome, they are usually inherited as a single unit. Genes
inherited in this way are said to be linked, and are referred to as
 SCIENCE LABORATORY TECHNOLOGY
"linkage groups." For example, in fruit flies the genes affecting eye
color and wing length are inherited together because they appear on
the same chromosome.
But in many cases, even genes on the same chromosome that are
inherited together produce offspring with unexpected allele
combinations. This results from a process called crossing over. At the
beginning of normal meiosis, a chromosome pair (made up of a
chromosome from the mother and a chromosome from the father)
intertwine and exchange sections or fragments of chromosome. The
pair then breaks apart to form two chromosomes with a new
combination of genes that differs from the combination supplied by the
parents. Through this process of recombining genes, organisms can
produce offspring with new combinations of maternal and paternal
traits that may contribute to or enhance survival.
Genetic linkage was first discovered by the British geneticists William
Bateson and Reginald Punnett shortly after Mendel's laws were
rediscovered.

Linkage mapping
The observations by Thomas Hunt Morgan that the amount of crossing
over between linked genes differs led to the idea that crossover
frequency might indicate the distance separating genes on the
chromosome. Morgan's student Alfred Sturtevant developed the first
genetic map, also called a linkage map.
Sturtevant proposed that the greater the distance between linked
genes, the greater the chance that non-sister chromatids would cross
over in the region between the genes. By working out the number of
recombinants it is possible to obtain a measure for the distance
between the genes. This distance is called a genetic map unit
(m.u.), or a centimorgan and is defined as the distance between
genes for which one product of meiosis in 100 is recombinant. A
recombinant frequency (RF) of 1 % is equivalent to 1 m.u. A linkage
map is created by finding the map distances between a number of traits
that are present on the same chromosome, ideally avoiding having
significant gaps between traits to avoid the inaccuracies that will occur
due to the possibility of multiple recombination events.
 SCIENCE LABORATORY TECHNOLOGY
Linkage mapping is critical for identifying the location of genes that
cause genetic diseases. In an ideal population, genetic traits and
markers will occur in all possible combinations with the frequencies of
combinations determined by the frequencies of the individual genes.
For example, if alleles A and a occur with frequency 90% and 10%, and
alleles B and b at a different genetic locus occur with frequencies 70%
and 30%, the frequency of individuals having the combination AB
would be 63%, the product of the frequencies of A and B, regardless of
how close together the genes are. However, if a mutation in gene B that
causes some disease happened recently in a particular subpopulation,
it almost always occurs with a particular allele of gene A if the
individual in which the mutation occurred had that variant of gene A
and there have not been sufficient generations for recombination to
happen between them (presumably due to tight linkage on the genetic
map). In this case, called linkage disequilibrium, it is possible to search
potential markers in the subpopulation and identify which marker the
mutation is close to, thus determining the mutation's location on the
map and identifying the gene at which the mutation occurred. Once the
gene has been identified, it can be targeted to identify ways to mitigate
the disease.

Linkage map
A linkage map is a genetic map of a species or experimental population
that shows the position of its known genes and/or genetic markers
relative to each other in terms of recombination frequency, rather than
as specific physical distance along each chromosome.
A genetic map is a map based on the frequencies of recombination
between markers during crossover of homologous chromosomes. The
greater the frequency of recombination (segregation) between two
genetic markers, the farther apart they are assumed to be. Conversely,
the lower the frequency of recombination between the markers, the
smaller the physical distance between them. Historically, the markers
originally used were detectable phenotypes (enzyme production, eye
color) derived from coding DNA sequences; eventually, confirmed or
assumed noncoding DNA sequences such as microsatellites or those
generating restriction fragment length polymorphisms (RFLPs) have
been used.
 SCIENCE LABORATORY TECHNOLOGY
Genetic maps help researchers to locate other markers, such as other
genes by testing for genetic linkage of the already known markers.
A genetic map is not a physical map or gene map.

LOD score method for estimating recombination frequency

The LOD score (logarithm (base 10) of odds, also called logit by
mathematicians) is a statistical test often used for linkage analysis in
human populations, and also in animal and plant populations. The test
was developed by Newton E. Morton. Computerized LOD score
analysis is a simple way to analyze complex family pedigrees in order to
determine the linkage between Mendelian traits (or between a trait and
a marker, or two markers).
The method is described in greater detail by Strachan and Read.
Briefly, it works as follows:

1. Establish a pedigree
2. Make a number of estimates of recombination frequency
3. Calculate a LOD score for each estimate
4. The estimate with the highest LOD score will be considered
the best estimate

The LOD score is calculated as follows:

NR denotes the number of non-recombinant offspring, and R denotes

the number of recombinant offspring. The reason 0.5 is used in the
denominator is that any alleles that are completely unlinked (e.g.
alleles on separate chromosomes) have a 50% chance of
recombination, due to independent assortment.
Theta is the recombinant fraction, that is to say it is equal to R / (NR +
R)
 SCIENCE LABORATORY TECHNOLOGY
In practice, LOD scores are looked up in a table which lists LOD scores
for various standard pedigrees and various values of recombination
frequency.
By convention, a LOD score greater than 3.0 is considered evidence for
linkage. (A score of 3.0 means the likelihood of observing the given
pedigree if the two loci are not linked is less than 1 in 1000). On the
other hand, a LOD score less than -2.0 is considered evidence to
exclude linkage. Although it is very unlikely that a LOD score of 3
would be obtained from a single pedigree, the mathematical properties
of the test allow data from a number of pedigrees to be combined by
summing the LOD scores.

Recombination frequency
Recombination frequency (θ) is the frequency that a chromosomal
crossover will take place between two loci (or genes) during meiosis.
Recombination frequency is a measure of genetic linkage and is used
in the creation of a genetic linkage map. A centimorgan (cM) is a
unit that describes a recombination frequency of 1%.
During meiosis, chromosomes assort randomly into gametes, such that
the segregation of alleles of one gene is independent of alleles of
another gene. This is stated in Mendel's Second Law and is known as
the law of independent assortment. The law of independent
assortment always holds true for genes that are located on different
chromosomes, but for genes that are on the same chromosome, it does
not always hold true.
As an example of independent assortment, consider the crossing of the
pure-bred homozygote parental strain with genotype AABB with a
different pure-bred strain with genotype aabb. A and a and B and b
represent the alleles of genes A and B. Crossing these homozygous
parental strains will result in F1 generation offspring with genotype
AaBb. The F1 offspring AaBb produces gametes that are AB, Ab, aB,
and ab with equal frequencies (25%) because the alleles of gene A
assort independently of the alleles for gene B during meiosis. Note that
2 of the 4 gametes (50 %)—Ab and aB—were not present in the
parental generation. These gametes represent recombinant
gametes. Recombinant gametes are those gametes that differ from
both of the haploid gametes that made up the diploid cell. In this
 SCIENCE LABORATORY TECHNOLOGY
example, the recombination frequency is 50% since 2 of the 4 gametes
were recombinant gametes.
The recombination frequency will be 50% when two genes are located
on different chromosomes or when they are widely separated on the
same chromosome. This is a consequence of independent assortment.
When two genes are close together on the same chromosome, they do
not assort independently and are said to be linked. Whereas genes
located on different chromosomes assort independently and have a
recombination frequency of 50%, linked genes have a recombination
frequency that is less than 50%.
As an example of linkage, consider the classic experiment by William
Bateson and Reginald Punnett. They were interested in trait
inheritance in the sweet pea and were studying two genes—the gene for
flower color (P, purple, and p, red) and the gene affecting the shape of
pollen grains (L, long, and l, round). They crossed the pure lines PPLL
and ppll and then self-crossed the resulting PpLl lines. According to
Mendelian genetics, the expected phenotypes would occur in a 9:3:3:1
ratio of PL:Pl:pL:pl. To their surprise, they observed an increased
frequency of PL and pl and a decreased frequency of Pl and pL (see
table below).

Bateson and Punnett experiment

Phenotype and genotype

Observed
Expected from 9:3:3:1
 SCIENCE LABORATORY TECHNOLOGY

ratio

Purple, long (P_L_) 284 216

Purple, round (P_ll) 21 72

Red, long (ppL_) 21 72

Red, round (ppll) 55 24

Their experiment revealed linkage between the P and L alleles and the
p and l alleles. The frequency of P occurring together with L and with p
occurring together with l is greater than that of the recombinant Pl and
pL. The recombination frequency cannot be computed directly from
this experiment, but intuitively it is less than 50%.
The progeny in this case received two dominant alleles linked on one
chromosome (referred to as coupling or cis arrangement).
However, after crossover, some progeny could have received one
parental chromosome with a dominant allele for one trait (eg Purple)
linked to a recessive allele for a second trait (eg round) with the
opposite being true for the other parental chromosome (eg red and
Long). This is referred to as repulsion or a trans arrangement. The
phenotype here would still be purple and long but a test cross of this
individual with the recessive parent would produce progeny with much
greater proportion of the two crossover phenotypes. While such a
problem may not seem likely from this example, unfavorable repulsion
linkages do appear when breeding for disease resistance in some crops.
When two genes are located on the same chromosome, the chance of a
crossover producing recombination between the genes is directly
related to the distance between the two genes. Thus, the use of
 SCIENCE LABORATORY TECHNOLOGY
recombination frequencies has been used to develop linkage maps or
genetic maps.

MUTATION AND VARIATION

Mutation is defined as a sudden or sponteneous change in the
amount , arrangement or structure of DNA of an organism .the effect
of these changes is that it produces a change in the genotype (genetic
constitution of an organism ) which may be inherited by cells derived
by mitosis or meiosis from the cells that underwent mutation (mutant
cells) . a mutant reffers to an organism or an individual cell which
exhibit a phenotypical characteristic which is as a result of a particular
mutation ..the term mutation reffers to both the changes in the
genetic material and also to the process by which the change occurs
Sponteneous mutation occurs in the natural environment event in
the absence of the agents called mutagents that are known to cause
mutation . Mutations are rare and they occur randomly, but each gene
mutates sponteneously at a characteristic frequency . The rate of
mutation is defined as the probability that a mutation will be
observed in a given gene each time a cell divides . Mutation rates of
different genes usually varies between 10 –4 to 10 –12 per cell
divisions . spontenous mutation could be due to inherent low level of
metabolic errors e.g. mistakes during DNA replication . such mistakes
could be a a result of changes in temperature , PH level , high
concentration of waste products etc or due to mutagenic agents
present in the environment . in 1920 , H.J muller showed that the
frequency of mutation could be increased or induced a bove a
sponteneous level . induced mutation results from exposure of
organisms to mutagenic agents that cause mutation such as ionizing
radiations e.g. UV light from sun, X –rays , gamma rays , alpha ,
beta and cosmic particles and some chemical substances such as
mastered gas , caffein , formaldehyde , choloroform , colchicine , some
drugs , food preservatives and pesticides .
there exist two types of mutation i.e. somatic mutation and
chromosomal mutation . somatic mutation or abberations are
mutations which occur in normal body cells (somatic cells). Such
 SCIENCE LABORATORY TECHNOLOGY
mutations are not passed from one cell to the daughter cell ,they are
not inheritable i.e. they are not passed from one generation to
another .
Chromosomal mutation or chromosomal abberetion reffers to
mutations resulting from a change in the amount of arrangement of
DNA in the cell ( a change that result to an extra or less the total
number of chromosomes ), or as a result of a part of a chromatid
breaking away and getting lost . the deletion or removal of one or
several nucleotides changes the nucleotide sequence, these affect the
translation of genes by shifting the order of gene arrangements from
their original positions especially when its transcribed to m RNA .
these type of mutation is called frame shift mutation. most common
types of mutation results from mistakes during DNA synthesis .
when an incorrect base is incoperated into a DNA , an event called
base substitution occurs , if only one base pair is changed , then the
mutation is called point mutation occurs . point mutation is a
term that is frequently being used when describing individual genes or
changes in the DNA sequence at a single locus or point . they involve
changes in a single base pair or substituting a base pair with
another or removing a particular base out of the DNA sequence or
repeating a particular base sequence. A mutation which results in the
substitution of different amino acids is reffered to as missence
mutation .in many cases , incoperation of an incorrect amino acid
results in the synthesis of a protein that is partially functional e.g.
mutation in the gene that codes for tryptophan biosynthesis may
resulting a strain that grows slowly in absence of tryptophan . a
mutation that changes a codon that normally encodes an amino acid
to a stop codon is called nonsense mutation and any mutation that
inactivate the gene resulting in a strain that is unable to grow at all
unless tryptophan is added is called null or knockout mutation
the concept of mutation as the cause of the sudden or sponteneous
appearance of new characteristics was first proposed by a Dutch
botanist Hugo de vries in 1901 and latter in 1910 by T.H Morgan who
investigated these concept using drosophila . it have now been
established that mutation is the ultimate source of all genetic
variations and that mutation provides a raw material for evolution .
advantageous mutation makes an organism to acquire advantageous
characteristics that will promote the survival of the organism in
 SCIENCE LABORATORY TECHNOLOGY
different environment .however , most mutation are disadvantageous
because they could disrupt the genetic material and its transmission
In multicellular organisms with dedicated reproductive cells,
mutations can be subdivided into germ line mutations, which can
be passed on to descendants through the reproductive cells, and
somatic mutations, which involve cells outside the dedicated
reproductive group and which are not usually transmitted to
descendants. If the organism can reproduce asexually through
mechanisms such as cuttings or budding the distinction can become
blurred. For example, plants can sometimes transmit somatic
mutations to their descendants asexually or sexually where flower buds
develop in somatically mutated parts of plants. A new mutation that
was not inherited from either parent is called a de novo mutation.
The source of the mutation is unrelated to the consequence, although
the consequences are related to which cells are affected.
Mutations create variation within the gene pool. Less favorable (or
deleterious) mutations can be reduced in frequency in the gene pool by
natural selection, while more favorable (beneficial or advantageous)
mutations may accumulate and result in adaptive evolutionary
changes. For example, a butterfly may produce offspring with new
mutations. The majority of these mutations will have no effect; but one
might change the color of one of the butterfly's offspring, making it
harder (or easier) for predators to see. If this color change is
advantageous, the chance of this butterfly surviving and producing its
own offspring are a little better, and over time the number of
butterflies with this mutation may form a larger percentage of the
population.
Neutral mutations are defined as mutations whose effects do not
influence the fitness of an individual. These can accumulate over time
due to genetic drift. It is believed that the overwhelming majority of
mutations have no significant effect on an organism's fitness. Also,
DNA repair mechanisms are able to mend most changes before they
become permanent mutations, and many organisms have mechanisms
for eliminating otherwise permanently mutated somatic cells.
Mutation is generally accepted by the scientific community as the
mechanism upon which natural selection acts, providing the
 SCIENCE LABORATORY TECHNOLOGY
advantageous new traits that survive and multiply in offspring or
disadvantageous traits that die out with weaker organisms.

Classification of mutations

By effect on structure
The sequence of a gene can be altered in a number of ways. Gene
mutations have varying effects on health depending on where they
occur and whether they alter the function of essential proteins.
Structurally, mutations can be classified as:

Small-scale mutations, such those as affecting a small gene in one or a

few nucleotides, including:

Point mutations, often caused by chemicals or malfunction of DNA

replication, exchange a single nucleotide for another. Most common is
the transition that exchanges a purine for a purine (A ↔ G) or a
pyrimidine for a pyrimidine, (C ↔ T). A transition can be caused by
nitrous acid, base mis-pairing, or mutagenic base analogs such as 5-
bromo-2-deoxyuridine (BrdU). Less common is a transversion,
which exchanges a purine for a pyrimidine or a pyrimidine for a purine
(C/T ↔ A/G). A point mutation can be reversed by another point
mutation, in which the nucleotide is changed back to its original state
(true reversion) or by second-site reversion (a complementary
mutation elsewhere that results in regained gene functionality). These
changes are classified as transitions or transversions. An example of a
transversion is adenine (A) being converted into a cytosine (C). There
are also many other examples that can be found. Point mutations that
occur within the protein coding region of a gene may be classified into
three kinds, depending upon what the erroneous codon codes for:
Silent mutations: which code for the same amino acid. Missense
mutations: which code for a different amino acid.

Nonsense mutations: which code for a stop and can truncate the
protein.
 SCIENCE LABORATORY TECHNOLOGY

Point Mutations in Coding

Sequences

Missense – changes amino acid Frameshift – alters

Nonsense – creates stop codon remainder of reading frame
results in completely
different amino acid
sequence.

Insertions add one or more extra nucleotides into the DNA. They are
usually caused by transposable elements, or errors during replication
of repeating elements (e.g. AT repeats). Insertions in the coding region
of a gene may alter splicing of the mRNA (splice site mutation), or
cause a shift in the reading frame (frameshift), both of which can
significantly alter the gene product. Insertions can be reverted by
excision of the transposable element.

Deletions remove one or more nucleotides from the DNA. Like

insertions, these mutations can alter the reading frame of the gene.
They are generally irreversible: though exactly the same sequence
might theoretically be restored by an insertion, transposable elements
able to revert a very short deletion (say 1–2 bases) in any location are
either highly unlikely to exist or do not exist at all. Note that a deletion
is not the exact opposite of an insertion: the former is quite random
 SCIENCE LABORATORY TECHNOLOGY
while the latter consists of a specific sequence inserting at locations
that are not entirely random or even quite narrowly defined. Large-
scale mutations in chromosomal structure, including:

Amplifications (or gene duplications) leading to multiple copies of

all chromosomal regions, increasing the dosage of the genes located
within them.

Deletions of large chromosomal regions, leading to loss of the genes

within those regions.

Mutations whose effect is to juxtapose previously separate pieces of

DNA, potentially bringing together separate genes to form functionally
distinct fusion genes (e.g. bcr-abl). These include:

Chromosomal translocations: interchange of genetic parts from

nonhomologous chromosomes.

Interstitial deletions: an intra-chromosomal deletion that removes

a segment of DNA from a single chromosome, thereby apposing
previously distant genes. For example, cells isolated from a human
astrocytoma, a type of brain tumor, were found to have a chromosomal
deletion removing sequences between the "fused in glioblastoma" (fig)
gene and the receptor tyrosine kinase "ros", producing a fusion protein
(FIG-ROS). The abnormal FIG-ROS fusion protein has constitutively
active kinase activity that causes oncogenic transformation (a
transformation from normal cells to cancer cells).

Chromosomal inversions: reversing the orientation of a

chromosomal segment.

Loss of heterozygosity: loss of one allele, either by a deletion or

recombination event, in an organism that previously had two different
alleles.
 SCIENCE LABORATORY TECHNOLOGY
By effect on function

Loss-of-function mutations are the result of gene product having

less or no function. When the allele has a complete loss of function
(null allele) it is often called an amorphic mutation. Phenotypes
associated with such mutations are most often recessive. Exceptions
are when the organism is haploid, or when the reduced dosage of a
normal gene product is not enough for a normal phenotype (this is
called haploinsufficiency).
Gain-of-function mutations change the gene product such that it
gains a new and abnormal function. These mutations usually have
dominant phenotypes. Often called a neomorphic mutation.
Dominant negative mutations (also called antimorphic
mutations) have an altered gene product that acts antagonistically to
the wild-type allele. These mutations usually result in an altered
molecular function (often inactive) and are characterised by a
dominant or semi-dominant phenotype. In humans, Marfan syndrome
is an example of a dominant negative mutation occurring in an
autosomal dominant disease. In this condition, the defective
glycoprotein product of the fibrillin gene (FBN1) antagonizes the
product of the normal allele.
 Lethal mutations are mutations that lead the death of the
organisms which carry the mutations.
 A back mutation or reversion is a point mutation that restores
the original sequence and hence the original phenotype.

 By inheritance

inheritable generic in pro-generic tissue or cells on path to be changed

to gametes.

non inheritable somatic (eg, cancerogenic mutation)

non inheritable post mortem aDNA mutation in decaing remains.

 By pattern of inheritance
The human genome contains two copies of each gene – a paternal and
a maternal allele.
 SCIENCE LABORATORY TECHNOLOGY
 A heterozygous mutation is a mutation of only one allele.
 A homozygous mutation is an identical mutation of both the
paternal and maternal alleles.
 Compound heterozygous mutations or a genetic compound is
two different mutations in the paternal and maternal alleles.
 A wildtype or homozygous non-mutated organism is one in
which neither allele is mutated. (Just not a mutation)

By impact on protein sequence

 A frameshift mutation is a mutation caused by insertion

or deletion of a number of nucleotides that is not evenly divisible by
three from a DNA sequence. Due to the triplet nature of gene
expression by codons, the insertion or deletion can disrupt the reading
frame, or the grouping of the codons, resulting in a completely
different translation from the original. The earlier in the sequence the
deletion or insertion occurs, the more altered the protein produced is.

 Missense mutations or nonsynonymous mutations are

types of point mutations where a single nucleotide is changed to cause
substitution of a different amino acid. This in turn can render the
resulting protein nonfunctional. Such mutations are responsible for
diseases such as Epidermolysis bullosa, sickle-cell disease, and SOD1
mediated ALS
 A neutral mutation is a mutation that occurs in an amino
acid codon which results in the use of a different, but chemically
similar, amino acid. This is similar to a silent mutation, where a codon
mutation may encode the same amino acid (see Wobble Hypothesis);
for example, a change from AUU to AUC will still encode leucine, so no
discernible change occurs (a silent mutation).

 A nonsense mutation is a point mutation in a sequence of

DNA that results in a premature stop codon, or a nonsense codon in
the transcribed mRNA, and possibly a truncated, and often
nonfunctional protein product.

 Silent mutations are mutations that do not result in a

change to the amino acid sequence of a protein. They may occur in a
 SCIENCE LABORATORY TECHNOLOGY
region that does not code for a protein, or they may occur within a
codon in a manner that does not alter the final amino acid sequence.
The phrase silent mutation is often used interchangeably with the
phrase synonymous mutation; however, synonymous mutations are a
subcategory of the former, occurring only within exons. The name
silent could be a misnomer. For example, a silent mutation in the
exon/intron border may lead to alternative splicing by changing the
splice site (see Splice site mutation), thereby leading to a changed
protein.

Special classes

 Conditional mutation is a mutation that has wild-type (or

less severe) phenotype under certain "permissive" environmental
conditions and a mutant phenotype under certain "restrictive"
conditions. For example, a temperature-sensitive mutation can cause
cell death at high temperature (restrictive condition), but might have
no deleterious consequences at a lower temperature (permissive
condition).

Causes of mutation
Two classes of mutations are spontaneous mutations (molecular decay)
and induced mutations caused by mutagens.
Spontaneous mutations on the molecular level include:

 Tautomerism – A base is changed by the repositioning of a

hydrogen atom.
 Depurination – Loss of a purine base (A or G) to form an
apurinic site (AP site).
 Deamination – Changes a normal base to an atypical base.
Examples include C → U and A → HX (hypoxanthine), which can be
corrected by DNA repair mechanisms; and 5MeC (5-methylcytosine) →
T, which is less likely to be detected as a mutation because thymine is a
normal DNA base.
 Transition – A purine changes to another purine, or a
pyrimidine to a pyrimidine.
 Transversion – A purine becomes a pyrimidine, or vice versa.
 SCIENCE LABORATORY TECHNOLOGY
Induced mutations on the molecular level can be caused by:

 Chemicals
o Hydroxylamine NH2OH
o Base analogs (e.g. BrdU)
o Alkylating agents (e.g. N-ethyl-N-nitrosourea) These
agents can mutate both replicating and non-replicating DNA. In
contrast, a base analog can only mutate the DNA when the analog is
incorporated in replicating the DNA. Each of these classes of chemical
mutagens has certain effects that then lead to transitions,
transversions, or deletions.
o Agents that form DNA adducts (e.g. ochratoxin A
metabolites)
o DNA intercalating agents (e.g. ethidium bromide)
o DNA crosslinkers
o Oxidative damage
 Radiation
o Ultraviolet radiation (nonionizing radiation). Two
nucleotide bases in DNA – cytosine and thymine – are most vulnerable
to radiation that can change their properties. UV light can induce
adjacent thymine bases in a DNA strand to pair with each other, as a
bulky dimer.
o Ionizing radiation
 Viral infections

DNA has so-called hotspots, where mutations occur up to 100 times

more frequently than the normal mutation rate. A hotspot can be at an
unusual base, e.g., 5-methylcytosine.
Mutation rates also vary across species. Evolutionary biologists have
theorized that higher mutation rates are beneficial in some situations,
because they allow organisms to evolve and therefore adapt more
quickly to their environments. For example, repeated exposure of
bacteria to antibiotics, and selection of resistant mutants, can result in
the selection of bacteria that have a much higher mutation rate than
the original population (mutator strains).
 SCIENCE LABORATORY TECHNOLOGY
Nomenclature
Nomenclature of mutations specify the type of mutation and base or
amino acid changes.

 Nucleotide substitution (e.g. 76A>T) - The number is the

position of the nucleotide from the 5' end, the first letter represents the
wild type nucleotide, and the second letter represents the nucleotide
which replaced the wild type. In the given example, the adenine at the
76th position was replaced by a thymine.
o If it becomes necessary to differentiate between
mutations in genomic DNA, mitochondrial DNA, and RNA, a simple
convention is used. For example, if the 100th base of a nucleotide
sequence mutated from G to C, then it would be written as g.100G>C if
the mutation occurred in genomic DNA, m.100G>C if the mutation
occurred in mitochondrial DNA, or r.100g>c if the mutation occurred
in RNA. Note that for mutations in RNA, the nucleotide code is written
in lower case.
 Amino acid substitution (e.g. D111E) – The first letter is the
one letter code of the wild type amino acid, the number is the position
of the amino acid from the N terminus, and the second letter is the one
letter code of the amino acid present in the mutation. If the second
letter is 'X', any amino acid may replace the wild type.
 Amino acid deletion – The Greek letter Δ (delta) indicates a
deletion. The letter refers to the amino acid present in the wild type
and the number is the position from the N terminus of the amino acid
were it to be present as in the wild type.

Harmful mutations
Changes in DNA caused by mutation can cause errors in protein
sequence, creating partially or completely non-functional proteins. To
function correctly, each cell depends on thousands of proteins to
function in the right places at the right times. When a mutation alters a
protein that plays a critical role in the body, a medical condition can
result. A condition caused by mutations in one or more genes is called a
genetic disorder. Some mutations alter a gene's DNA base sequence but
do not change the function of the protein made by the gene. Studies in
the fly Drosophila melanogaster suggest that if a mutation does
 SCIENCE LABORATORY TECHNOLOGY
change a protein, this will probably be harmful, with about 70 percent
of these mutations having damaging effects, and the remainder being
either neutral or weakly beneficial.
If a mutation is present in a germ cell, it can give rise to offspring that
carries the mutation in all of its cells. This is the case in hereditary
diseases. On the other hand, a mutation can occur in a somatic cell of
an organism. Such mutations will be present in all descendants of this
cell, and certain mutations can cause the cell to become malignant, and
thus cause cancer.
Often, gene mutations that could cause a genetic disorder are repaired
by the DNA repair system of the cell. Each cell has a number of
pathways through which enzymes recognize and repair mistakes in
DNA. Because DNA can be damaged or mutated in many ways, the
process of DNA repair is an important way in which the body protects
itself from disease.
Beneficial mutations
Although many mutations are deleterious, mutations may have a
positive effect given certain selective pressures in a population.
For example, a specific 32 base pair deletion in human CCR5 (CCR5-
Δ32) confers HIV resistance to homozygotes and delays AIDS onset in
heterozygotes. The CCR5 mutation is more common in those of
European descent. One theory for the etiology of the relatively high
frequency of CCR5-Δ32 in the European population is that it conferred
resistance to the bubonic plague in mid-14th century Europe. People
who had this mutation were able to survive infection; thus, its
frequency in the population increased. It could also explain why this
mutation is not found in Africa where the bubonic plague never
reached. Newer theory says the selective pressure on the CCR5 Delta
32 mutation has been caused by smallpox instead of the bubonic
plague.

CHROMOSOMAL MUTATIONS
Chromosomal mutations results to changes in the number of
chromosomes . sudden forms of chromosome mutation may affect
several genes and they may have adverse effect on the phenotype
 SCIENCE LABORATORY TECHNOLOGY
even more than the gene mutation . changes in the number of
chromosome are usually as a result of errors during meiosis and
mitosis . These changes may involve the loss or gain of a single
chromosome , a condition called aneuploidy . the changes may also
cause an increase in the entire haploid set of chromosomes i.e. a
condition called euploidy or polyploidy .
Aneuploidy
In these condition , half of the daughter cells produced have an extra
( i.e. n+I , 2n+1
extra ). While the other half have a chromosome missing (i.e. n-1 or 2n
–1 ). Aneuploidy can arise from the failure of homologous
chromosomes pairs separating during anaphase 1 of meiosis . in such
cases ,both sets of homologous chromosomes move to the same pole
of the cell and separation of homologous chromosomes during
anaphase 11 may lead to the formation of gametes cells containing
either one or more chromosome s . These phenomenon is known as
non – disjunction
 SCIENCE LABORATORY TECHNOLOGY

Meiotic Nondisjunction Generates Aneuploidies

abnormal
gametes

Zygotic Ploidy Zygotic Ploidy

Zygotes containing less than diploid number of chromosomes

usually fail to develop but those with extra chromosomes in most case
develop to maturity .These is probably due to the zygote loosing very
important genes that were needed for early development of that
organism
In most cases where trysomy occurs , it produces severe
abnormalities e.g. conditions like down syndrome , trysomy 13 ,
 SCIENCE LABORATORY TECHNOLOGY
klinefelter syndrome , turners synndrome etc . below is a table which
gives a summery of some trysomy in humans

Table showing trysomy in humans

Chromosome Chromosome Clinical syndrome Estimated frequency
Main phenotypical characteri
nomenclature formular
47 ,+21 non disjunction
2n +1 Down syndrome 1/700 Have short broad hands with
type palmer creases
of two chromosome 21
-short in height
 SCIENCE LABORATORY TECHNOLOGY
hyper flexib
mental reta
broad face w
round face
wide bridge
open mouth
protruding large tongue
47,+13 2n +1 Trysomy 13 1/2000 -mental deficiency
deafness
minor muscle siezures
cleft lip or pallet
many phalenges
have cardiac abnormalities

47,+18 2n +1 Trysomy 18 1/8000 Multiple congenital deformat

many organs
Low set malformed ears
Reciding multiples
Small mouth and nose menta
retardation deficiency
Malformed kidneys
Short chest bone 90% die wi
1st 6 months
45, x 2n-1 Tunners syndrome 1/3000 Females with retarded sexua
development
Usualy sterile
Short in height
Web neck due to a flap of th
Heart problems
Hearing impairment
47, XXY 2n +1 Klinefelter syndrome
1/500 All are male (due to y )
48 XXXY 2n +2 They are sub fertile with sma
49 XXYY 2n +3 They develop breast . they hav
feminine voice and talkative
 SCIENCE LABORATORY TECHNOLOGY
50 XXXXY 2n +3 They have long limps
Have knock knees
47,XXX 2n+1 Tripple X 1/700 Slight mental retardation
Limited fertility
Females with normal genital o

EUPLOIDY (POLYPLOIDY )
Gametes and somatic cells containing multiples of haploid number of
chromosomes are called polyploids. The predixes tri, tetra , penta ,
hexa etc indicates the extend of polyploidy i.e. triploidy ( 3n ),
tetraploidy(4n), pentaploidy(5n)

Polyploidy
 Polyploids with odd #’d chromosome sets are
usually sterile
 produce mostly aneuploid gametes
 rare a diploid & haploid gamete are produced

Each cell receives

one copy of some
chromosomes
and two copies of
other chromosomes

Figure 8.23

Polyploidy is much common in plants than in animals e.g. more than

50% of all known angiosperms are polyploids . These is because in
animals , increased number of chromosomes in polyploids makes
gametes formation during meiosis to have very many errors . since
most plants are capable of propagating themselves vegetatively , they
are able to reprduce despite being polyploids. incease in size,
production , hardness and resistance to diseases . These is called
hybrid vigour or heterosis. Most domestic plants are polyploids
producing large fruits , food storage Polyploids is often associated
with advantageous features such as organs e.g. stem tubbers , large
flowers etc .
 SCIENCE LABORATORY TECHNOLOGY
There are two forms of polyploidy i.e. autoploidy and alloploidy
(a) autopolyploidy

Generation of Polyploids
 Autopolyploidy
 Complete nondisjunction of both gametes can produce an
individual with one or more sets of chromosomes

Figure 8.27

(b) allopolyploidy

Interspecies Crosses can Generate Alloploids

 Alloploidy
 Offspring generally sterile

Figure 8.27
 SCIENCE LABORATORY TECHNOLOGY
GENETIC ENGINEERING

Genetic engineering, also called genetic modification, is the

direct human manipulation of an organism's genetic material in a way
that does not occur under natural conditions. It involves the use of
recombinant DNA techniques, but does not include traditional animal
and plant breeding or mutagenesis. Any organism that is generated
using these techniques is considered to be a genetically modified
organism. The first organisms genetically engineered were bacteria in
1973 and then mice in 1974. Insulin producing bacteria were
commercialized in 1982 and genetically modified food has been sold
since 1994.
The most common form of genetic engineering involves the insertion of
new genetic material at an unspecified location in the host genome.
This is accomplished by isolating and copying the genetic material of
interest, generating a construct containing all the genetic elements for
correct expression, and then inserting this construct into the host
organism. Other forms of genetic engineering include gene targeting
and knocking out specific genes via engineered nucleases such as zinc
finger nucleases or engineered homing endonucleases.
Genetic engineering techniques have been applied in numerous fields
including research, biotechnology, and medicine. Medicines such as
insulin and human growth hormone are now produced in bacteria,
experimental mice such as the oncomouse and the knockout mouse are
being used for research purposes and insect resistant and/or herbicide
tolerant crops have been commercialized. Genetically engineered
plants and animals capable of producing biotechnology drugs more
cheaply than current methods (called pharming) are also being
developed and in 2009 the FDA approved the sale of the
pharmaceutical protein antithrombin produced in the milk of
genetically engineered goats.
 SCIENCE LABORATORY TECHNOLOGY
Definition
Genetic engineering alters the genetic makeup of an organism using
techniques that introduce heritable material prepared outside the
organism either directly into the host or into a cell that is then fused or
hybridized with the host.[1] This involves using recombinant nucleic
acid (DNA or RNA) techniques to form new combinations of heritable
genetic material followed by the incorporation of that material either
indirectly through a vector system or directly through micro-injection,
macro-injection and micro-encapsulation techniques. Genetic
engineering does not include traditional animal and plant breeding, in
vitro fertilisation, induction of polyploidy, mutagenesis and cell fusion
techniques that do not use recombinant nucleic acids or a genetically
modified organism in the process. Cloning and stem cell research,
although not considered genetic engineering, are closely related and
genetic engineering can be used within them. Synthetic biology is an
emerging discipline that takes genetic engineering a step further by
introducing artificially synthesized genetic material from raw materials
into an organism.
If genetic material from another species is added to the host, the
resulting organism is called transgenic. If genetic material from the
same species or a species that can naturally breed with the host is used
the resulting organism is called cisgenic. Genetic engineering can also
be used to remove genetic material from the target organism, creating a
knock out organism.[6] In Europe genetic modification is synonymous
with genetic engineering while within the United States of America it
can also refer to conventional breeding methods.

Transformation (genetics)
An unrelated process called malignant transformation occurs in the
progression of cancer.

In molecular biology transformation is the genetic alteration of a cell

resulting from the direct uptake, incorporation and expression of
exogenous genetic material (exogenous DNA) from its surrounding and
taken up through the cell membrane(s). Transformation occurs most
commonly in bacteria and in some species occurs naturally.
Transformation can also be effected by artificial means. Bacteria that
 SCIENCE LABORATORY TECHNOLOGY
are capable of being transformed, whether naturally or artificially, are
called competent. Transformation is one of three processes by which
exogenous genetic material may be introduced into bacterial cell, the
other two being conjugation (transfer of genetic material between two
bacterial cells in direct contact), and transduction (injection of foreign
DNA by a bacteriophage into the host). Transformation may also be
used to describe the insertion of new genetic material into nonbacterial
cells including animal and plant cells, however, because transformation
has a special meaning in relation to animal cells indicating progression
to a cancerous state, the term should be avoided for animal cells when
describing introduction of exogenous genetic material. Introduction of
foreign DNA into eukaryotic cells is usually called "transfection".
Transformation was first demonstrated in 1928 by British
bacteriologist Frederick Griffith. Griffith discovered that a harmless
strain of Streptococcus pneumoniae could be made virulent after being
exposed to heat-killed virulent strains. Griffith hypothesized that some
"transforming principle" from the heat-killed strain was responsible for
making the harmless strain virulent. In 1944 this "transforming
principle" was identified as being genetic by Oswald Avery, Colin
MacLeod, and Maclyn McCarty. They isolated DNA from a virulent
strain of S. pneumoniae and using just this DNA were able to make a
harmless strain virulent. They called this uptake and incorporation of
DNA by bacteria "transformation." See Avery-MacLeod-McCarty
experiment.
The results of Avery et al.'s experiments were at first sceptically
received by the scientific community and it was not until the
development of genetic markers and the discovery of other methods of
genetic transfer (conjugation in 1947 and transduction in 1953) by
Joshua Lederberg that Avery's experiments were accepted.
Transformation did not become routine procedure in laboratories until
1972 when Stanley Cohen, Annie Chang and Leslie Hsu successfully
transformed Escherichia coli by treating the bacteria with calcium
chloride. This created an efficient and convenient procedure for
transforming bacteria and opened the way for molecular cloning in
biotechnology and research.
Transformation using electroporation was developed in the late 1980s
thus increasing the efficiency and number of bacterial strains that
could be transformed.Transformation of animal and plant cells was
 SCIENCE LABORATORY TECHNOLOGY
also investigated with the first transgenic mouse being created by
injecting a gene for a rat growth hormone into a mouse embryo in
1982. In 1907 a bacterium that caused plant tumors, Agrobacterium
tumefaciens, was discovered and in the early 1970s the tumor inducing
agent was found to be a DNA plasmid called the Ti plasmid. By
removing the genes in the plasmid that caused the cancer and adding
in novel genes researchers were able to infect plants with A.
tumefaciens and let the bacteria insert their chosen DNA into the
genomes of the plants. Not all plant cells are susceptible to infection by
A. tumefaciens so other methods were developed including
electroporation and micro-injection. Particle bombardment was made
possible with the invention of the Biolistic Particle Delivery System
(gene gun) by John Sanford in 1990.

Mechanisms

Bacteria
Bacteria transformation may be referred to as a stable genetic change
brought about by the uptake of naked DNA (DNA without associated
cells or proteins) and competence refers to the state of being able to
take up exogenous DNA from the environment. Two forms of
competence exist: natural and artificial.

Natural competence
About 1% of bacterial species are capable of naturally taking up DNA
under laboratory conditions; many more are able to take it up in their
natural environments. Such bacteria carry sets of genes that provide
the protein machinery to bring DNA across the cell membrane(s).
DNA material can be transferred between different strains of bacteria,
in a process called horizontal gene transfer.

Artificial competence
Artificial competence is induced by laboratory procedures and involves
making the cell passively permeable to DNA by exposing it to
conditions that do not normally occur in nature.
Calcium chloride transformation is a method of promoting
competence. Chilling cells in the presence of divalent cations such as
 SCIENCE LABORATORY TECHNOLOGY
Ca2+ (in CaCl2) prepares the cell membrane to become permeable to
plasmid DNA. The cells are incubated on ice with the DNA and then
briefly heat shocked (e.g., 42°C for 30–120 seconds) thus allowing the
DNA to enter the cells. This method works very well for circular
plasmid DNA. An excellent preparation of competent cells will give
~108 colonies per microgram of plasmid. A poor preparation will be
about 104/μg or less. Good, non-commercial preparations should give
105 to 106 transformants per microgram of plasmid. The method,
however, usually does not work well for linear DNA, such as fragments
of chromosomal DNA, probably because the cell's native exonuclease
enzymes rapidly degrade linear DNA. Interestingly, cells that are
naturally competent are usually transformed more efficiently with
linear DNA than with plasmid DNA.
Electroporation is another method of promoting competence. In the
method the cells are briefly shocked with an electric field of 10-20
kV/cm that creates holes in the cell membrane through which the
plasmid DNA enters. This method is amenable to the uptake of large
plasmid DNA. After the electric shock the holes are rapidly closed by
the cell's membrane-repair mechanisms.
The efficiency with which a competent culture can take up exogenous
DNA and express its genes is known as Transformation efficiency.

Plasmid transformation
In order to be stably maintained in the cell a plasmid DNA molecule
must contain an origin of replication, which allows it to be replicated in
the cell independent of the replication of the cell's own chromosome.
Because transformation usually produces a mixture of relatively few
transformed cells and an abundance of non-transformed cells a
method is needed to identify the cells that have acquired the plasmid.
The method usually consists of using a plasmid that contains a gene
that gives the bacterial cells resistance to an antibiotic that they are
naturally sensitive to. The mixture of cells are then plated on media
that contains the antibiotic thus only the transformed cells are able to
grow. Cells that did not take up the plasmid are killed in the media.
Another selection method called blue-white screen uses a plasmid that
contains an antibiotic resistance gene and the lacZ gene. The lacZ gene
codes for the lacZ-α subunit of the enzyme β-galactosidase, a homo-
 SCIENCE LABORATORY TECHNOLOGY
tetramer with each monomer composed of one lacZ-α subunit and one
lacZ-ω subunit. The method also requires an E. coli strain that
possesses in its genome the code for only the lacZ-ω subunit and not
the lacZ-α subunit. One of the first steps in any transformation is the
production of a recombined plasmid obtained by the successful ligation
of the gene of interest into its corresponding vector, which in this
method results in the disruption of lacZ because the gene of interest is
inserted within the lacZ code. A cell that takes up a recombined
plasmid would thus not be able to express the lacZ-α subunit and
would, in turn, not be able to produce a functional β-galactosidase.
Conversely, a cell that has taken up non-recombined plasmid (perhaps
one formed by the ligation of the vector's own two ends) will express
the lacZ-α subunit and thus produce a functional β-galactosidase. A cell
that does not take up any plasmid is not conferred with antibiotic
resistance and will die upon plating. Consequently, the blue-white
screen method allows for the ready detection of not just transformed
cells, but, most importantly, cells that have been transformed by a
successfully recombined plasmid. Selection occurs as a result of the
action of β-galactosidase on its substrate X-gal, which is included in the
media along with the appropriate antibiotic. X-gal is a colorless,
modified galactose sugar whose hydrolysis by β-galactosidase produces
galactose and the pre-chromophore 5-bromo-4-chloro-3-
hydroxyindole. The latter is subsequently oxidized to 5,5'-dibromo-
4,4'-dichloro-indigo, an insoluble, blue product that is readily seen by
the naked eye. Colonies of cells that have been transformed by a
successfully recombined plasmid will thus appear white whereas those
that have been transformed by non-recombined plasmid will appear
blue.

Plants
A number of mechanisms are available to transfer DNA into plant cells:

 Agrobacterium mediated transformation is the easiest and

most simple plant transformation. Plant tissue (often leaves) are cut
into small pieces, e.g. 10x10mm, and soaked for 10 minutes in a fluid
containing suspended Agrobacterium. Some cells along the cut will be
transformed by the bacterium, that inserts its DNA into the cell. Placed
on selectable rooting and shooting media, the plants will regrow. Some
 SCIENCE LABORATORY TECHNOLOGY
plants species can be transformed just by dipping the flowers into
suspension of Agrobacterium and then planting the seeds in a selective
medium. Unfortunately, many plants are not transformable by this
method.
 Particle bombardment: Particles of gold or tungsten are
coated with DNA and then shot into young plant cells or plant
embryos. Some genetic material will stay in the cells and transform
them. This method also allows transformation of plant plastids. The
transformation efficiency is lower than in agribacterial mediated
transformation, but most plants can be transformed with this method.
 Electroporation: make transient holes in cell membranes
using electric shock; this allows DNA to enter as described above for
Bacteria.
 Viral transformation (transduction): Package the desired
genetic material into a suitable plant virus and allow this modified
virus to infect the plant. If the genetic material is DNA, it can
recombine with the chromosomes to produce transformant cells.
However genomes of most plant viruses consist of single stranded RNA
which replicates in the cytoplasm of infected cell. For such genomes
this method is a form of transfection and not a real transformation,
since the inserted genes never reach the nucleus of the cell and do not
integrate into the host genome. The progeny of the infected plants is
virus free and also free of the inserted gene.

Transduction (genetics)
Transduction is the process by which DNA is transferred from one
bacterium to another by a virus. It also refers to the process whereby
foreign DNA is introduced into another cell via a viral vector. This is a
common tool used by molecular biologists to stably introduce a foreign
gene into a host cell's genome.
When bacteriophages (viruses that infect bacteria) infect a bacterial
cell, their normal mode of reproduction is to harness the replicational,
transcriptional, and translation machinery of the host bacterial cell to
make numerous virions, or complete viral particles, including the viral
DNA or RNA and the protein coat.
 SCIENCE LABORATORY TECHNOLOGY
Lytic and lysogenic (temperate) cycles
Transduction happens through either the lytic cycle or the lysogenic
cycle.
If the lysogenic cycle is adopted, the phage chromosome is integrated
into the bacterial chromosome, where it can remain dormant for
thousands of generations. If the lysogen is induced (by UV light for
example), the phage genome is excised from the bacterial chromosome
and initiates the lytic cycle, which culminates in lysis of the cell and the
release of phage particles. The lytic cycle leads to the production of new
phage particles which are released by lysis of the host.

Transduction as a method of transfer genetic material

The packaging of bacteriophage DNA has low fidelity and small pieces
of bacterial DNA, together with the bacteriophage genome, may
become packaged into the bacteriophage genome. At the same time,
some phage genes are left behind in the bacterial chromosome.
There are generally three types of recombination events that can lead
to this incorporation of bacterial DNA into the viral DNA, leading to
two modes of recombination.

Generalized transduction
Generalized transduction may occur in two main ways, recombination
and headful packaging.
If bacteriophages undertake the lytic cycle of infection upon entering a
bacterium, the virus will take control of the cell’s machinery for use in
replicating its own viral DNA. If by chance bacterial chromosomal DNA
is inserted into the viral capsid used to encapsulate the viral DNA, the
mistake will lead to generalized transduction.
If the virus replicates using 'headful packaging', it attempts to fill the
nucleocapsid with genetic material. If the viral genome results in spare
capacity, viral packaging mechanisms may incorporate bacterial
genetic material into the new virion.
The new virus capsule now loaded with part bacterial DNA continues to
infect another bacterial cell. This bacterial material may become
recombined into another bacterium upon infection.
 SCIENCE LABORATORY TECHNOLOGY
When the new DNA is inserted into this recipient cell it can fall to one
of three fates

1. The DNA will be absorbed by the cell and be recycled for

spare parts.
2. If the DNA was originally a plasmid, it will re-circularize
inside the new cell and become a plasmid again.
3. If the new DNA matches with a homologous region of the
recipient cell’s chromosome, it will exchange DNA material similar to
the actions in conjugation.

This type of recombination is random and the amount recombined

depends on the size of the virus being used.

Specialized transduction
The second type of recombination event is called specialized
transduction and occurs as a result of mistakes in the transition from a
virus' lysogenic to lytic cycle. If a virus incorrectly removes itself from
the bacterial chromosome, bacterial DNA from either end of the phage
DNA may be packaged into the viral capsid. Specialized transduction
leads to three possible outcomes:

1. DNA can be absorbed and recycled for spare parts.

2. The bacterial DNA can match up with a homologous DNA in
the recipient cell and exchange it. The recipient cell now has DNA from
both itself and the other bacterial cell.
3. DNA can insert itself into the genome of the recipient cell as
if still acting like a virus resulting in a double copy of the bacterial
genes.

Example of specialized transduction is λ phages in Escherichia coli.

RNA, DNA
Viruses with RNA genomes are not able to package DNA and so do not
usually make this mistake.
Upon lysis of the host cell, the mispackaged virions containing
bacterial DNA can attach to other bacterial cells and inject the DNA
 SCIENCE LABORATORY TECHNOLOGY
they have packaged, thus transferring bacterial DNA from one cell to
another. This DNA can become part of the new bacterium's genome
and thus be stably inherited.

More general uses of the term

More generally, transduction is the process by which genetic
material, e.g. DNA or siRNA, is inserted into a cell by a virus.
Common techniques in molecular biology are the use of viral vectors
(including bacteriophages), electroporation, or chemical reagents that
increase cell permeability. Transfection and transformation are also
common ways to insert DNA into a cell.

Discovery
Transduction was discovered by Norton Zinder and Joshua Lederberg
at the University of Wisconsin–Madison in 1951.

Bacterial conjugation
Bacterial conjugation is the transfer of genetic material between
bacterial cells by direct cell-to-cell contact or by a bridge-like
connection between two cells.[1] Discovered in 1946 by Joshua
Lederberg and Edward Tatum,[2] conjugation is a mechanism of
horizontal gene transfer as are transformation and transduction
although these two other mechanisms do not involve cell-to-cell
contact.
Bacterial conjugation is often incorrectly regarded as the bacterial
equivalent of sexual reproduction or mating since it involves the
exchange of genetic material. During conjugation the donor cell
provides a conjugative or mobilizable genetic element that is most
often a plasmid or transposon. Most conjugative plasmids have
systems ensuring that the recipient cell does not already contain a
similar element.
The genetic information transferred is often beneficial to the recipient.
Benefits may include antibiotic resistance, xenobiotic tolerance or the
ability to use new metabolites. Such beneficial plasmids may be
considered bacterial endosymbionts. Other elements, however, may be
 SCIENCE LABORATORY TECHNOLOGY
viewed as bacterial parasites and conjugation as a mechanism evolved
by them to allow for their spread.

Mechanism

Schematic drawing of bacterial conjugation. Conjugation diagram 1-

Donor cell produces pilus. 2- Pilus attaches to recipient cell
and brings the two cells together. 3- The mobile plasmid is
nicked and a single strand of DNA is then transferred to the
recipient cell. 4- Both cells synthesize a complementary
strand to produce a double stranded circular plasmid and
also reproduce pili; both cells are now viable donors.
 SCIENCE LABORATORY TECHNOLOGY
The prototypical conjugative plasmid is the F-plasmid, or F-factor.[1]
The F-plasmid is an episome (a plasmid that can integrate itself into
the bacterial chromosome by homologous recombination) with a
length of about 100 kb. It carries its own origin of replication, the oriV,
and an origin of transfer, or oriT.[4] There can only be one copy of the F-
plasmid in a given bacterium, either free or integrated, and bacterium
that possess a copy are called F-positive or F-plus (denoted F+). Cells
that lack F plasmids are called F-negative or F-minus (F-) and as such
can function as recipient cells.
Among other genetic information the F-plasmid carries a tra and trb
locus, which together are about 33 kb long and consist of about 40
genes. The tra locus includes the pilin gene and regulatory genes, which
together form pili on the cell surface. The locus also includes the genes
for the proteins that attach themselves to the surface of F- bacteria and
initiate conjugation. Though there is some debate on the exact
mechanism of conjugation it seems that the pili are not the structures
through which DNA exchange occurs. This has been shown in
experiments where the pilus are allowed to make contact, but then are
denatured with SDS and yet DNA transformation still proceeds.
Several proteins coded for in the tra or trb locus seem to open a
channel between the bacteria and it is thought that the traD enzyme,
located at the base of the pilus, initiates membrane fusion.
When conjugation is initiated by a signal the relaxase enzyme creates
a nick in one of the strands of the conjugative plasmid at the oriT.
Relaxase may work alone or in a complex of over a dozen proteins
known collectively as a relaxosome. In the F-plasmid system the
relaxase enzyme is called TraI and the relaxosome consists of TraI,
TraY, TraM and the integrated host factor IHF. The nicked strand, or
T-strand, is then unwound from the unbroken strand and transferred
to the recipient cell in a 5'-terminus to 3'-terminus direction. The
remaining strand is replicated either independent of conjugative action
(vegetative replication beginning at the oriV) or in concert with
conjugation (conjugative replication similar to the rolling circle
replication of lambda phage). Conjugative replication may require a
second nick before successful transfer can occur. A recent report claims
to have inhibited conjugation with chemicals that mimic an
intermediate step of this second nicking event.[7]
 SCIENCE LABORATORY TECHNOLOGY
If the F-plasmid that is transferred has previously been integrated into
the donor’s genome some of the donor’s chromosomal DNA may also
be transferred with the plasmid DNA.[3] The amount of chromosomal
DNA that is transferred depends on how long the two conjugating
bacteria remain in contact. In common laboratory strains of E. coli the
transfer of the entire bacterial chromosome takes about 100 minutes.
The transferred DNA can then be integrated into the recipient genome
via homologous recombination.
A cell culture that contains in its population cells with non-integrated
F-plasmids usually also contains a few cells that have accidentally
integrated their plasmids. It is these cells that are responsible for the
low-frequency chromosomal gene transfers that occur in such cultures.
Some strains of bacteria with an integrated F-plasmid can be isolated
and grown in pure culture. Because such strains transfer chromosomal
genes very efficiently they are called Hfr (high frequency of
recombination). The E. coli genome was originally mapped by
interrupted mating experiments in which various Hfr cells in the
process of conjugation were sheared from recipients after less than 100
minutes (initially using a Waring blender). The genes that were
transferred were then investigated.

Inter-kingdom transfer
The nitrogen fixing Rhizobia are an interesting case of inter-kingdom
conjugation.[8] For example, the tumor-inducing (Ti) plasmid of
Agrobacterium and the root-tumor inducing (Ri) plasmid of A.
rhizogenes contain genes that are capable of transferring to plant cells.
The expression of these genes effectively transforms the plant cells into
opine-producing factories. Opines are used by the bacteria as sources
of nitrogen and energy. Infected cells form crown gall or root tumors,
respectively. The Ti and Ri plasmids are thus endosymbionts of the
bacteria, which are in turn endosymbionts (or parasites) of the infected
plant.
The Ti and Ri plasmids can also be transferred between bacteria using
a system (the tra, or transfer, operon) that is different and independent
of the system used for inter-kingdom transfer (the vir, or virulence,
operon). Such transfers create virulent strains from previously
avirulent strains.
 SCIENCE LABORATORY TECHNOLOGY
Genetic Engineering Applications
Conjugation is a convenient means for transferring genetic material to
a variety of targets. In laboratories successful transfers have been
reported from bacteria to yeast,[9] plants, mammalian cells[10][11] and
isolated mammalian mitochondria.[12] Conjugation has advantages over
other forms of genetic transfer including minimal disruption of the
target's cellular envelope and the ability to transfer relatively large
amounts of genetic material (see the above discussion of E. coli
chromosome transfer). In plant engineering, Agrobacterium-like
conjugation complements other standard vehicles such as tobacco
mosaic virus (TMV). While TMV is capable of infecting many plant
families these are primarily herbaceous dicots. Agrobacterium-like
conjugation is also primarily used for dicots, but monocot recipients
are not uncommon.

CHAPTER TWENTY NINE

HISTOLOGICAL
TECHNIQUES
Histology is a microscopical study of normal body tissues, while
histopathology is the microscopical study of diseased body tissues.
Biopsy is the excision of living tissues from the body and
microscopically examining them to establish diagnosis whereas
autopsy is the examination of dead body tissues for the purpose of
diagnosis
Histology and histopathology is a study whose aim is to
(i) Establish a disease and therefore deciding on the method
of prevention ,control and treatment
(ii) To establish crime e.g. in police cases dealing with
accidents ,murder ,rape ,suicide etc
 SCIENCE LABORATORY TECHNOLOGY
(iii) To establish cause of death i.e. autopsy
(iv) For research purposes and teaching .
The tissues normally studied include ; liver , lungs ;intestines
,kidneys ,skin ,blood ,brain ,heart, rectum, placenta etc
TISSUE PROCESSING
Tissue processing involves all those preparation done on the tissue
after removal from the organism until when it will be mounted on a
microscope for examination . It’s the treatment of tissues with various
agents so as to enable the production of thin sections . These thin
sections allows light to pass through thus enabling the examination of
tissues under the microscope . tissue processing can be permanent or
temporary . Permanent preparation remains unchanged for many years
if properly stored while temporary preparation are only useful for a
very short e.g. few hours they go bad quickly. Tissue processing can be
done in either manually using hands or mechanically using automatic
tissue processor .Thestages involved in tissue processing include ;
(i) Fixation
(ii) Dehydration
(iii) Clearing
(iv) Impregnation
(v) Embedding
(vi) Sectioning
(vii) Dewaxing
(viii) Hydration
(ix) Staining
(x) Mounting
(xi) Examination
 SCIENCE LABORATORY TECHNOLOGY
TEMPORARY SLIDE PREPARATION

(a) Tissue preparation by squashing and maceration

Requirements
-razor blade/scalpel
-root tips
Alcohol 70%
Microscope
Acetic acid
Squashing
This is where the cellular content of the small pieces of tissue not
exceeding 1mm in diameter can be examined by placing the tissue in the
centre of the microscopic slide and forcefully apply a cover slip i.e. squize
the tissue to the slide by a cover slip

Maceration
Is the process used in breaking the middle lamellae which lies between
adjacent cells cementing them together.
The cell may be separated by squashing so as to enable individual
examination.
Root tip lamellae is broken by soaking in hydrochloric acid at 60oc for 6-
10 min
Frost sectioned delicate herbaceous tissues is soaked in 5% chromic (iv)
acid for 24hours then washed in running tap water

Methods of preparation
First , about 5mm of the top of the root is removed by razor blade and left
in alcohol or acetic acid mixture for 4hours. After that, they are stored in
70%alcohol until required preferably in a refrigerator until the required
side is achieved.

Transfer the root tip to cold normal hydrochloric acid heated at 60oc and
left for 12 minute.
 SCIENCE LABORATORY TECHNOLOGY
This process hydrolyses the substance holding the cells together so that
the roots can be squashed on the slide.
Then transfer the root to distilled water
The root is next stained by use of a fenlegen stain for 1-2 hours in a dark
cupboard which stains the root red. Then the tissue is removed from the
stain and put in 45% of acetic acid on a slide.
Then the tissue is teased out with a scalpel and covered with a cove slip
then excess acetic acid is blotted out.
Finally the slide is heated until the acid blown at the edge of the cover slip.
The squashing is done and the slide is placed on the microscope for
examination and observation.

Use of temporary mountants (glycerine, saline and water)

Observation of materials on a microscope can be mounted
temporary by use of drop of water, saline or glycerine on a slide then
cover slip is added.
An aesthetic fluid fixative and stain may be introduced by a method called
irrigation.
A drop of reagent is placed on the slide so that it first touché the edge of
the cover slip.
Fluid is then withdrawn from the opposite side of the cover slip by means
of a filter paper or bloating paper and reagent flows to replace the fluid
taken out.
NB Care should be taken as there is always secure fluid touching the cover
slip to replace that removed.

Staining using iodine and methyl blue

Iodine
Epidermal cell staining with iodine solution will give a clear picture of
the cell which will enable us make the observation to identify the
distribution of the cytoplasm valuable granular

Requirements
- Iodine solution,
- Microscope slide,
- Water
- Cover slip,
- Pipette
 SCIENCE LABORATORY TECHNOLOGY
- Microscope
Procedure
- Strip off a piece of epidermis from one of the the inner fleshly scale of the
onion
- Mount it on a slide
- Add one to 2 drops of iodine solution.
- Put a coverslip on top of the slide
- Mount the slide on the microscope
- Identify by observing.

Methylene blue
This is used to bring out a clear picture of the specimen(thick cell)

Requirement
-Scalpel/ blade
- Cover slip
- Microscope
-Water
- Glass slide
- Stain(methyline blue)
- Pipette

Procedure
- Gently scrap the inside of your check with a sterilized spatula.
- Mount the scrapping in a drop of water on the slide
- To get best picture of the nucleus, stain with methylne blue to observe
the structure clearly
- Cover the slide with a cover slip placed at an angle of 45o to avoid
trapping air bubbles
- Place the specimen on the stage of a microscope and observe the
specimen starting with objective x40
- Finally, locate a single cell and examine it under high power

Preparation of tissues for microscopic examination

The following governs the selection of techniques
a) Structure
b) The amount and nature of tissue to be examined
 SCIENCE LABORATORY TECHNOLOGY
c) Weather the specimen is fresh or dry/preserved
d)The urgency of examination or investigation.
Histological tissues may be prepared as fresh tissue preparations or
permanent tissue preparation
(a) Fresh tissue preparation.
They are prepared when they are still fresh and are disposed off
immidiately after they are used.
They may be examined as teased and squash preparation smears, frozen

Teased
This are prepared by carefully dissecting by mounted middle lamellae to
be examined.
Dissecting is carried when the specimen is immersed in isotonic solution
such as ringer solution in a Petri dish on watch glass.
The selected pieces are transferred to a microscopic slide and mounted as
wet preparation.
Care should be taken to ensure that no air bubble is trapped between the
cover slip and the slide.
The preparation is then examined by bright filled microscope.
The amount of light reduced by either the Irish diaphragm or lowering the
substage condenser.
The use of phase contrast microscopy greatly increases the structural
details of the cells examined and allows movement and mitotic divisions
to be observed.
Stains such as methylene can also be used on teased tissues

Advantage
This method permits the cell to be examined in the living state

Squash preparation
The method is suitable for preparation of small tissues not exceeding
1mm in diameter.
The method is suitable for preparation of slides from soft tissue, however
it can also be used to prepare tissues which are hard e.g. plant tissue
however, the hard tissue must undergo maceration to soften it.Maceration
is done using macerating fluid .The tissue is placed at the centre of the
microscopic slide and forcefully apply a cover slip and slide.
Capillarity can be enhanced by placing a filter paper at the opposite edge
of the cover slip.
 SCIENCE LABORATORY TECHNOLOGY

Smears
In smearing , cellular materials are spread lightly on a slide and examined
under the microscope .

Smears can be prepared from specimen drawn from cultures in Petri

dishes. It can also be prepared for blood with thin or thick blood smears.
It can also be made by making a position smear to the edge of the second
slide.
Smears can be observed as fresh preparation or by using a sub vital
staining technique.

FIXATION

Fixation is the preservation of tissue in as much life state as it was using a

fixative (the process of making the excised tissue remain just exactly as it
was in the life form ) the purpose of fixation are ;
(i)To arrest postmortem changes i.e. these are those changes that take
place on tissue cells after the death of the tissue or after the tissue have
been removed from the body. there are two changes that normally take
place i.e. petrifaction and autolysis.
Putrefaction is the processes of breakdown of tissue cells after their death
due to bacterial action often accompanied with the formation of gas. these
bacteria normally originates from the alimentary canal and the
surrounding organs , some of these bacteria may be the actual cause of the
disease that resulted into these death (parasitic bacteria ) or those that
only feed on already dead matter (saprophytic bacteria)
Autolysis is the process of dissolving or lysing of the cells and tissues due
to enzymatic action after the death . it should be noted that enzymatic
action s in cells do not stop after the death of these cells but their action or
behavior become altered .these changes in enzymatic actions are most
prominent in specialized organs e.g. the liver , brain , kidneys and the
alimentary canal
(ii) fixing helps in preventing the destruction of tissues
(iii) it also allows for subsequent treatment
(iv) it also helps in hardening the tissues
(v) it also help to prevent infections
 SCIENCE LABORATORY TECHNOLOGY
Types or methods of fixation
Fixation can be done using any of the following methods ;
(i)Heat treatment – done by passing the tissue over a flame
(ii) Cold treatment done by keeping the tissue in a deep freezer
(iii) irradiation – done by passing some radiation’s through the
tissue
(iv) chemical treatment – done using chemicals called fixatives ,
these the most commonly used method .

Criteria for a good fixative

A fixative is a chemical substance used to preserve the shape , structure
and the chemical constituency of the tissue and cells after death in a life
–like manner as much as possible .
Fixation is the foundation for processing of tissues for diagnosis because
there is no remedy for already well fixed tissues.
Higher temperatures increase the rate of putrefaction and Autolysis but
reduce fixation time. The optimum temperatures for Autolysis and
putrefaction are 36. 5 oc . low temperature in deep freezes and
refrigerators only arrest Autolysis and petrifaction

A good fixative therefore is the one which ;

(i) Must cause sudden death to tissue cells.
(ii) Must be antibacterial .
(iii) Must inactivate enzymes .
(iv) must penetrate the tissue quickly and evenly .
(v) Must render all substances in the cell insoluble .
(vi) Must preserve cells in as life-like manner as possible.
(vii) Must harden tissues and enable easy manipulation of naturally
soft tissues or organs e.g. the brain , liver and the spleen .
(viii) It must solidify colloidal material to irreversible semisolid state
that is easily demonstrated
(ix) It must alter to varying degrees the refractive indices of
different cells structures so that they are made more visible
(x) It must fortify the tissue against harsh effect of solutions that
will be used during subsequent tissue processing i.e. it must allow
subsequent changes
 SCIENCE LABORATORY TECHNOLOGY
(xi) It must have good effect on staining
in order to obtain good results in tissue processing , the tissue to be
processed or demonstrated must always be born in mind because these
will help in deciding the type of fixative to use .
In order to achieve the best fixation effects of the fixative used , the
following points must be remembered
(i) The tissue must be fixed as soon as possible after removal from
the body
(ii) The amount of fixative used should be 15-20 the size of the
tissue to be fixed
(iii) Thin slices of the tissue should be made to enable quick
penetration and even fixation of tissue

Classification of fixatives

Fixatives are classified into simple and compound fixatives

(a) Simple fixatives – they consist of one chemical substance in
solution.
examples include;
Formaldehyde
Mercuric chloride
Potassium dichromate
Chromic acid
Osmium tetra oxide
Picric acidAcetone
Trichloroacetic acid
(b) Compound fixatives
They are composed of two or more fixatives . they are divided into
(i) Micro-anatomical fixatives
(i) Cytological fixatives
(i) Micro –anatomical fixative

These fixatives preserves the micro –anatomical structures of tissues with

correct
relationship of the tissue layers and cells . they include ;

Formal –sublimate
Zenkers fluid
 SCIENCE LABORATORY TECHNOLOGY
Helly‘s fluid
Bouin‘s fluid
Cytological fixative
Cytological fixatives generally preserves
the intercellular structures and inclusions of the cell . Cytological fixatives
are divided into two i.e. nuclear fixatives and cytological fixatives.

(a) Nuclear fixative

This preserves the nucleus and its inclusions. some common nuclear
fixatives are ;
Carnoys fluid
Flemmings fluid
Clarks fluid
(b) Cytoplasmic fixative
This preserves the cell cytoplasm and cytoplasmic inclusions . common
cytoplasmic inclusions are
Mullers fluid
Orths fluid
Schaudins fluid
NB; a good histochemical fixative should preserve the constituent to be
demonstrated . it should not react with the reagents to be used in the
process of visualization .
Vapour fixatives
These are fixatives used for fixing cut sections of fresh tissues ,thin blocks
of freeze dried tissues and cytological smears . They include;
Acetaldehyde
Gluteraldehyde
Formaldehyde
Treatment of tissues after fixation
Tissues should be treated as follows after fixation ;
(i) Tissues fixed in formal saline should be taken straight away into 70%
ethanol to commence parafin wax processing .
(ii) Tissues fixed in heidenhein susa should be taken to 95% ethanol
(iii) Tissues fixed in canoys fluid should .be taken straight to absolute
ethanol
(iv) Tissues fixed in K2Cr2O7 containing fixatives such as flemming
solution ,orths fluids and zenkers fluid must be runned in tap water
overnight and then transferred to 70% ethanol to commence processing .
 SCIENCE LABORATORY TECHNOLOGY
Preservation and storage of tissuesTissues meant for future
processing may be preserved using any of the methods below.
(i) It may be left in buffered formal saline
(ii) It may be stored in 70% ethanol after fixation in formal saline and
washed in tap wate
(iii) It may be stored in 30% aqueous glycerin after fixation and washed
in tap water
(iv) Museum tissues and specimens may be prepared and mounted
in jars , they may be stored in 10-20% diethyl glycol
(v) It may be stored in 1% phenoxytal
(vi) It may be processed and
embedded by paraffin wax method and kept in a paraffin wax tissue
block

Importance of tissue storage

(i) For future reference while assessing the progress of the patient
(ii) For teaching purposes
(iii) For general demonstration
(iv) For research
(v) For enabling the laboratory staff to attend to more urgent work

Fixation terminologies

Secondary fixation
After fixation with formal salin , the tissue is treated in another
appropriate fixative e.g. formal mercury chloride , heidehein susa or
zenker fluid for abot 4 hrs . These second treatment improoves the
preservationn , staining of specific tissues constituents and inclussions

Post fixation

Some proteins are masked by fatty substances which do ot allow fixatives

to penetrate the tissue , the tissue is fixed , dehydrated , cleared and then
latter fixed in ethanol to preserve the protein . the tissue is then
recleaed ,impregnated in wax ,embedded and sectioned .

Factors to consider prior to further tissue processing

 SCIENCE LABORATORY TECHNOLOGY
(i) Precise identification of the specimen – these can be labeled using
,aspecial pencil
,type written on carbon linked slip and is to be carrried together in the
specimen throughout processing .
(ii) Small fragments of tissues should be carried or placed in a
piece of thin filter paper or curetin basket to avoid losing them during
processing or contaminating other specimens
(iii) Glass stoppered containers must be used in order to avoid
fumes during manual processing procedures

(iv) The volume of the reagent to be used should be 20 –50 times

the size of the specimensthe surface of the blocks to be cut should be
identified
(v) the duration of the processing should be
know

DEHYDRATION
Dehydration is the complete removal of water from the tissue.
Dehydration is necessary because many embeding media are immiscible
with water . dehydration is achieved by use of reagents or by freeze drying
Dehydrating agents
The common dehydrating agents are ;
Acetone ,
Butyl alcohol
Dioxane Isopropyl alcohol
Ethyl alcohol
Ethyl alcohol is the best dehydrant ,its used in concentration of
70%,80% ,90%and 100% (absolute). Its however expensive .
Dehydration is carried out in stoppered glass jars or glass beakers .
Graded alcohols i.e.( 70-100 % )are used to avoid rapid removal of water
which can cause rapid distortion of the tissue .
Complete dehydration is indicated by change of an hydrous copper
sulphate which will change from white to blue on presence of traces of
water .Also CaO may also be used as accessory agent during dehydration
i.e. CaO dehydrates the alcohol by removing water from alcohol.
CLEARING
Most dehydrating agents used are not miscible with the embedding
mediums that shall be used in the subsequent stages ahead , These, they
 SCIENCE LABORATORY TECHNOLOGY
have to be therefore cleared from the tissues after they have been used so
as to enable the use of these embedding mediums .
Clearing is a process of replacing the dehydrating fluid with a substance
that is miscible to the embedding medium to be employed . some
clearing agents also have some additional advantage of making the tissues
clear and transparent i.e. they improve or increase their refractive index .
Clearing agents must mix freely with the dehydrating agents and the
embedding agent but must also be eliminated or removed in the process
of infiltration of the embedding agent . clearing agents commonly used
are , toluene , xylem , benzene , cedar wood oil chloroform carbon
tetrachloride
and aniline oil. most clearing agents are usually volatile ,toxic and
flammable .the most commonly used clearing agents include;
Xylene
Toluene
Benzene
Cedar wood oil
Chloroform
Carbon tetrachloride
Aniline oil
Carbon disulphide
Carbon tetrachloride
Paraffin oil
Celloslove
Methyl benzoic

INFILTRATION OR IMPREGNATION

Infiltration or impregination is the process of allowing the parrafin wax to

penetrate or enter into the tissue during procesing . its function is to give
support to a tissue block from which thin sections can be cut and stained
for microscopic examination .
These is done by completely soaking the tissue blocks in molten parafin
wax at 56-58 oc and then subsequently allowed to cool and solidify to
produce parafin wax blocks . impregination therefore is the process of
completely saturating the thissues with the medium to be used for
embeding .
Paraffin wax and ester wax is the most commonly used impregnating
media for routine purposed because it gives sufficient surport for easy
 SCIENCE LABORATORY TECHNOLOGY
sectioning of tissues .Waxes used in histology for impregnating tissues
should be soluble in tissues .
Paraffin wax
Parrafine wax is a mixture of hydrocarbons produced in the cracking of
mineral oils . its properties vary considerably .its melting point ranges
from 40-70oc
Parrafin wax contain additives which hardens the wax in order to facilitate
the production of thin serial sections . the common paraffin wax additives
are bees wax ,rubber , stearic acid and diethylene gycol diestearic .
Other forms of paraffin wax used in histology are paraplast, paraplast
plus ,bioloid and tissue mat.
(a) paraplast
It’s a mixture of highly puriffied parafin wax and several plastic polymers
. its more elastic than parafin wax because of its plastic content .
Its sold in form of pellets and its used in the same way as paraffin wax

(b) paraplast plus-

these is a compound of purified paraffin wax and plastic polymers of

regulated molecular weight . it contain dimethyl sulfoxide and it gives
more penetrating power than paraplast .its also supplied in form of
pellets and its used in the same way as parafin wax .

(c) Bioloid

Its similar to parafin wax but contain more plastic polymers .its
recommended as an embeding media for thin walled and circular
specimens to be regained after processing and sectioning

(d)Tissue mat
these is a product of parafin wax which contains rubber . its used in the
same way as paraffin wax and paraplast . its supplied in form of sheets or
mats.
Other impregnating agents are colliding, agar ,gelatin and low viscosity
nitrocellulose wax (LVN) .
Poor impregnation of tissues causes excessive shrinkages and hardening
of tissues hence causing difficulties in cutting of sections . It may also
make the blocks to become too soft that they crumble during cutting and
gives
 SCIENCE LABORATORY TECHNOLOGY
poor sections which breaks out during floating out on water bath

Factors affecting impregnating time

1. Tissue density
Dense tissue require immersion in molten paraffin wax to ensure compete
impregnation. E.g bone tissues.
2. Size of the block of tissue
vacuum embedding / impregnating techniques
Vacuum embedding is a special technique used in preparing special
tissues . there are two types of vacuum embedding techniques
(i) Vacuum embedding
(ii) Peterfis double embedding techniques
These technique depends on the production of negative pressure inside
the embedding oven which hastens the extrusion of air bubbles and the
remains of the clearing agents from the tissue blocks thereby facilitating
rapid penetration of paraffin wax into the blocks.
The time the tissue may remain in the paraffin wax is greatly reduced
compared to ordinary method . this will also minimize the chances of
tissues becoming brittle
These technique is useful for the following tissues .

(i) tough fibrous tissues such as uterus

(ii) dense tissues e.g. brain
(iii) hard tissues e.g. bones
(iv) tissues containing air spaces e.g. lungs
(v) blood containg tissues such as liver and spleen
(vi) poorly penetrated tissues like skin
(vii) tissues made of delicate structure and different consistencies
e.g. the eye
Vacuum embedding oven consist of a heavy brass chamber with a lid of
thick plate glass which rest on a rubber washer to effect an air tight seal
when the washer is in use . It have two screw valves in the upper part of
the chamber to allow exhaustion and admission of air . The greater part of
the chamber is enclosed in a water jacket , the temperature of which is
thermostatically controlled at 2-4 oc above melting point of paraffin wax
being used ..
procedure for vacuum embedding
(i) Fix and dehydrate the tissue
(ii) Clear in xylene for 1hr
 SCIENCE LABORATORY TECHNOLOGY
(iii) Place the tissue in molten wax in vacuum chamber and make the
chamber air tight exhaust the air slowly by means of a vacuum pump
until there is negative pressure of 400-500mmHg
(iv) leave for 15 min then slowly readmit air until normal
atmospheric pressure is reached
(v) place the tissue in fresh paraffin wax
(vi) repeat stage 4 and 5
(vii) place the tissue in another fresh paraffin wax and leave for 30 –
45 min
(viii) bring to normal atmospheric pressure and embed the tissue

EMBEDING
Embedding is the process of allowing the already infiltrated impregnating
material e.g. celloidin to harden inside the tissue celloidin is supplied in
shreds –It gives so much suport that large tissues can be processed , its
also useful for hard and fragile specimens e.g. brain and nerves ,does not
require heat and causes minimum shrinkages and hardening of tissues
Its however extremely slow and only thick sections can be cut , these
blocks must be stored in 70% ethanol and the blocks and the knives to be
used must be continuously moistened with 70% ethanol during cutting .
celloidin is also highly flammable
Gelatin is also another substance that can be used for embedding
especially when cutting frozen sections .all impregnation in it is done at
37oc
Agar can also be used for embedding unfixed tissues which should be
cut on freezing microtomes

Celloidin
Celloidine is the trade name given to purified form of nitro cellulose. Its
used mainly for embedding hard tissues of mixed consistency, cutting very
thick sections, or when minimum shrinkage required and the frozen
section technique is not practicable. It is also important in situations
where heat is not required in the procedure of handling tissues
Evaporation is a constant problem when using celloidin and working
solution should always be stored in bottles treated with brown glass
stopper
N/B Use of celloidine can lead to fire in the laboratory due to ether
vapour which is highly flammabl
 SCIENCE LABORATORY TECHNOLOGY
Celloidin Impregnation and embedding

Impregnation
1. Dehydrate tissue through ascending grades of alcohol. Complete
the dehydration by use of a bath of absolute alcohol.

Complete the dehydration by use of a bath of absolute alcohol containing

CUSO4
2. Transfer the tissue to a mixture of equal parts of alcohol or
ether for24hours. The purpose of this step is to speed up subsequent
impregnation
3. Transfer tissue to a thin(2%) solution of celloidin for 5-7 hours
4. transfer tissue to a media(4%) solution of celloidin for 5-7 days
5. Transfer tissue to a thick (8%) solution of celloidine for 3day

Embedding
1. Half fill the a suitable embedding mould with a thick celloidin 8% and
place tissue in position withsurface to be cut outermost.
Top up the mould with more of the embed solution. The mould should be
considerably deeper than the chickness of the tissue in order to prevent
tissue from becoming exposed as the celloidine shrinks on hardening.
Paper moulds are ideal embedding moulds for this purpose.
1. Place the mould in a dessicator containing ether vapour in order
to remove all the air bubbles. Immediately all the air bubbles are are
removed from embedding media invert the tissue so that the surface to be
cut is facing downwards in the mould. This prevents any air bubbles from
being trapped beneath the tissue.
2. Transfer mould to second desicator containing chloroform
vapour until celloidine is hardened to the required consistency. This can
be tested by pressing the back of the thumb (not nail) against the surface
of the block. The celloidin being hard enough when no impression is left
on the surface.
3. Remove the block from the mould and place it in pure
chloroform. The block floats at first but eventually sinks to bottom of the
solution. When block has sink, transfer it to solution of70% alcohol until
required for cutting
Advantage of celloidin as embedding media
1. It permits thicker sections to be cut
 SCIENCE LABORATORY TECHNOLOGY
2. Its rubbery consistency makes it of great value when sections
are required from blocks of tissue that are either very hard or composed of
a number of tissues of varying consistency.
3. As heat is not required during impregnation, the tissue, less
shrinkage occurs in celloidine sections than in those prepared by paraffin
wax.

Disadvantage of celloidin as an embedding media

1. This method is too slow (impregnation takes several weeks and

canot be used in situations where time is an important factor
2. The blocks and sections must be stored in 70% ethyl- alcohol
otherwise drying will occur.
3. Sections of less than 10mm can not be easily cut
4. Serial sections are difficult to prepare owing to each one having
to be handled individually during cutting and staining.

Advantage of paraffin wax as an embedding media

1.It permits thin individual and serial sections to be cut with ease from
majority of tissues
2. It allows a multitude of staining technique to be used
3. It facilitates storage of blocks and unstained mounted sections

Attaching of bock to holder

The celloidin blocks are attached to wooden or vulcanised holders with
deep serrations contained on to them. The block holder is coarted with
medium(4%) celloidin and the trimmed block pressed firmly into position.
Pressing is done by a lead weight or thread around the holder and the
block. After about 1hour during which time the block and holder can be
returned to the chloroform dessicator, once it is firm , the block and the
holder should be reimersed in 70% alcohol for 30min. The cutting surface
of block may be trimmed with a hard sharp razor
N/B;The wood or block should be soaked before placing in 70% alcohol to
ensure that decolourization of alcohol and block does not occur.
Hardening can also be done by allowing vapour to evaporate and celloidin
to thicken
Low viscosity nitrocellulose (LVN)
 SCIENCE LABORATORY TECHNOLOGY
This is an embedding media which is preffered to celloidin because it
forms a harder block than celloidin and thinner section can be cut. The
section have a tendancy to crack but a plasticizers can be inco-operated
into LVN to overcome this problem,
The addition of 5% castor oil is recommended for embedding chrom
mordanted tissues
Procedure for LVN
Solution-1
LVN…………………………………7GM
Ethyl alcohol absolute………42ml
Ether…………………………………50ml
Oil resin…………………………….0.5ml

Solution-2
LVN………………………………… 14GM
Ethyl alcohol absolute……………… 42ml
Ether………….………………………50ml
Oil resin………………………....……0.5ml

Solution-3
LVN……………………………...... …28gm
Ethyl alcohol absolute..... ....... 42ml
Ether…………………………… …50ml
Oleum…………………………...…0.5ml

Mode of preparation
For solution 1,2,3, dissolve the LVN in alcohol and ether ADD the oleum
resin , mix well and label

Procedure
1. Dehydrate tissue according to celloidin technique
2. Place n solution 1 for 4-7 days
3. Place in solution2 for 4-7 days
4. Embed in solution 3 and continue according to celloidine
technique
Sections should be cut dry and collected in 70% alcohol
Peterfis double impregnation method
This method is valuable and for preparation of sections from blocks of
tissues of varying consistence
 SCIENCE LABORATORY TECHNOLOGY
Composition
Celloidin dry …………….1gm
Methyl benzoate ………………100ml
Place celloidine in 200mldry flask and add benzoate and stop at the flask
firmly
Shake several times daily occasionally
inverting the flask until the solution of the celloidin is effected.

Procedure for use

1. Dehydrate according to normal schedule for celloidin
2. Transfer to methyl benzoate to absolute alcohol and impregnate
for 24-72 hours.
3. Pass 3 changes of toluene over a period of 24 hours
4. Impregnate and embedded in
paraffin wax according to normal
procedure of paraffin wax impregnation.

Procedure for embedding in cellodin sections

Position the specimen in a paper boat and fill to the brim with cello din
wax.
place the boat under a bell jar with one edge of the jar slightly raised by
means of a march stick or an applicator stickthe boat may be placed in a
desiccators to harden
hardening is complete when a finger makes no impression on the surface
store the blocks in 70 % ethanol until when ready for cutting

MOUNTING OF THE BLOCKS FOR SECTIONING

The block is trimmed to the required size using wooden blocks .a

generous amount of 2% cello din is applied on the block and the cello din
block is firmly raised into position and kept in these position for at least I
hour. The seal is hardened by placing it on 70% ethanol exactly 30 min
before cutting
CASTING OF PROCESSED TISSUES
Casting is the arranging the impregnated tissue in a precise position in a
mould containing the embedding medium and allowing the medium to
solidify. It’s simply referred to as tissue embedding
 SCIENCE LABORATORY TECHNOLOGY
ORIENTATION OF TISSUES
These is arranging of tissues in a mold. The position of tissues in the mold
should be decided before processing so that the opposite side is marked
with Indian ink or by making a groove or knot.
The soft parts should be cut first.
Orientation also refers to positioning of the blocks on the microtome
prior to sectioning

TYPES OF MOLDS
A variety of moulds are used for blocking out tissue or embedding after
impregnation. They include:
1. Paper boards
Have the advantage of being cheap to make and blocks to be stored
without being removed. A successful method of making the position of
minute pieces of tissue in the paraffin wax is to draw across with a soft
lead pencil or tie the inner surface of the bottom of the board. The mark is
clearly visible on the wax block when it’s removed from the paper board
and the position of the tissue is easily known

1. Watch glass
watch glass are ideal for embedding fragmented specimens smeared with
glycerin to make removal of glass block easier from the watch glass.
2. Plastic ice tray
This forms convenient moulds for busy laboratories, one block being
embedded in each compartment of the ice tray when set the wax block are
easily removed by flexing the ice tray. This can be facilitated by smearing
the inside of the ice tray with glycerin or liquid paraffin
3. Leuckhard embedding boxes
This is convenient molds for team work and is widely used. They are two l-
shaped pieces of metal .e.g brass and are arranged on glass or metal
plates to form a mould of the desired size.
When wax has solidified, the mould and the encased block are removed
from the base of the block and the two L- pieces comes out of the block
and ready for use
other types of molds used are
(i) Petri dishes
(ii) Cover glass boxes
(iii) Tissue –tek II
Technique for using embedding moulds
 SCIENCE LABORATORY TECHNOLOGY
1. Fill the mould with paraffin wax.
2. Warm a paire of blurnt nosed forceps and use to transfer the
tissue from the paraffin bath to the mould
3. Warm the forceps and oriented the tissue until its inclined in
desired place.
4. Run the warm forceps around the tissue to ensure any solidified
wax which may have solidified during transfer from the paraffin bath to
the mould is melted
5. Remove the corresponding label of the specimen from the
paraffin and place it adjacent to the tissue
6. Transfer the mould to gcold water andimmerse it gently. The
mould should remain submerged until wax is hard
7. Transfer the wax in running water to solidify further

Gelatine embedding method

Frozen tissues as a general tissue, from which frozen tissues are
prepared is not embedded, freezing of tissues provides sufficient support
for sections but its advantageus to ensure the tissue IS in surportive
media to prevent the tissue from fragmentation.

The embedding media is gelatin for this purpose. After embedding the
block of tissues are transferred to formaline in order to harden them. The
formaline changes structure of gelatine from hydrosol to hydrogel
Aschoff Gelatin Embedding Method
Solution-1
Gelatine…………………..125gm
Distilled water……… ….87ml
Phenol(preservative)……...…1gm

Solution -2
Gelatine …………………………25gm
Distilled water………………….75ml
Phenol crystal…………… …1gm

Solution -3
Concentrated formaldehyde..5ml
Distilled water………………… 95ml

Mode of preparation
 SCIENCE LABORATORY TECHNOLOGY

Heat distilled water to 37oc and dissolve the phenol. Add gelatine and
incubate at 37oc until solution is effected. Fill that through surgical gause
bottle and leather
Add concentrated formaline solution. Mix well and label

Preparation for use

Melt the gelatine by heating in a water bath

Procedure

1. Place thoroughly washed formaline fixed tissues not

exceeding 3mm thick in solution-1 and incubate at 37oc for 12hours
2. Transfer to solution 2 for 12-24 hours at 37oc
3. Embed in solution2 using a loop of hair embedding in cool and
trimmed( excess gelatine inhibits trimming)
4. Place the trimmed block in solution-3 for 24hours. Cut the
sections by use of a microtome.

MICROTOMY
Microtomy is the process of cutting or sectioning tissues into thin slices or
sections after tissue processing on a microtome. The thickness of the
tissues sectioned will depend on the type of the tissue and the
embedding media used.
There are about six types of microtomes used in histology i.e.
(i) Rotary microtome: This is the most common type of
microtome. It is the best in cases where serial sections are required.
(ii) Cambridge rocker microtome. It is the simplest and oldest
type of microtome
(iii) sliding microtome : it differs with other types of microtomes
as in sliding microtome blocks remains stationery while microtome knife
moves during the process of sectioning. it’s the best microtome for cutting
tissues embedded in celloidin
(iv) Base sledge microtome :
Suitable for sectioning tissues embedded in all forms of media. Its
excellent for cutting sections from blocks of tough tissues especially large
block which offer resistance to the knife

(v) Freezing microtome:

 SCIENCE LABORATORY TECHNOLOGY
Used for cutting sections when:
a) Speed is of utmost importance.
b) When it is required to demonstrate fats histologically.
c) When neurological structures are to be studied.
The cooling of the freezing microtome stage is done by use of liquid
carbon dioxide in a cylinder connected by means of flexible lead which is
hollow and perforated around the perimeter. This perforations are
essential part of the cooling, allowing the gas to flow and freely escape,
thereby producing even freezing of the tissue. A second freezing facility
for the knife is also inco-operated. A thermoelectric cooling unit may also
be used in place of Carbon dioxide gas to freeze the knife and this is
called Thermomodils

(vi) Cryostat microtome:

The best method of preparing sections from unfixed tissues is by use of a
cryostat. This consist of microtome housed in in a deep freezer cabinet
maintained at a temperature of approximately -15oc-30oc. to avoid the
formation of Large disruptive ice crystals when freezing fresh tissues, a
rapid freezing is necessary, a cryostat usually provides a rapid freezing
attachment for this purpose and for attaching blocks of tissue to the
block holder. Blocks of fresh tissue not to be sectioned immediately
should be quenched and stored at temperature of -20 oc in air tight
containers or aluminium foil.

Microtome knives
This are classified according to their cross sections as follows.
a) plano concave it has a hollow ground on one sid
b) wedge shaped: This is plain on both sides
c) biconcave: this is hollow on both sides

Cutting Sections Using A Microtome

Preparation of paraffin wax sections

If several sections of tissues are embedded in the same mould, it must be
divided into several blocks, this may be done by cutting a v – shaped
groove on the intervening wax and breaking it along the line.
The individual paraffin wax blocks should then be trimmed with a hard
razor to be 1/8 of an inch of the tissue, taking care by the side of the blocks
if parallel. Excess wax on the side of the block should be paved off
 SCIENCE LABORATORY TECHNOLOGY
attaching the block to the holderHeat a wooden handled spatula over a
Bunsen burner and hold it on the surface of the block holder.Place the
paraffin waxed block on the spatula, the hot spatula melts the wax on the
block holder and base of the block.After few second the spatula is
withdrawn and the block placed firmly on the holder. The section between
the two is now sealed by heating the spatula and running it around the
base of the holder and paraffin wax on.
Orientation of the blocks on the microtome
- Fix the holder on to the position on the microtom
-Turn Back the feed mechanism( open the knife holder)
-Insert a suitable side the microtome and secure it to the tightening
screws
-move the block holder forward until the paraffin block is almost touching
the knife edge
N/B Make sure that the surface to be cut at the lower edge to the block are
parallel to the knife edge
-Check all the tightening screws on the microtome
-Set the gauge controlling thicknessto 15 um and extreme end of the knife
is used to trim the block until the whole surface is cut , the tissue is now
ready for sectioning.

Cutting Of Tissues
Set the gauge to the required thickness and position the knife so that the
centre of the block is positioned for cutting.Screw back the screw
mechanism slightlyOperate the microtome until the complete section on
being cut and maintain a regular cutting rhythmThe cutting gauge varies
with the nature of the tissue and size of the block if the block face and
upper and lower edges are parallel to the knife the section will form a
ribbon
This ribboning is due to slight heat generated between the block and the
the knife edge which joins the cut sections together to form a ribbon
.cutting continues until the ribbon evolved is about 6 inches or15 cm .
-Moisten a second hair brush and gently raise the last section cut thereby
freeing the ribbon placed surface uppermost to a section board or sheet of
black paper
when serial sections are being prepaired the section from drought and
dust

Factors that can lead to poor cutting of section on microtom

 SCIENCE LABORATORY TECHNOLOGY
1.Poor trimming of the block
2. incorrect orientation of the block of wax on the microtome
3. inadequate impregnation.
If dehydration, clearing of wax, impregnation are inadequate cramping of
sections may occur. This fault is easily detected as the block usually smells
of clearing agent
N/B To correct this, the block should be trimmed down as near to the
tissue as possible. Return to the embedding oven to melt the remaining
wax and then transfer to the clearing agent
-If the dehydration is suspected as being a fault, the tissue should be taken
up to absolute alcohol.
4. imperfect knife edge
More failure can be attributed to badly prepared knife than to any other
single factor
-Nicks In the knife edge result in scouring of the sections with vertical
lines. To overcome this the knife has to be resharpened
-If the knife is blunt, the section may cut alternately thick and thin. To
solve this problem, the knife has to be resharpened
5. Incorrect setting of the knife The knife is set at atilt on the microtome
to allow clearance angle between the cutting face and the block of tissues
NB Clearing angles should be 1-6 faults due to incorrect tilt

Faults due to incorrect tilt

i) chattering
This is a term used to describe horizontal lines or furrows across the
section. This can be reduced by reducing the angle of tilt
ii) Intermittent cutting
If the angle of tilt of the knife is too small the block is comprest by cutting
facts and sections are not cut at the same point. The degree of
compression increases until the tissue expands suddenly and results in
cutting of thick section.This can be solved by increasing the angle of the
tilt.
1.Brittle and hard tissues
When possible such tissues should be cut on the base of base sledge
microtome
2. Dirt in the impregnating wax
When dirt is in the impregnating wax, the embedding of tissue in freshly
filtered wax is necessary.
Other factors
 SCIENCE LABORATORY TECHNOLOGY
presence of calcium in the tissue due to poor decalcification.

ATTACHING SECTIONS TO THE SLIDE

As the sections tend to shrink slightly on cutting, and they must be
flattened by gently heating before being attached to the slide,
Two methods are commonly used
-Water bath method
-hot plate method
Before flattening, the glass slide should thoroughly cleared and be
inscribed by diamond with appropriate name and identification before use

.WATER BATH METHOD

A cut Section or short ribbon of section is gently lowered by means of a
carmel hair brush or fine forceps on to the surface of a warm water in a
water bath
The temperature of water should be approximately 10oc below melting
point of wax.
When the section is flat and fully expanded a prepaired clean grease free
slide is deeped obliquely(parallel) into the water as close to the section as
possible
Slowly withdraw the slide allowing the surface to touch the edge of the
section and then completely remove the slide with the attached section
and adjust the section to the suitable position on the slide with the needle.
Drain off excess water and transfer the slide to an incubator or hot plate at
45-50oc for atleast 1 hour to ensure the section is thoroughly dried before
being stained

HOT PLATE METHOD

A clean grease free slide is placed on a warm hot plate and floated with
distilled water. A section or a short ribbon is laid on the surface of the
water and any major greases removed by stretching the wax amounted
needle. As the water worm up the section will flatten up and when its fully
extended remove the slide from hot plate and drain off any excess fluid.
Dry the section as in the previous method. In incubator at 45-50oc
Terminologies used in microtomy
(i) Ribbon – these are sections or slices of tissues which are
attached to each other cutting
 SCIENCE LABORATORY TECHNOLOGY
(ii) Serial sections – these are sections or slices of a tissue
produced consecutively during section cutting . this is achieved
progressively through an entire tissue block
(iii) Class sections – these are 50-200 sections cut serially and
mounted singly on a microscope slide . they are used for class teaching
purpose
(iv) Slant of a microtome knife – this is the angle the knife edge
makes with the line of section or direction of travel of the object
(v) The tilt of the microtome knife – these is the angle between the
surface of he block and the line bisecting the edge of the knife

Points to consider when sharpening (horning) knife

(i) Never use the same glass plate for knifes of different length
(ii) Always center the knife precisely
(iii) Use one surface of the glass horn plate for coarse horning and
the other for fine horning and mark them according
(iv) Use proper oil base abbrassive for each type of horning and
wash both knifes and plate well after each phase
(v) Follow the manufacturers instructions
NB horns are also called oil stones because oil is commonly used as a
lubricant

SECTION CUTTTING
Requirements
(i) Microtome in proper working condition
(ii) Well processed block tissue
(iii) Readily sharpened knife
(iv) Albuminized slides
(an adhesive )
(v) Water bath at 2-30c bellow the melting point of wax
(vi) Camel hair brash
(vii) 20 –30 % alcohol

Sectioning is done in two stages ;

(i)Trimming – this is the process of cutting off the excess wax untill full
piece of cutting section is exposed or obtained , these is done using a
blunt knife
(ii)Actual section cutting
Set the microtome gauge to the required thickness
 SCIENCE LABORATORY TECHNOLOGY
· Put the knife on the knife holder and tighten
· Put the blocks on the block holder and tighten
· Swing the microtome in a smooth systematic manner
· Pick up the sections from the knife by means of camel hair brush
· Run 20 –30 % alcohol underneath
Put the section on a clean slide leave the sections until no microtome
folds are seen and until the sections are straight and foldless.

Floating Out Sections

(i) Now pick out sections .
(ii) Put the sections on the slide .
(iii) Float the sections in warm water bath until its microscopically
foldless
(iv) Attach the sections on the slideB(albuminize ).
(v)Label the patient number on the slide and leave it in the molten
paraffin wax oven
(vi)stain the tissue

Section adhesives

Section adhesives is a sticky syrupy fluid used to attach the tissue onto a
microscope slide so that the sections does not wash off during the
subsequent vigorous treatment i.e. stages of staining
A section adhesive is especially essential for techniques which employ
certain reagents like ammonia with a tendency of detaching the sections
from the slide (also other alkaline reagents ).,
Section adhesives are also important in the techniques which involves
long periods of immersion in dyes or reagents .
However section adhesives may not be strictly necessary in
haematoxylene and eosin methods of staining but its applied to save the
previous sections from any dangers of detaching
Section adhesives include ;

(i) Mayers egg albumin

It consist of the following;
Glycerine pure
White of egg
Distilled water
Crystals of preservatives e.g.
 SCIENCE LABORATORY TECHNOLOGY
thymol and phenol

(ii) Starch paste

consist of;
Powdered starch
Cold water
Concentrated HCL
Thymol
(iii) Glycerin jelly
Consist of;
Gelatin
Glycerin
Distilled water
NB- The preparation gives a completely clear background.
Albumen retains some of the stains thereby giving a dirty background .
Some thymol resistant microorganism may be found to grow on these
adhesives .
After preparation, the adhesives are stored at a freezing temperature and
melted when required for use. Glycerin jelly is mostly routinely used.

PREPARATION OF FROZEN SECTIONS

Method of Cutting Frozen Sections
1. Clamp the microtome to the bench and connect the carbon dioxide
cylinder by means flexible pipes provided.
2. Close the release valve on the microtome and open the valve on the
cylinder
3. Insert the microtome blade into the knife clamp and tighten the locking
screws.
4. Place a piece of filter paper soaked on gum syrup 0n the stage of
microtome and open the stage release valve and allow short bursts of
carbon dioxide gas to escape freezing the filter paper to the stage.
5. Position the block of the tissue on the stage and apply a few drops of
gum syrup on it.
6. Adjust the microtome until the surface of tissue is just about the level
with the edge of microtome.
7. Set the thickness gauge to the required gauge and cut the sections

Floating Out Frozen Sections

The cut sections are placed in a deep glass dish of distilled water.
 SCIENCE LABORATORY TECHNOLOGY
A slide is inserted beneath the section and slowly withdrawn to remove
the attached section from the water. Finally drain the excess water from
the slide.

Attaching Frozen Sections to the Slide

Frozen sections may be attached to the slide by celloidinization, albumen
or starch adhesive. They can also be attached by simply floating then onto
celloidinized slide.
Celloidinization
Float the section onto clean grease, free slide.
Draw off excess water and bloat using filter paper, bloat with a section
filter paper soaked in absolute alcohol.
Coat the slide in 1% cello dine in ether alcohol, when celloidine has harden
the section can be stain in normal manner.

Albuminized Or Starch Slide

Float the section on a starch slide spread with starch paste and drain off
excess water.
Bloat the sections using fielter paper and cover with a few drops of
mixture of equal parts of clove and aniline oil. Allow the mixture to
coagulate for 3 minutes and then remove it from the slide with xylene
followed by alcohol and stain in normal manner

Gelatinized Slide
Float the sections on a slide smeared with 0.2% gelatine and allow to dry
drain off excess water and transfer the slide to a dish of formaline vapour.
The action of which converts to an irreversible gel and hold the sections in
place. Remove the slide and wash in running tap water. Stain the sections
in the normal manner.

Preparation Of Celloidine Sections

The microtome most suitable for sectioning celloidine embedded tissues is
the sliding type. To avoid dehydration and shrinkage during cutting of
celloidine
The sections and the blocks are kept wet all the times with 70% alcohol.
When cutting knife sections and block are kept wet by applying 70%
alcohol to the knife by means of a large carmel hair brush. When cut, the
 SCIENCE LABORATORY TECHNOLOGY
section and the block are stored in a similar solution in a jar with is tightly
fitting lids.

Technique for cutting celloidine sections

Clamp the vulcanized block securely into the object holder of thew
microtome and turn the feed mechanism. Secure the aplano concave knife
into the the knife holder and adjust the angle of slant to about 4o.
oriented the block until the surface to cut and the edge of the knife are
parallel. Setb the thickness gauge to about 15um. Float the knife and block
with 70% alcohol and trim block until complete section are cut to the
required thicknes are refloat with 70% alcohol and continue to cut the
sections quickly. Flatten the section by transfer into 70% alcohol. When
serial sections are required small pieces of paper are placed over each
section and each piece should be numbered

STAINING
Staining properties of dyes
Stains may be considered as having micro-anatomical or cytological
properties.
Micro-anatomical are used for demonstrating the general relationship of
tissues to each other. Nucleus and cytoplasm are differentiated but their
included structures are not necessary emphasized.
Cytological stains demonstrate minute structures in the nucleus and
cytoplasm of the cell without necessarily aiding in the general
differentiation of the various tissues types.
Staining brought about by the aid of a mordant is called indirect staining
e.g
haematoxylin, conversely where a mordant is unnecessary as in the
majority of aqueous or alcoholic stain , the term direct staining is used.
Mordants are metallic substances which act as a a link between stain
and tissue to be stained. They may be used in three ways;
1.Before application of the stain( pre- mordanting) e.g in weigers ion
haematoxylin where iron chloride is added before staining
2. In conjuction with stain ( metachrome staining) e.g in ehrlich’s acid
alum haematoxylin.
3 .After application of the stain (postmordanting) e.g grams stain.
 SCIENCE LABORATORY TECHNOLOGY
Substances when inco-operated into staining solution increases staining
power of the solution without acting as a mordant are termed as
accelerators.
Stains which colour tissues elements at in a definite order are termed as
progressive stains.
Those which colour all tissue elements at same time and necessitate
washing out(differentiation) before individual elements can be studied
are termed as regressive stains.
Staining of inclusions in live cells is reffered to as vital staining. Living
cells may be stained after removal from organism (supervital staining)
or while still part of the body(Intra vital staining) .
Some tissue assume different colour from that of the solution in which
they are immersed, this is called metachromatic staining. this is seen
in basic anilin stain e.g methyl violet.
Negative staining is used for examination of bacteria morphology.
The organism and substance of choice are mixed on a slide when the
organism are examined microscopically the un stained organism are
sharply contrasted against a black background.
Certain tissues and organism are demonstrated by a process called
Impregnation. The solution used in the impregnation technique are not
stains but are solution of metallic salts. They differ from stains in being
colourless. A stain is absorped by tissue but impregnating agent is
deposited on its surface.
This makes certain organism appear larger than they actually are e.g
spirochaetes.

Basic, Acid And Neutral Stains

Basic stains
In basic stains such as methylene blue the coloured substance contained
in the basic part of the compound.
The colourless acid radical is usually derived from HCL, H2SO4 or acetic
acid
Acid stains
In this e.g Eosin the coloured substance is contained in the acid
component and acid base is usually sodium
Neutral stains
Obtained by combination of equal solution of basic and acid stains.The
resultant precipitate are usually insoluble in water but soluble in alcohol.
E.g Leishman’s stain.
 SCIENCE LABORATORY TECHNOLOGY
Nuclei are usually stained by basic stain and cytoplasm by acid stain.
Neutral stains will colour both nuclei and cytoplasm.

NB;
Certain parts of tissues are acid in character e.g the nuclei of cells while
other parts such as cytoplasm have basic reaction as mentioned above, the
coloured substance in a basic stain is contained in the basic part of the
compound. The acid radical being colourless, conversely with the acid
stain, the colour substance is contained in acid component while the basic
component is colourless
Differential stain reaction is due critical combination being formed
between the tissue and the stains inv olved, thus acid tissues elements e.g
nucleus will have high affinity for basic stains while cytoplasm(basic in
character)_ will have an affinity for acid stains
Bacteria which are rich in ribonucleic acid therefore have an affinity for
basic stains and tend to be un affected byacid stains in bacteriology the
acid stain are used mainly in negative stain technique in which the
bacteria are seen unstained against a stained background

Staining procedure
There are three methods of staining slides in common use
1. using staining dishes
2. using staining rags
3. using staining machines

Staining dishes
Avariety of this staining dishes is available. Small jars used for staining
single single slides coplain jars that hold 5-10 slides and also large staining
troughs with separate baskets that are able upto 20 slides to be stained at
the same time.

Staining rags
Oftenly used in medical laboratories. Two glass rods 2 inches apart are
fixed across the sink. The slides are laid across this rods and the solution
(stain) poured onto the slides using a drop bottle

Staining machines
Used for staining large numbers of slides they have greater application in
cytology and haematology for staining smears than for staining sections.
 SCIENCE LABORATORY TECHNOLOGY

Procedure for staining

1. staining of paraffin wax sections
Before sections preparation by paraffin wax technique can be stained the
surrounding coax must be removed and the sections transferred through
grades of alcohol to distilled water in order for them to be hydrated.
Steps ( steps in dewaxing)

a) Dewaxing
Free the section from paraffin wax by immersing the slide in xylene for 2-
3 minutes. This process may be speeded by first gentle worming the
section over a Bunsen burner until paraffin wax just begins to melt
b) Transfer the slide to absolute alcohol for 30 seconds to remove xylene
c) Transfer the slide to section dish of absolute alcohol for further 30
seconds to ensure all all xylene is removed and not carned over to lower
grades of alcohol
d) Transfer the slide to 90% acohol for 30 seconds
e) Transfer the slide to 70% alcohol for 30 second
f) wash the slide thouroughly in distilled water.
After staining the section is passed through the graded alcohol to
dehydrate it, that is70%, 90% and two changes of absolute alcohol. Wash
thoroughly in
absolute alcohol

Clearing
After staining and dehydration the tissue is cleared in two changes of
zylene. The main reason for this is to ;
1- The sections after having been immersed in zylene is miscible wuth
xylene balsam(DPX)
2- The refractive index of tissues is raised after a 2nd clearing so that its
approximately the same as that of the glass slide to which its attached.
This is an important factor as the refraction of light is reduced to a
minimum when the section is examined under the microscope

Staining frozen sections

 SCIENCE LABORATORY TECHNOLOGY
Frozen section may be attached to the slide before staining or may be
stained separately . Some sections in which fat is to be demonstated is
normally stained by being passed through small quantities of staining
solution by means of glass rod. After staining the sections are floated out
in a dish of water, picked on a slide and mounted in aqueous mounting
media.

Control and test slides

No positive slide should be used as a control with all specialized staining

procedures. The control slide / test slide will enable the worker to
determine the end point of a given staining procedure. A control slide can
also be to test quality of new batches of stains that have been purchased
before being used for routine staining purposes in histological laboratories

Mounting of sections

After stains have been stained it must be prepared as a permanent

preparation for microscopic examination. This is accomplished by
mounting the sections in a suitable medium under a glass coverslip. The
mountant most commonly used for mounting stained sections are Canada
balsam which may be acidic or neutral, DPXThe choice of medium
depends entirely upon the the staining procedure used.

Method of mounting sections

1. Clean a cover slip of appropriate size and place it on the bench on a

sheet of filter paper.
2. wipe off excess xylene from the slide with a dust free cloth.
3. lie the slide on the filter paper in front of cover slip.
4. Gently bloat the section with a folded sheet of filter paper5. Place the
necessary amount of mounting media on the section.
5. Quickly invert the slide and lower it on the cover slip applying gentle
pressure until the mounting media flows gently to the edges of the cover
slip
6. Turn the slide over and if necessary squire up the cover slip by means of
a mounted needle
 SCIENCE LABORATORY TECHNOLOGY
7. After the section have been mounted to the slide should be transferred
to an incubator at 37oc for12-24 hours to harden the mounting media but
if slide are required for examination quickly , they can be placed in a hot
plate to dry fasterNB;
The reason for bloating the sections is to remove all excess zylene. If this is
not done,. The xylene will mix with mounting media and form air bubbles
which becomes trapped beneath the cover slip. On no account should
sections be bloated so hard that it becomes dry and shrinkens and
cracking results.
Excess balsam may be removed by wiping with a clean duster dipped in
zylene
If DPX is used as a mounting media excess amount should be placed on
the slide, the supplirs being stopped off 24hrs later when it has hardened.
This is necessary in order to counteract the shrinkage which it undergoes
on drying. the cover slip to be used should be stored in absolute alcohol to
prevent contamination with moisture and water

MOUNTING MEDIA
Media for mounting microscopic preparation may be divided into two
groups-
(i) Aqueous media
(ii) Resinous media
Aqueous media
This is designed to make temporary or permanent mount of water
miscible preparations. the formulae consist of solidifying agent such as
gelatine or gum arabic to which is added glycerol to prevent drying and
cracking. Various sugars to bring about increase in refractive index and
preservative.
Example of aqueous media are : glycerine jelly, and hard corn syrup
Resinous media
This may be divided into natural and synthetic mediaq. The most
important of natural resins is Canada balsam
Examples of aqueous mountants are;

Glycerine jelly
Formulae

Gelatin……….....................….10gm
Glycerol……......................…..70ml
 SCIENCE LABORATORY TECHNOLOGY
Distilled water…................…..60ml
Phenol.Crystal( preservative).0.25gm

Mode of preparation
Weigh the gelatine into distilled water and incubate at 60oc. Until the
solution is effected, add glyceral and phenal crystal. Mix well, label and
store in a fridge at 4oc
Preparation for use

Melt the glycerine jelly by heating in a water bath or incubator at 60 oc. To

media prior to use

2. Card corn syrup

Formulae
Card corn syrup(clear volume)
Distilled water ........2 volume
Thymol as preservative(1 crystal)

Mode of preparation

Dilute the corn syrup with distilled water and add the thymal, mix well
and store in a refrigerator at 4oc
(ii) Resinous mountants

1. Neutral balsam
Dissolve Canada balsam in xylene to form a firmly thin solution. (40_%-
50)%). Add calcium carbonate in excess and stir thoroughly.
Allow the mixture to settle. Decant the fluid into a bottle and discard the
residue.Record date and label

2. Canada balsam
Dissolve Canada balsam in xylene to form a fairely thin solution (50%) .
add Nalycylic acid to excess and stir thoroughly.Allow the mixture to
settle. Decant the mixture and store in a bottle

Synthetic resins
 SCIENCE LABORATORY TECHNOLOGY
Prepared by dissolving polystyrene in a romatic hydrocarbon solvent and
adding aplasliesers e.g dibutyphthalate to prevent formation of airspaces
on drying
Examples of synthetic resins-
Formulae
Disterine………...........................10gms
Dibutyphthalate……...................5mL
Xylene……………….....................35ml

Combine the dibutyphthalate with xylene and mix well. Add disterine
and record date and label
NB;To remove the cover glass from preparation mounted with DPX
immerse in trichloro xylene preparation mounted in DPX should be
cleared in xylene free from paraffin wax.

DECALCIFICATION
Decalcification is the process of removal of calcium from a tissue to
facilitate cuting it into the required sections. Decalcification is necessary
for the following specimens
(i) Bones:blocks suitable for sectioning are selected from the gross
specimen by a means of a fine fret saw. It should not exceed 5mm in
thickness. Damage to the edges can be remedied by trimming the
decalcified tissue with a hand razor
(ii) Teeth
(iii) Calcified tissues e.g. –calcified lymph nodes, calcified arteries,
calcified thyroid glands, chronic tuberculosis foci ,cacified scar formed as
a result of deposition of necrotic tissue whereby the calcium salt in it
accumulate and hardens bone marrow

Technique Of Decalcification
1. a thin slice of the tissue is suspended in the decalcifying solution by
means of a waxed thread( to protect from acid) the solution should have
free access to the tissu
2. The degree of decalcification should be checked daily and the final
stages more frequent tests are made, e.g hourly. The fluid should be
changed following each positive test
3. when decalcification is complete the tissue may be neutralized by 5%
sodium sulphate and then washed overnight in running tap water after
certain decalcifying
 SCIENCE LABORATORY TECHNOLOGY
agent the tissue is transferred directly to 90% alcohol followed by several
changes of the alcohol
4. the tissue is then dehydrated, cleared , vacuum impregnated and
embedded in paraffin wax

Degree of decalcification

There are three methods of checking the end point of decalcification

1. Mechanical examinationThis involves probing with a fine needle an

bending of tissue. The the needling procedure is liable to cause artefacts
and foci of decalcification may not be detected

2. x- rays
This is the method of choice but can not be used if the tissue has been
fixed in mercuric acid (radio -opaque)

3. Chemical test
It is simple , reliable and convenient, it is carried out as follows
(i) Measure 5ml of used decalcifying solution into a clean test tube and
add a small piece of litmus paper. It changes to red owing to used acid
(ii) Add strong ammonia drop by drop until the litmus paper turns blue
again indicating alkalinity
(iii)if the solution becomes cloudy, calcium
is present in considerable amounts and the tissue should be placed in
fresh decalcifying solution
If the solution remains clear add 0.5ml of saturated aqueous solution of
ammonium oxalate and allow to stand for 30 minutes. If trace of
cloudiness occurs at this point due to formation of calcium oxalate,
decalcification is incomplete and emersion into fresh declassifying
solution is required.
If the solution remains clear, decalcification is regarded complete. It is
important to use distilled water to prepare decalcification solution to
avoid false positive test
Decalcification solution
(i) Formic acid(HCOOH)
It s recommended for post morterm and research. The time required for
decalcification is 2-7 days
Formulae:
 SCIENCE LABORATORY TECHNOLOGY
Formic acid-( s.g 1.20)...........................5ml
Distilled water ................................... 90ml
Formaldehyde(40%) ............................5ml
This solution permits excellent staining results . the disadvantage is that
decalcification is very slow owing to the above strength
This can be speeded byby increasing content of formic acid to 25ml
however the disadvantage of using more than 8% of formic acid is the
opacity that interfeares with the chemical test for decalcification
Nitric acid formaldehyde
Its required for urgent biopsies. Time for decal. Is 1-6 days
Formulae: nitric acid-(s.g 1.41) ........................10ml
Formaidehyde(40%)……… ..........................…5-10ml
Distilled water………...............................…….10-100ml

Advantages
It’s a rapid acting decalcifying Solution which permits good nuclear
staining
Disadvantage
Nuclear staining is not as good as that obtau=ined following slow acting
solution. Nitric acid frequently develops a yellow colour owing to the
formation of nitrous acid. This increases the speed of decal. But impaires
the subsequent staining results. 0.1% urea when added to the pure
concentration of nitric acid temporally arrests the discolouration and
does not appear to affect the efficiency of the acid

Aqueous nitric acid

It’s a rapid decalcifying solution used for routine work
Formulae:
Nitric acid(s.g 1.41)………5-10ml
Distilled wate…………….to 100ml

Advantage
Causes very little hydrolysis and staining results are very good

Disadvantages
Refer to nitric acid formaidehyde
Pereny”s fluid
Its good for routine use. Time is 2-10 days
Formulae:
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Nitric acid 10% aq. Solution……………....................…40ml
Absolute ethyl alcohol…………………......................…30ml
Chromic acid 0.5% aq. Solution……………………….30ml
When frequently mixed with the solution, its yellow but it rapidly assumes
a yellow clear colour

Advantage
No hardening of the tissueCellular details are well preserved Subsequent
staining is good when decalcification is complete there is no washing and
the tissue may be changed directly to 70% alcohol ( several changes)

Disadvantages
It is rather slow for decalc. Dense bones
Chemical test can not be used to to determine end point of decalcification
due to precipitate formed when ammonia is added to perenys
fluid.However a simple modification can be used.
Transfer 5ml of used decal. Solution to a chemically clean test tube and
add a small piece of litmus paper
Add ammonium hydroxide solution drop by drop mixing between drops
until the solution is alkalineAdd glacial acetic acid drop by drop until the
precipitate is dissolved
Add 0.5ml saturated aqueous of ammonium oxalate
The appearance of a white precipitate within 30min indicates the presence
of calcium

Von-Ebners fluid
The use of this fluid is recommended for teeth. The time is 3-5 days. There
are various formulae given but this gives the best results
Distilled water………………............................................50ml
Saturated aq NACL(36%…….........................................50ml
HCL……… …………................................................……. 8ml

Advantage
It is faily rapid
Has good staining results
Excess acid is removed byseveral changes of 90% alcohol for 24hours.
Dehydration is therefore hastened

Disadvantages
 SCIENCE LABORATORY TECHNOLOGY
Nuclear staining is not as good as that of formic acid.
Chemical test can not be used to asses decalcification

Trichloracetic acid
It is recommended for small pieces of delicate tissues which require
decalcification. The number of times is 4-5 days
Formulae
10% formal saline………………..........95ml
Trichloracetic acid………………………5g
Advantages
It permits good nuclear staining
Excess acid is removed by washing in several changes of 90% alcohol
Disadvantage
It’s a slow decalcifying Solution

Citrate citric acid buffer(ph 4.5)

It’s a recommended when speeds is not an important factor. The time is
approximately 6 days during which the solution should be changed daily.
Formulae :
Citric acid(monohydrate) 7% aqueous solution…...................5.0ml
Ammonium citrate(anhydrous) ……………………..………………….7.4%
aqueous solution………… …………………………………………….…..95.0ml
Zinc sulphate 1% aqueous solution...........................................0.2ml
Chloroform as a preservative.................................a few drops

Advantages
There is no cell or tissue damage
It permits excellent staining results
Disadvantages
The method is too slow for routine work

Ion exchange resins

Incoperation of ion exchange resin(ammonium for of polystyrene resin)
into the decalcification.
Solution speeds up the process of decalcification as ions are removed from
the solution hence increasing the rate of solubility of calcium ions from
the tissue and deposited on the bottom.
 SCIENCE LABORATORY TECHNOLOGY
DETERMINATION OF END POINT OF DECALCIFICATION
The end point of decalcification is determined by Radiological
examination.
Chelating agents -This is very slow decalcification solution
recommended only for detailed microscopical work .
The time required is approximately 3weeks during which the solution can
be changed at intervals of 3 days reducing to 1 day in the final stages
Formulae
EDTA Disodium salt…................................................…..5.5g
10% neutral formalin…...........................................……100ml

Advantages
histological artefacts are minimized.
Subsequent staining results are excellent.
Disadvantages
It is very slow and unsuitable for urgent work.
The chelating agent also tends to harden the tissue slightly

Qualities of a good decalcifying agent

(i) It should remove all the traces of calcium
(ii) It should cause no damage to the cells or tissue fibre
(iii) Should not impaire staining and impregnation procedures.
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER TWENTY SEVEN

MICROBIOLOGICAL
TECHNIQUES
Bacteria
Bacteria were first observed by Antonie van Leeuwenhoek in 1676, using a
single-lens microscope of his own design. The name bacterium was
introduced much later, by Christian Gottfried Ehrenberg in 1838.
Bacteria display a wide diversity of shapes and sizes, called morphologies.
Bacterial cells are about one tenth the size of eukaryotic cells and are
typically 0.5–5.0 micrometres in length
Most bacterial species are either spherical, called cocci (singular. coccus, )
or rod-shaped, called bacilli (singular. bacillus, ). Some rod-shaped
bacteria, called vibrio, are slightly curved or comma-shaped; others, can
be spiral-shaped, called spirilla, or tightly coiled, called spirochaetes. A
small number of species even have tetrahedral or cuboidal shapes.. The
large surface area to volume ratio conferred by this morphology may give
these bacteria an advantage in nutrient-poor environments. This wide
variety of shapes is determined by the bacterial cell wall and cytoskeleton,
and is important because it can influence the ability of bacteria to acquire
nutrients, attach to surfaces, swim through liquids and escape predators.
Many bacterial species exist simply as single cells, others associate in
characteristic patterns: Neisseria form diploids (pairs), Streptococcus
form chains, and Staphylococcus group together in "bunch of grapes"
clusters. Bacteria can also be elongated to form filaments, for example the
Actinobacteria. Filamentous bacteria are often surrounded by a sheath
 SCIENCE LABORATORY TECHNOLOGY
that contains many individual cells; certain types, such as species of the
genus Nocardia, even form complex, branched filaments, similar in
appearance to fungal mycelia.
Bacteria often attach to surfaces and form dense aggregations called
biofilms or bacterial mats. These films can range from a few micrometers
in thickness to up to half a meter in depth, and may contain multiple
species of bacteria, protists and archaea. Bacteria living in biofilms display
a complex arrangement of cells and extracellular components, forming
secondary structures such as microcolonies, through which there are
networks of channels to enable better diffusion of nutrients

Intracellular structures
The bacterial cell is surrounded by a lipid membrane, or cell membrane,
which encloses the contents of the cell and acts as a barrier to hold
nutrients, proteins and other essential components of the cytoplasm
within the cell. As they are prokaryotes, bacteria do not tend to have
membrane-bound organelles in their cytoplasm and thus contain few
large intracellular structures. They consequently lack a nucleus,
mitochondria, chloroplasts and the other organelles present in eukaryotic
cells, such as the Golgi apparatus and endoplasmic reticulum
Bacteria do not have a membrane-bound nucleus, and their genetic
material is typically a single circular chromosome located in the cytoplasm
in an irregularly shaped body called the nucleoid. The nucleoid contains
the chromosome with associated proteins and RNA

Like all living organisms, bacteria contain ribosomes for the production of
proteins, but the structure of the bacterial ribosome is different from those
of eukaryotes

Extracellular structures
Around the outside of the cell membrane is the bacterial cell wall.
Bacterial cell walls are made of peptidoglycan (called murein in older
sources), which is made from polysaccharide chains cross-linked by
unusual peptides containing D-amino acids
Bacterial cell walls are different from the cell walls of plants and fungi,
which are made of cellulose and chitin, respectively. The cell wall is
 SCIENCE LABORATORY TECHNOLOGY
essential to the survival of many bacteria, and the antibiotic penicillin
produced by bacteria cell wall for instance , is able to kill bacteria by
inhibiting a step in the synthesis of peptidoglycan.
There are broadly speaking two different types of cell wall in bacteria,
called Gram-positive and Gram-negative. The names originate from the
reaction of cells to the Gram stain, a test long-employed for the
classification of bacterial species.
Gram-positive bacteria possess a thick cell wall containing many layers of
peptidoglycan and teichoic acids. In contrast, Gram-negative bacteria
have a relatively thin cell wall consisting of a few layers of peptidoglycan
surrounded by a second lipid membrane containing lipopolysaccharides
and lipoproteins. These differences in structure can produce differences in
antibiotic susceptibility; for instance, vancomycin can kill only Gram-
positive bacteria and is ineffective against Gram-negative pathogens, such
as Haemophilus influenzae or Pseudomonas aeruginosa.
Flagella are rigid protein structuresthat are used for motility. Flagella are
driven by the energy released by the transfer of ions down an
electrochemical gradient across the cell membrane.
Fimbriae are fine filaments of protein.They are distributed over the
surface of the cell, and resemble fine hairs when seen under the electron
microscope. Fimbriae are believed to be involved in attachment to solid
surfaces or to other cells and are essential for the virulence of some
bacterial pathogens.
Pili (sing. pilus) are cellular appendages, slightly larger than fimbriae,
that can transfer genetic material between bacterial cells in a process
called conjugation
Capsules or slime layers are produced by many bacteria to surround
their cells, and vary in structural complexity: ranging from a disorganised
slime layer of extra-cellular polymer, to a highly structured capsule or
glycocalyx. These structures can protect cells from engulfment by
eukaryotic cells, such as macrophages. They can also act as antigens and
be involved in cell recognition, as well as aiding attachment to surfaces
and the formation of biofilms.
Endospores
 SCIENCE LABORATORY TECHNOLOGY
Certain genera of Gram-positive bacteria, such as Bacillus, Clostridium,
Sporohalobacter, Anaerobacter and Heliobacterium, can form highly
resistant, dormant structures called endospores.
In almost all cases, one endospore is formed and this is not a reproductive
process, although Anaerobacter can make up to seven endospores in a
single cell. Endospores have a central core of cytoplasm containing DNA
and ribosomes surrounded by a cortex layer and protected by an
impermeable and rigid coat.
Endospores show no detectable metabolism and can survive extreme
physical and chemical stresses, such as high levels of UV light, gamma
radiation, detergents, disinfectants, heat, pressure and desiccation. In this
dormant state, these organisms may remain viable for millions of years,
and endospores even allow bacteria to survive exposure to the vacuum
and radiation in space.
Endospore-forming bacteria can also cause disease: for example, anthrax
can be contracted by the inhalation of Bacillus anthracis endospores, and
contamination of deep puncture wounds with Clostridium tetani
endospores causes tetanus.
Classification of bacteria
Bacteria can be classified based on many factors including
(a) classification according to nutritional requirements
Bacterial metabolism is classified into nutritional groups on the basis of
three major criteria: the kind of energy used for growth, the source of
carbon, and the electron donors used for growth. An additional criterion
of respiratory microorganisms are the electron acceptors used for aerobic
or anaerobic respiration. ] Carbon metabolism in bacteria is either
heterotrophic, where organic carbon compounds are used as carbon
sources, or autotrophic, meaning that cellular carbon is obtained by fixing
carbon dioxide. Heterotrophic bacteria include parasitic types. Typical
autotrophic bacteria are phototrophic cyanobacteria, green sulfur-bacteria
and some purple bacteria, but also many chemolithotrophic species, such
as nitrifying or sulfur-oxidising bacteria. Energy metabolism of bacteria is
either based on phototrophy, the use of light through photosynthesis, or
on chemotrophy, the use of chemical substances for energy, which are
mostly oxidised at the expense of oxygen or alternative electron acceptors
(aerobic/anaerobic respiration
 SCIENCE LABORATORY TECHNOLOGY

Nutritional types in bacterial metabolism

Source of
Nutritional type Source of carbon Examples
energy

 Organic compounds Cyanobacteria, Green sulfur

 Phototrophs  Sunlight
(photoheterotrophs) or carbon
bacteria, Chloroflexi, or Purple
fixation (photoautotrophs)
bacteria 

 Organic compounds Thermodesulfobacteria,
Inorganic
 Lithotrophs (lithoheterotrophs) or carbon
Hydrogenophilaceae, or
compounds
fixation (lithoautotrophs)
Nitrospirae 

 Organic compounds
Organic (chemoheterotrophs)  Bacillus,
or Clostridium or
 Organotrophs
compounds carbon fixation Enterobacteriaceae 
(chemoautotrophs)  

FUNGI
A fungus is a eukaryotic organism that is a member of the kingdom
Fungi . Most fungi are largely invisible to the naked eye, are
heterotrophic organisms possessing a chitinous cell wall, with the majority
 SCIENCE LABORATORY TECHNOLOGY
of fungal species growing as multicellular filaments called hyphae forming
a mycelium; some fungal species also grow as single cells.

Characteristics
The fungi have a range of features defining the fungal kingdom, some of
which are shared with other organisms while others are unique to the
fungi.
Shared features:

 With eukaryotes: All fungi are eukaryotic, containing

membrane-bound nuclei with chromosomes. Fungal cells contain
membrane-bound cytoplasmic organelles, DNA with noncoding regions
called introns. Fungi have a characteristic range of soluble carbohydrates
and storage compounds, including mannitol and other sugar alcohols,
trehalose and glycogen the latter of which is also found in animals.
 With animals: Fungi lack chloroplasts and are heterotrophic
organisms, requiring preformed organic compounds as energy sources
and also as carbon skeletons for organic synthesis.
 With plants: Fungi possess a cell wall. They reproduce by both
sexual and asexual means, and like some basal plant groups, such as ferns
and mosses produce spores. Similar to mosses and algae, fungi typically
have haploid nuclei.
 With prokaryotes: As in some bacteria, biosynthesis of the
amino acid, L-lysine, is via the α-aminoadipate pathway.

Other features:

 Fungi typically grow as hyphae, which extend at their tips. This

apical growth form is shared with the morphologically similar oomycetes
and in contrast with other filamentous organisms, like filamentous green
algae, which grow by repeated cell divisions within a chain of cells
(intercalary growth).
 Some fungi grow as single-celled yeasts which reproduce by
budding, and some dimorphic fungi can switch between a yeast phase and
a hyphal phase in response to environmental conditions.
 The fungal cell wall contains glucans also found in plants, but
also chitin not found in the Plant kingdom, but in some animals. In
 SCIENCE LABORATORY TECHNOLOGY
contrast to plants and the oomycetes, fungal cell walls do not contain
cellulose.
 Fungal hyphae may have several nuclei within each hyphal
compartment, and many budding yeasts are diploid.

Classification of Fungi
Over 60,000 species of fungi are known. Fungi are classified by their
method of reproduction (both sexual and asexual). It seems likely that
fungi are not a monophyletic group. Historically they have been divided
into four taxonomic divisions: Zygomycota, Ascomycota, Basidiomycota,
and Deuteromycota.

Chytridiomycota
The Chytridiomycota are commonly known as chytrids. These fungi are
ubiquitous with a worldwide distribution. Chytrids produce zoospores that
are capable of active movement through aqueous phases with a single
flagellum, leading some early taxonomists to classify them as protists.
Molecular phylogenies, inferred from rRNA sequences in ribosomes,
suggest that the Chytrids are a basal fungal group divergent from the other
fungal divisions, consisting of four major clades with some evidence for
paraphyly or possibly polyphyly.

Blastocladiomycota
The Blastocladiomycota were previously considered a taxonomic clade
within the Chytridiomycota. Recent molecular data and ultrastructural
characteristics, however, place the Blastocladiomycota as a sister clade to
the Zygomycota, Glomeromycota, and Dikarya (Ascomycota and
Basiomycota). The blastocladiomycetes are fungi that are saprotrophs and
parasites of all eukaryotic groups and undergo sporic meiosis unlike their
close relatives, the chytrids, which mostly exhibit zygotic meiosis. [45]

Neocallimastigomycota
The Neocallimastigomycota were earlier placed in the phylum
Chytridomycota. Members of this small phylum are anaerobic organisms,
living in the digestive system of larger herbivorous mammals and possibly
in other terrestrial and aquatic environments. They lack mitochondria but
contain hydrogenosomes of mitochondrial origin. As the related chrytrids,
 SCIENCE LABORATORY TECHNOLOGY
neocallimastigomycetes form zoospores that are posteriorly uniflagellate
or polyflagellate.

Zygomycota
The Zygomycota contain the taxa, Zygomycetes and Trichomycetes, and
reproduce sexually with meiospores called zygospores and asexually with
sporangiospores. Black bread mold (Rhizopus stolonifer) is a common
species that belongs to this group; another is Pilobolus, which is capable of
ejecting spores several meters through the air. Medically relevant genera
include Mucor, Rhizomucor, and Rhizopus. Molecular phylogenetic
investigation has shown the Zygomycota to be a polyphyletic phylum with
evidence of paraphyly within this taxonomic group.

Bread mould

Glomeromycota
Members of the Glomeromycota are fungi forming arbuscular
mycorrhizae with higher plants. Only one species has been observed
forming zygospores; all other species solely reproduce asexually. The
symbiotic association between the Glomeromycota and plants is ancient,
with evidence dating to 400 million years ago.

Ascomycota
The Ascomycota, commonly known as sac fungi or ascomycetes, constitute
the largest taxonomic group within the Eumycota. These fungi form
 SCIENCE LABORATORY TECHNOLOGY
meiotic spores called ascospores, which are enclosed in a special sac-like
structure called an ascus. This division includes morels, a few mushrooms
and truffles, single-celled yeasts (e.g., of the genera Saccharomyces,
Kluyveromyces, Pichia, and Candida), and many filamentous fungi living
as saprotrophs, parasites, and mutualistic symbionts. Prominent and
important genera of filamentous ascomycetes include Aspergillus,
Penicillium, Fusarium, and Claviceps. Many ascomycetes species have
only been observed undergoing asexual reproduction (called anamorphic
species), but analysis of molecular data has often been able to identify
their closest teleomorphs in the Ascomycota. Because the products of
meiosis are retained within the sac-like ascus, several ascomycetes have
been used for elucidating principles of genetics and heredity (e.g.
Neurospora crassa).

Basidiomycota
Members of the Basidiomycota, commonly known as the club fungi or
basidiomycetes, produce meiospores called basidiospores on club-like
stalks called basidia. Most common mushrooms belong to this group, as
well as rust and smut fungi, which are major pathogens of grains. Other
important Basidiomycetes include the maize pathogen Ustilago maydis,
human commensal species of the genus Malassezia, and the opportunistic
human pathogen, Cryptococcus neoformans.
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Lichens and Mycorrhizae 

Lichens are a symbiosis between a photosynthetic organism (alga or

cyanobacterium) and a fungus (sac or club). Mycorrhizae are fungi
(usually a zygomycete or basidiomycete) symbiotic with the roots of
plants. Both relationships are mutualistic: both parties benefit. Fungi
provide nutrients from the substrate, the phototroph provides food. Plants
with mycorrhizae grow better: the plant gets nutrients from the fungus in
exchange for carbohydrates.

lichens

Reproduction

Sexual and asexual reproduction of the fungi is commonly via spores,

often produced on specialized structures or in fruiting bodies.

(i) Asexual reproduction

Asexual reproduction via vegetative spores or through mycelial
fragmentation is common in many fungal species and allows more rapid
dispersal than sexual reproduction. In the case of the "Fungi imperfecti"
or Deuteromycota, which lack a sexual cycle, it is the only means of
propagation. Asexual spores, upon germination, may found a population
that is clonal to the population from which the spore originated, and thus
colonize new environments.
 SCIENCE LABORATORY TECHNOLOGY
(ii) Sexual reproduction
Most fungi have both a haploid and diploid stage in their life cycles.
Sexual reproduction with meiosis exists in all fungal phyla, except the
Deuteromycota. It differs in many aspects from sexual reproduction in
animals or plants. Many differences also exist between fungal groups and
have been used to discriminate fungal clades and species based on
morphological differences in sexual structures and reproductive
strategies. The major fungal clades have initially been delineated based on
the morphology of their sexual structures and spores; for example, the
spore-containing structures, asci and basidia, can be used in the
identification of ascomycetes and basidiomycetes, respectively. Many
fungal species have elaborate vegetative incompatibility systems that
allow mating only between individuals of opposite mating type, while
others can mate and sexually reproduce with any other individual or itself.
Species of the former mating system are called heterothallic, and of the
latter homothallic.
Some species however, have lost the ability to form reproductive
structures, and propagate solely by vegetative growth. Yeasts, molds, and
mushrooms are examples of fungi.

Spore dispersal
Both asexual and sexual spores or sporangiospores of many fungal species
are actively dispersed by forcible ejection from their reproductive
structures. This ejection ensures exit of the spores from the reproductive
structures as well as travelling through the air over long distances. Many
fungi thereby possess specialized mechanical and physiological
mechanisms as well as spore-surface structures, such as hydrophobins, for
spore ejection

Habitat and economical importance

most fungi are living for the most part in soil, dead matter, and as
symbionts of plants, animals, or other fungi. They perform an essential
role in all ecosystems in decomposing organic matter and are
indispensable in nutrient cycling and exchange. Some fungi become
noticeable when fruiting, either as mushrooms or molds. Many fungal
 SCIENCE LABORATORY TECHNOLOGY
species have long been used as a direct source of food, such as mushrooms
and truffles and in fermentation of various food products, such as wine,
beer, and soy sauce. More recently, fungi are being used as sources for
antibiotics used in medicine and various enzymes, such as cellulases,
pectinases, and proteases, important for industrial use or as active
ingredients of detergents. Many fungi produce bioactive compounds
called mycotoxins, such as alkaloids and polyketides that are toxic to
animals including humans. Some fungi are used recreationally or in
traditional ceremonies as a source of psychotropic compounds. Several
species of the fungi are significant pathogens of humans and other
animals, and losses due to diseases of crops (e.g., rice blast disease) or
food spoilage caused by fungi can have a large impact on human food
supply and local economies.

VIRUS
A virus (from the Latin virus meaning toxin or poison) is a sub-
microscopic infectious agent that is unable to grow or reproduce outside a
host cell. Viruses infect all cellular life. The first known virus, tobacco
mosaic virus, was discovered by Martinus Beijerinck in 1899, and now
more than 5,000 types of virus have been described. The study of viruses
is known as virology, and is a branch of microbiology.
Viruses consist of two or three parts: all viruses have genes made from
either DNA or RNA, long molecules that carry genetic information; all
have a protein coat that protects these genes; and some have an envelope
of fat that surrounds them when they are outside a cell. Viruses vary in
shape from simple helical and icosahedral shapes, to more complex
structures. They are about 100 times smaller than bacteria.
Viruses spread in many ways; plant viruses are often transmitted from
plant to plant by insects that feed on sap, such as aphids, while animal
viruses can be carried by blood-sucking insects. These disease-bearing
organisms are known as vectors. Influenza viruses are spread by coughing
and sneezing, and others such as norovirus, are transmitted by the faecal-
oral route, when they contaminate hands, food or water. Rotaviruses are
often spread by direct contact with infected children. HIV is one of several
viruses that are transmitted through sex.
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Not all viruses cause disease, as many viruses reproduce without causing
any obvious harm to the infected organism. Some viruses such as HIV can
cause life-long or chronic infections, and the viruses continue to replicate
in the body despite the hosts' defence mechanisms. However, viral
infections in animals usually cause an immune response, which can
completely eliminate a virus. These immune responses can also be
produced by vaccines that give lifelong immunity to a viral infection.
Microorganisms such as bacteria also have defences against viral
infection, such as restriction modification systems. Antibiotics have no
effect on viruses, but antiviral drugs have been developed to treat life-
threatening and more minor infections

Structure
Viruses display a wide diversity of shapes and sizes, called morphologies.
Viruses are about 100 times smaller than bacteria. Most viruses which
have been studied have a diameter between 10 and 300 nanometres. Some
filoviruses have a total length of up to 1400 nm, however their diameters
are only about 80 nm. Most viruses are unable to be seen with a light
microscope so scanning and transmission electron microscopes are used
to visualise virus particles. To increase the contrast between viruses and
the background, electron-dense "stains" are used. These are solutions of
salts of heavy metals such as tungsten, that scatter the electrons from
regions covered with the stain. When virus particles are coated with stain
(positive staining), fine detail is obscured. Negative staining overcomes
this problem by staining the background only.
A complete virus particle, known as a virion, consists of nucleic acid
surrounded by a protective coat of protein called a capsid. These are
formed from identical protein subunits called capsomers. Viruses can
have a lipid "envelope" derived from the host cell membrane. The capsid is
made from proteins encoded by the viral genome and its shape serves as
the basis for morphological distinction. Virally coded protein subunits will
self-assemble to form a capsid, generally requiring the presence of the
virus genome. However, complex viruses code for proteins which assist in
the construction of their capsid. Proteins associated with nucleic acid are
known as nucleoproteins, and the association of viral capsid proteins with
viral nucleic acid is called a nucleocapsid. In general, there are four main
morphological virus types:
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Some Virus Structures

Phage

Capsid
protein

TMV

Helical
Helical capsids are composed of a single type of capsomer stacked around
a central axis to form a helical structure which may have a central cavity,
or hollow tube. This arrangement results in rod-shaped or filamentous
virions: these can be short and highly rigid, or long and very flexible. The
genetic material, generally single-stranded RNA, but ssDNA in some
cases, is bound into the protein helix, by interactions between the
negatively-charged nucleic acid and positive charges on the protein.
Overall, the length of a helical capsid is related to the length of the nucleic
acid contained within it and the diameter is dependent on the size and
arrangement of capsomers. The well-studied Tobacco mosaic virus is an
example of a helical virus.
 SCIENCE LABORATORY TECHNOLOGY
Icosahedral
Most animal viruses are icosahedral or near-spherical with icosahedral
symmetry. A regular icosahedron is the optimum way of forming a closed
shell from identical sub-units. The minimum number of identical
capsomers required is twelve, each composed of five identical sub-units.
Many viruses, such as rotavirus, have more than twelve capsomers and
appear spherical but they retain this symmetry. Capsomers at the apices
are surrounded by five other capsomers and are called pentons.
Capsomers on the triangular faces are surround by six others and are call
hexons.

Enveloped
Some species of virus envelope themselves in a modified form of one of
the cell membranes, either the outer membrane surrounding an infected
host cell, or internal membranes such as nuclear membrane or
endoplasmic reticulum, thus gaining an outer lipid bilayer known as a
viral envelope. This membrane is studded with proteins coded for by the
viral genome and host genome; the lipid membrane itself and any
carbohydrates present are entirely host-coded. The influenza virus and
HIV use this strategy. Most enveloped viruses are dependent on the
envelope for their infectivity.

Complex
These viruses possess a capsid which is neither purely helical, nor purely
icosahedral, and which may possess extra structures such as protein tails
or a complex outer wall. Some bacteriophages have a complex structure
consisting of an icosahedral head bound to a helical tail which may have a
hexagonal base plate with protruding protein tail fibres.
The poxviruses are large, complex viruses which have an unusual
morphology. The viral genome is associated with proteins within a central
disk structure known as a nucleoid. The nucleoid is surrounded by a
membrane and two lateral bodies of unknown function. The virus has an
outer envelope with a thick layer of protein studded over its surface. The
whole particle is slightly pleiomorphic, ranging from ovoid to brick shape.
Mimivirus is the largest known virus, with a capsid diameter of 400 nm.
Protein filaments measuring 100 nm project from the surface. The capsid
 SCIENCE LABORATORY TECHNOLOGY
appears hexagonal under an electron microscope, therefore the capsid is
probably icosahedral.

Reproduction/Replication
Viral populations do not grow through cell division, because they are
acellular; instead, they use the machinery and metabolism of a host cell to
produce multiple copies of themselves, and they assemble in the cell.

Life cycle of
phage T2
Figure 9.5
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Replication cycle

 Attachment is a specific binding between viral capsid proteins

and specific receptors on the host cellular surface. This specificity
determines the host range of a virus. For example, HIV infects only
human T cells, because its surface protein, can interact with CD4 and
receptors on the T cell's surface. This mechanism has evolved to favour
those viruses that only infect cells in which they are capable of replication.
Attachment to the receptor can induce the viral-envelope protein to
undergo changes that results in the fusion of viral and cellular
membranes.
 Penetration follows attachment; viruses enter the host cell
through receptor mediated endocytosis or membrane fusion. This is often
called viral entry. The infection of plant cells is different to that of animal
cells. Plants have a rigid cell wall made of cellulose and viruses can only
get inside the cells following trauma to the cell wall. Viruses such as
tobacco mosaic virus can also move directly in plants, from cell-to-cell,
through pores called plasmodesmata. Bacteria, like plants, have strong
cell walls which a virus must breach to infect the cell. Some viruses have
evolved mechanisms which inject their genome into the bacterial cell
while the viral capsid remains outside.

 Uncoating is a process in which the viral capsid is degraded by

viral enzymes or host enzymes thus releasing the viral genomic nucleic
acid.
 Replication involves synthesis of viral messenger RNA (mRNA)
for viruses except positive sense RNA viruses (see above), viral protein
synthesis and assembly of viral proteins and viral genome replication.
 Following the assembly of the virus particles, post-translational
modification of the viral proteins often occurs. In viruses such as HIV, this
modification, (sometimes called maturation), occurs after the virus has
been released from the host cell.
 Viruses are released from the host cell by lysis—a process that
kills the cell by bursting its membrane. Enveloped viruses (e.g., HIV)
typically are released from the host cell by budding. During this process
the virus acquires its envelope, which is a modified piece of the host's
plasma membrane.
 SCIENCE LABORATORY TECHNOLOGY
DNA viruses
The genome replication of most DNA viruses takes place in the cell's
nucleus. If the cell has the appropriate receptor on its surface, these
viruses enter the cell by fusion with the cell membrane or by endocytosis.
Most DNA viruses are entirely dependent on the host cell's DNA and RNA
synthesising machinery, and RNA processing machinery. The viral
genome must cross the cell's nuclear membrane to access this machinery.

RNA viruses
RNA viruses are unique because their genetic information is encoded in
RNA. Replication usually takes place in the cytoplasm. RNA viruses can be
placed into about four different groups depending on their modes of
replication. The polarity (whether or not it can be used directly to make
proteins) of the RNA largely determines the replicative mechanism, and
whether the genetic material is single-stranded or double-stranded. RNA
viruses use their own RNA replicase enzymes to create copies of their
genomes.

Reverse transcribing viruses

Reverse transcribing viruses replicate using reverse transcription, which is
the formation of DNA from an RNA template. Reverse transcribing
viruses containing RNA genomes use a DNA intermediate to replicate,
whereas those containing DNA genomes use an RNA intermediate during
genome replication. Both types use the reverse transcriptase enzyme to
carry out the nucleic acid conversion. Retroviruses often integrate the
DNA produced by reverse transcription into the host genome. They are
susceptible to antiviral drugs that inhibit the reverse transcriptase
enzyme, e.g. zidovudine and lamivudine. An example of the first type is
HIV which is a retrovirus. Examples of the second type are the
Hepadnaviridae, which includes Hepatitis B virus.

Effects on the host cell

The range of structural and biochemical effects that viruses have on the
hosts cell is extensive. These are called cytopathic effects. Most virus
infections eventually result in the death of the host cell. The causes of
death include cell lysis, alterations to the cell's surface membrane and
apoptosis. Often cell death is caused by cessation of its normal activities
 SCIENCE LABORATORY TECHNOLOGY
due to suppression by virus-specific proteins, not all of which are
components of the virus particle.
Some viruses cause no apparent changes to the infected cell. Cells in
which the virus is latent and inactive show few signs of infection and often
function normally. This causes persistent infections and the virus is often
dormant for many months or years. This is often the case with herpes
viruses. Viruses, such as Epstein-Barr virus often cause cells to proliferate
without causing malignancy, but viruses, such as papillomaviruses are an
established cause of cancer.

STERILIZATION
Sterilization is the total killing of all living microorganisms on an item
both pathogenic and non-pathogenic. There are several methods of
sterilization which include
Heating,
Incineration,
Filtration
UV light
Use of disinfectants

A . HEAT METHOD
All living things are killed when exposed to heat . there are two methods
method of sterilization by heat these include
(a) sterilization by dry heat
(b) sterilization by moist heat

( a) sterilization by dry heat

These method is further divided into
Red heat method (flaming)
Flaming is done by directly exposing an item an open flame to sterilize
things like inoculating loops , needles , mouth of culture tubes and flasks
. The apparatus made of metal are flamed until red hot and cooed either
by dipping them in 70% alcohol or leaving them in the air to cool. These
apparatus should be sterilized these way before and after being used.

Heating in hot air or oven

 SCIENCE LABORATORY TECHNOLOGY
These is done by subjecting items to be sterilized indirectly to the source
of heat e.g. by placing them in an oven where they gain heat which will
kill the microorganisms by oxidizing their cell constituents . Hot air ovens
are used for
sterilizing dry apparatus e.g. glass wares like petri dishes , culture tubes ,
flasks , pipettes glass syringes , unwanted cultures etc at 160 oc for 60
min
Media are not sterilized by dry heat because these may destroy or
denature the organic composition of the media .

(b) Sterilization by moist heat

These method involves subjecting the items and media to be sterilized to

heat through a liquid medium , these methods include
Boiling
Pasturilization
Steam under pressure
Tyndilization
(i) Boiling method
These is whereby the items are placed in the normal boiling water and
heated upto the boiling point of water .its believed that most
microorganisms especially the vegetative forms shall killed under these
temperatures
(ii) Pasturilization
These is the actual method of subjecting a liquid e.g. milk to high
temperatures for a very brief period of time then cooling .pasturilization
has for long been regarded as a heat process applied to
milk.pasturilization is by itself not really sterilization since these method
does not kill thermophilic microorganism but it only reduces the
microorganisms to a safe level

(iii) Tyndilization
Tyndilizaton is a method used to sterilize fluids e.g. blood , serum etc
which would be denatured if autoclaved or put in a hot air oven . it
involves heating the fluid at 56- 60 oc for about one hour in a water bath
for three consecutive days . these fluids should be cooled and incubated
after each heating session .
It is assumed that the media will show growth of microorganisms that
had survived after the first heating session . These will again be killed
 SCIENCE LABORATORY TECHNOLOGY
during the second heating session and the media shall again be incubated
the second time . During these second incubation period those
microorganisms especially the spores that had survived will germinate
and these are the ones to be killed by heating during the third heating
session the media will be finally incubated the third time and observation
made on them to determine whether there is any growth of
microorganisms still persisting

(iv)Steam under pressure (autoclaving)

Steam kills microorganisms by coagulating and denaturing proteins .
These can be achieved by boiling water for 30 min at 100oc . however
some bacteria develop spores and hence resist boiling temperatures .
Therefore for such cases they can be sterilized under pressure in an
autoclave or pressure cooker .
Autoclaves are made of stainless steel , copper or gun metal .its lids are
fasten by screws and bolts ,they operates depending on the pressure
inside a closed vessel where both pressure and temperature increases are
experienced . Any air inside the autoclave must be removed because if it’s
left inside , then less heat shall be produced .
The steam is generated by either adding life steam under pressure from
the main vent or by adding few volumes of water into an autoclave and
heating it by charcoal , gas or electricity.
Autoclaves have pressure and temperature gauges mounted on them
which measure pressure and temperatures they also have perforated
shelves for placing equipments.
Media and apparatus placed on an autoclave should be wrapped to
prevent moisture from entering the apparatus hence reducing the
temperatures attained in the autoclave . these apparatus should be
wrapped as follows
Glass containers e.g. flasks , test tubes etc should be wrapped in cotton
wool , aluminum foil or Kraft papers
petri dishes are wrapped in Kraft steam tight papers or aluminum foil and
sealed with special tape
Graduated pipettes should have cotton wool put in the ends and
wrapped in Kraft papers or aluminum foil
Empty test tubes should be put upside down in cupboard container s or
wire metal containers
Dissecting instruments should be wrapped in an aluminum foil but
sometimes instruments in an autoclave are not sterilized completely
 SCIENCE LABORATORY TECHNOLOGY
these could be due to the development of cold spots in the autoclaves
and errors in the pressure readings. These therefore mean that there is
need to check and confirm sterilization . these can be done using various
types of sterilization indicators which include ;
(i) Sulfur pellets – they are put in small test tubes and placed in
an autoclave .
They often change color when the temperatures reached inside the
autoclave are above 120 oc
(ii) Brown tubes – these are small glass tubes with indicators
that are normally red but changes color to green when correct
temperatures are reached . they must be stored at temperatures below 20
o
c so as to prevent deterioration
(iii) Spore strips – these are pieces of adsorbent materials soaked
in bacterial spores which are killed only after heating to 121oc for 12 min .
they are wrapped and placed in an autoclave during sterilization then
removed and incubated for several days and if they grow then that means
that sterilization was not effective in that particular autoclave
(iv) Dry earth - they work just the sane way as spore strip and are
sometimes used instead of spore strip .

B . INCINERATION METHOD
These method is almost similar to the heat method but however it
involves complete burning of the item or media to ash in a special
container called an incinerator. It’s often used as a method of disposal of
laboratory waste
( C) FILTRATION METHOD
bacteria can be removed by passing the liquid medium containing
bacteria through special filters under negative pressure. The filters used
have small pores and are made of cellulose acetate or other cellulose
esters . These filters can be re-sterilized and used many times .
(D) UV METHOD
The UV light is used to sterilize laboratories and theatre rooms and
sometimes to sterilize inoculating loops . the best UV light is that below
300 Ao but it is dangerous to the eyes and therefore should be used under
strict control
(E) DISINFECTANTS
Disinfection is the removal of some or all pathogenic organisms from the
surface of an item using chemicals called disinfectants . unlike
sterilization , disinfection suggest that some viable microbes persists .
 SCIENCE LABORATORY TECHNOLOGY
Most disinfectants seldomly destroy spores of bacteria. And therefore
they are less effective than heat method . they work in several ways which
include ; protein denaturalization which results in precipitation and
coagulation of cell membranes hence the affected cells dies . Chemical
agents also result in the destruction of cell membranes by oxidizing them
.
The factors that determine the effectiveness of chemical disinfectants are ;
(a) the concentration of the chemical
(b) the time for which it is applied
(c) presence of inactivating materials on the cell surface
(d) the pH of the chemical disinfectant .

Examples of common disinfectants are

(i) Alcohol
Alcohol can kill a wide range of vegetative bacteria including HIV but do
not kill bacterial spores and fungi. They serve as disinfectants at a
concentration of 70% alcohol in water NB / absolute or 100% alcohol is
not an effective disinfectant .
70 % alcohol is prepared by dissolving 70 ml of ethanol ,methylated
spirit, ethanol or isopropyl alcohol in 30ml of distilled water . and a few
drops of gentian violet solution is added and the container is tightly
capped

(ii) Phenol
Phenol kills a wide range of bacteria and some viruses including HIV .
phenols retain their activity even in presence of organic matter . They do
not kill bacterial spores and hepatitis B virus. They are used as
disinfectants at the following concentration ;
Lysol 5% in water
sudol 2% in water
Lysol is always recommended because it is cheap and readily available to
prepare of 5% Lysol solution ,add 100ml in 1900 ml of distilled water
while to prepare 10 % Lysol solution , add 100ml to 900 ml of distilled
water . Lysol is used for washing body fluids and hands
(iii) Halogens
They kill virus , including HIV , bacteria spores and fungi. Usually
chlorine releasing compounds e.g. hypochlorides are usually used as
disinfectants at a concentration of 0.5% for heavy contamiations and
 SCIENCE LABORATORY TECHNOLOGY
0.1% for light contamination . Iodine solutions are used for light
contamination at a concentration of 0.25% .
0.05% hypochloride solution is prepared by dissolving 300 ml of house
hold jik in 1800mls of distilled water or 10g of calcium hypochloride in
2000mls of distilled water.it is used for cleaning gloves , spillage and
broken glassware .
chlorine realizing compounds are easily inactivated by organic
compounds e.g. blood ,pus ,stool ,milk etc .These problem can largely be
overcooked at a concentration of 0.5%. They also loose their activity
when left standing in air and these therefore requires preparation of fresh
solutions on daily basis . it is corrosive to metals especially after
prolonged contact . Iodine solution may stain the skin and may couse
hypersensitivity reaction therefore use gloves

(iv) Aldehydes
Some aldehydes e.g. formaldehyde and glutaraldehydes solution are
highly lethal to all microorganisms including their spores . However
glutaraldehydes are affected by pH of the environment. They must
therefore be alkaline for reliable activity .
Formalin is used as a disinfectant at a concentration of 4% its however
not recommended for general use because formalin fumes are irritating to
the skin , eyes and lungs and may cause hypersensitivity reaction .
glutaraldehyde solution may be used for all chemical disinfection
procedures described above if Lysol and hypochlorides are not
available . However they are expensive and not easily available
(v) Soaps and detergents
They form the cheapest and the most easily available disinfectants used
for cleaning. They are however not very much effective against several
classes of bacteria , fungi and virus
Methods of cleaning disinfecting and sterilization
Cleaning, disinfection and sterilization is applied to ;
(i) The skin before and after laboratory practical
Rub skin using 70% alcohol or 0.25 % iodine solution using a cotton wool
and leave it to dry
(ii) Reusable syringes , needles and lancets
Clean them first before autoclaving wrap them in porous brown paper .
Never boil lancets ,syringes and needles together with contaminated
items . always use distilled water for final rinsing , autoclaving etc in
order to prevent salt deposits
 SCIENCE LABORATORY TECHNOLOGY
(iii) Contaminated reusable items e.g. glass slides , coversips ,
pipettes ,testtubes , glassrods and corks and centrifuge tubes . Soak slides
and coverslips in 5% lysol or hypochlorite or any detergent e.g. omo
solution prior to washing and dry them on wire basket before storage
(iv) Re- usable specimen containers . Soak reusable specimen
containers used for collection of stool, urine , blood ,skin etc in 5% lysol
or 0.5% hypochlorite prior to washing . boil them if disinfectants are not
available. Autoclave specimen containers that have been used for
collection of sputum
(v) Reusable gloves and other protective clothing . Wash in
detergent before use
(vi) Laboratory wares e.g. glass and plastic ware .Wash in
detergent before use
(vii) Contaminated reusable gloves
Soak in 5% lysol or 0.5 hypochlorite , wash in hot water at 70-80oc , rinse
in distilled water and allow to dry
(viii) Laboratory surfaces e.g. benchtops , shelves and floors
Wipe with cotton wool soaked in 0.5% hypochloride solution or 5% lysol
(ix) Laboratory clothing e.g. labcoats , gowns , sheets ,towels etc
(x) Wash in hot water with detergent , if heavily contaminated ,
first soak in 0.5% hypochloride or 2% lysol and allow to dry
(xi) Broken glasswares body fluids
Soak in o. 5% hypocloride or 5 % Lysol before disposal
(xii) Metal items e.g. wire loops , forceps , blades etc .Flame them
until red- hot
(xiii) Disposable materials e.g. cotton wool .soak in 0.5%
hypocloride or 5% lysol and dispose
(xiv) Sterile swabs , bottles , transport media etc sterilize them in
an autoclave before use
(xv) Hands wash in warm soapy water then 1% hypochloride or 2% lysol
and wipe with clean towel

MICROBIAL CULTIVATION IN LABORATORIES

When microorganisms are cultivated in the laboratory, a growth
environment called a medium is used. The medium may be purely
 SCIENCE LABORATORY TECHNOLOGY
chemical (a chemically defined medium), or it may contain organic
materials, or it may consist of living organisms such as fertilized eggs.
Microorganisms growing in or on such a medium form a culture. A
culture is considered a pure culture if only one type of organism is
present and a mixed culture if populations of different organisms
are present. When first used, the culture medium should be sterile,
meaning that no form of life is present before inoculation with the
microorganism.

CULTURE MEDIA
A culture media is a substrate or nutrient solution upon which
microorganisms grow in the laboratory. For the cultivation of bacteria, a
commonly used medium is nutrient broth, a liquid containing
proteins, salts, and growth enhancers that will support many bacteria.
To solidify the medium, an agent such as agar is added. Agar is a
polysaccharide that adds no nutrients to a medium, but merely
solidifies it. The medium that results is nutrient agar.
Different microorganisms require different nutrient media that are
composed of different compounds some simple compounds while some
complex compounds . There is therefore no universal media

Many media for microorganisms are complex, reflecting the growth

requirements of the microorganisms. For instance, most fungi require
extra carbohydrate and an acidic environment for optimal growth. The
medium employed for these organisms is potato dextrose agar, also
known as Sabouraud dextrose agar. For protozoa, liquid media
are generally required, and for rickettsiae and viruses, living tissue
cells must be provided for best cultivation.
For anaerobic microorganisms, the atmosphere must be oxygen free.
To eliminate the oxygen, the culture media can be placed within
containers where carbon dioxide and hydrogen gas are generated and
oxygen is removed from the atmosphere. Commercially available
products achieve these conditions. Anaerobic chambers can also be
used within closed compartments, and technicians can manipulate
culture media within these chambers. To encourage carbon dioxide
 SCIENCE LABORATORY TECHNOLOGY
formation, a candle can be burned to use up oxygen and replace it with
carbon dioxide.

Basic requirements of a culture media

An ideal culture medium should be the one which should contain the
following compounds
(a) Energy source
(b) Carbon source
(c) Nitrogen source
(d) Mineral source
(e) Have adequate redox potential
(f) Must have satisfactory pH
(g) Must have necessary growth factors e.g. vitamins and nucleic
acids

(a) Energy source

Microorganisms require energy so as to power biochemical reactions in
their cells. The most common energy source is carbohydrates e.g. glucose ,
lactose , sucrose and xylose . microorganisms differ adversely on the type
of carbohydrates that they can utilize i.e. some can utilize glucose while
others cannot. Such differences about
the type of microorganisms can utilize can be can be used for their
identification and isolation . It should also be noted that carbohydrates is
not the only source of energy to microorganisms since other
microorganisms can as well utilize other compounds .

(a) Carbon source

besides carbon being one of the chemical compound in the carbohydrate
structure which is important energy source, carbon is also required for
structural formation of the cell
(b) Nitrogen source
Nitrogen is one of the constituent of cells , its found cells and in the
intracellular structure.its required f0r the synthesis of enzymes , amino
acids and proteins .
 SCIENCE LABORATORY TECHNOLOGY
nitrogen source for many microorganisms differs . some can utilize
nitrogen in form of nitrates while others in the form of nitrogen e.g.
nitrogen fixing bacteria .
In culture media’s . nitrogen is provided by peptones.
(a) Mineral salts
Mineral salts e.g. phosphorus and sulfates form cell components, others
e.g. magnesium, copper, calcium etc are enzyme co-factors. They also
promote transport of soluble complex.
(b) Growth factors
These are organic compounds which a cell must contain in order to grow
but which it is unable to synthesis e.g. vitamins , purines ,pyrimidines .
Coenzyme ,fatty acids etc . different microbial species vary widely in
their growth factors while others do not require growth factors at all .

Common ingredients of a culture media

The following are among the ingredients of a culture media
(i) Peptone
(ii) Meat extract
(iii) Yeast extract
(iv) Mineral salts
(v) Carbohydrates
(vi) Agar
(vii) Water

(aPeptones
Peptone is a soluble product obtained by hydrolysis of animals and plant
proteins which is commonly obtained from meat, milk ,and soybeans by
acid or enzymes into free amino acids , peptides and proteosis . Peptones
provide nitrogen , nucleic acids ,mineral salts and vitamins . plant
products also provides carbohydrates
(b)Carbohydrates
Microorganisms require some form of carbon as an element . these are
provided by simple or complex sugars . microorganisms are very
sensitive and specific to the type of carbohydrates they can utilize .
carbohydrates could therefore be added in the media to aid in
differentiation and isolation of bacteria
(c)Meat extract
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Meat extract provides mineral salts , amino acids , and vitamins . It’s an
essential ingredient of many media including nutrient agar and nutrient
broth

(d)Yeast extract
Yeast extract contain essential growth stimulants for bacteria
(e) Mineral salts
Minerals are important for cell growth and as co- factors for several
enzymes
(f)Agar
Agar is an inert polysaccharide extracted from seaweeds. It consist of two
main polysaccharide namely Agaros – 70% Agaro pectin –30%Agar
is used to solidify culture media due to its high gelling structure , it have
a melting point of the
90-95oc and a setting temperature of 40 –45oc . Most agar used in
microbial work produce a firm gel at a concentration of about 1.5 –2% .
Agar is also thought to provide mineral salts to microorganisms .. It’s
also preferred because it is usually resistant to microbial attack
(g)Water
Water is essential for growth of microorganisms . It must be free from any
chemicals which might inhibit microbial growth. Deionised or distilled
water must always be used

Types of culture media

Basic media
these are simple media that contain only the necessary constituents for
growth e.g. meat extract , peptones and mineral salts . Examples are
peptone broth ,nutrient broth, and blood agar base .
Many microorganisms are capable of growing on basic media . They are
also referred to as general type media.Basic media are often used in the
lab to prepare enriched media and to maintain stock cultures , and for
subculturing of microorganisms from differential or selective media
prior to accomplishing serological and biochemical test . Basic media are
cheap ,

Enriched media
these are media enriched with whole blood , lysed blood ,serum, extra
peptones even amino acids so as to support the growth of pathogens
 SCIENCE LABORATORY TECHNOLOGY
that require additional nutrients or growth stimulants e.g. sheep blood
agar, horse blood agar,chocolate blood agar ,horse blood agar.
Sheep blood and horse blood is most preferred because they do not
contain inhibiting factors . chocolate blood is made by heating blood at
60oc which turns to chocolate brown color

Selective media

these are nutrient agar media which have been added specific chemicals
which will inhibitor prevent growth of one group of bacteria without
inhibiting the other group e.g. addition of crystal violet at specific
concentration will prevent the growth of gram positive without affecting
growth of gram negative bacteria.
These media retard the growth of unwanted organisms while
encouraging the growth of the organisms desired. For example,
mannitol salt agar is selective for staphylococci because most other
bacteria cannot grow in its high-salt environment. Another selective
medium is brilliant green agar, a medium that inhibits Gram-
positive bacteria while permitting Gram-negative organisms such as
Salmonella species to grow.

Differential media
These is a nutrient agar media in which certain reagents or chemical have
been incorporated which will result in the kind of growth change in the
bacteria present which will permit the observer to differentiate between
types of bacteria
These media provide environments in which different bacteria can be
distinguished from one another. For instance, violet red bile agar is
used to distinguish coliform bacteria such as Escherichia coli from
noncoliform organisms. The coliform bacteria appear as bright pink
colonies in this media, while noncoliforms appear a light pink or clear.
NB; Certain media are both selective and differential. For instance,
MacConkey agar differentiates lactose-fermenting bacteria from
nonlactose-fermenting bacteria while inhibiting the growth of Gram-
positive bacteria. Since lactose-fermenting bacteria are often involved
 SCIENCE LABORATORY TECHNOLOGY
in water pollution, they can be distinguished by adding samples of
water to MacConkey agar and waiting for growth to appear.
Enriched medium.
Such a medium provides specific nutrients that encourage selected
species of microorganisms to flourish in a mixed sample. When
attempting to isolate Salmonella species from fecal samples, for
instance, it is helpful to place a sample of the material in an enriched
medium to encourage Salmonella species to multiply before the
isolation techniques begin.

Assay media
These are media used for assay of vitamins, amino acids and antibiotics.
They are also used for testing antibiotics .

Enumeration media
These are media used for determining bacterial population or number .

Media for characterization

These are media used to determine the type of growth produced by
organisms as well as their ability to produce chemical changes

Maintenance media
they are media used to maintain viability and physiological characteristic
of culture. To preserve microbial cultures, they may be placed in the
refrigerator to slow down the metabolism taking place. Two other
methods are deep-freezing and freeze-drying. For deep-freezing, the
microorganisms are placed in a liquid and frozen quickly at
temperatures below –50°C. Freeze-drying (lyophilization) is
performed in an apparatus that uses a vacuum to draw water off after
the microbial suspension has been frozen. The culture resembles a
powder, and the microorganisms can be preserved for long periods in
this condition.
Preparation of culture media
 SCIENCE LABORATORY TECHNOLOGY
Some naturally occurring substance e.g. skimmed milk is used for
activation of bacteria . They are dispensed into suitable containers e.g. test
tubes , flasks and sterilized before use . Nutrient broth or media are
prepared by compounding the required individual ingredients and
hydrating them (adding water) . Practically all media are available
commercially in powder form .
To grow bacteria successfully in the laboratory , one should inoculate
bacteria in a media of suitable nutritional content and incubate the
inoculated media in an appropriate physical condition.
One must also know whether thee bacteria to be inoculated are aerobic or
anaerobic , whether the media contain the autotrophic or heterotrophic
bacteria or both .he should also know their temperature requirements i.e.
whether they are psychophylic , mesophylic or thermophylic .
Bacteria are typically grown in broth contained in test tubes or flasks or
on agarplate .these are often considered as batch or closed systems
because the nutrients in them are not renewed nor are waste generated by
the growing microorganisms are removed .under such conditions the
microorganisms increases I population in a predictable fashion and then
eventually declines . such growth pattern can be illustrated clearly in a
growth curve .
Nutrients must therefore be constantly added and waste products
removed from these systems in order to maintain the cells in a state of
continuos growth called open system or continuos culture .

PURE CULTURE
In nature bacteria often grow in close association with many other kind
of microorganisms and they jointly contribute to the numerous
activities and processes in their surrounding . They even depend on each
other for many survival needs e.g. some of the metabolic waste of some
microorganisms may serve as nutrients or energy source for another
microorganisms . but in the laboratory , bacteria can be isolated and
grown in pure cultures . These enables easy study of characteristics of
particular species .a pure culture is defined as a population of organisms
descended from a single cell and is therefore separated from all other
species.
There are a variety of techniques that can be used to obtain pure cultures .
but first one should ensure that all apparatus and media used are sterile
prior to use and should again be handled using aseptic techniques .Pure
 SCIENCE LABORATORY TECHNOLOGY
cultures can be grown on solid mediums or broths , the environment in
which they are grown and the method of isolation should be in aseptic
conditions. A single bacteria grown in such conditions will multiply to
form a colony which is a mass of cells descended from the original one .

Isolation methods.
To obtain separated colonies from a mixed culture, various isolation
methods can be used. One is the streak plate method,

The streak plate method

These is the simplest and the most commonly used technique for
isolating bacteria . A sterilized inoculating loop is dipped into the
solution containing the organisms of interest and then lightly spread
several times across an agar plate , creating a partern of parallel streaks
that covers approximately one third of the plate . The loop is again
sterilized and other several parallel streaks made at angles to the
previous streaks covering other remaining portion of the plate. These
action drugs some of the cells streaked over the first portion of the plate
to uninoculated portion thus creating a region containing a more dilute
inoculum. The objective is to reduce the number of cells being spread
with each successive series of streaks hence effectively diluting the
concentration of the cells .

STOCK CULTURE
Stock culture are cultures stored for use as inoculum in later procedures
or reference . stock culture are stored in refregirator on an agar slant .
agar slants are are agar mediums in a tube that was held at a shallow
angle as the media solidifies creating a larger surface are . Cultures can
be stored for a long time in frozen state at -70oc in a glycerol solution
.the glycerol prevents ice crystals from damaging cells .the cells can also
be lympholized

METHODS OF DETECTING AND MEASURING BACTERIAL

GROWTH
There are many techniques used to study bacteria growth these includes
; determining the bacterial population , detecting their products or
measuring their weight (biomas ). The choice of which technique to use
 SCIENCE LABORATORY TECHNOLOGY
depend on the various characteristics of the sample and the objectives of
the measurements .some of these methods includes

DETERMINING BACTERIAL POPULATION

(i) Direct cell count .

These technique is useful for determining the numbers of those bacteria
that cannot be grown in the culture . these method however do not
distinguish between living and dead cells . these method include

(a) Direct microscopic count .

these method requires relatively high bacterial concentration in the
sample being examined . the number of bacteria in the sample is counted
using special glass slides i.e. counting chambers , which hold a known
volume of liquid . these is normally viewed under the light microscope
and the number of bacteria contained
in the liquid can be counted precisely

(b) Cell- counting instrument

These is an electronic instrument that counts cells in a suspension as
they pass single file through a minute aperture. The suspending fluid
must be saline or an electrolyte because the machine actually detects
and subsequently counts brief changes in resistance that occur when
non- conducting particles such as bacteria passes by .

(ii) Viable cell count

These method is used to quantify the number of cells that are capable of
multiplying .these method is very invaluable for monitoring bacterial
growth in samples such as food and water that often contain low
concentration of bacterial that cannot be seen using direct microscopic
count .These method conprises the following techniques.

(a)Plate count
The method measures the number of viable bacterias in a sample by
exploiting the fact that when an isolated cell is allowed to grow in on a
nutrient agar it will give rise to one colony . the number of colonies
therefore found growing on a nutrient agar will tell the number of cells
that were initially present in the sample .There are two different plating
methods i.e. ;
 SCIENCE LABORATORY TECHNOLOGY
(i)Pour plate method
(ii) Spread plate method
Both methods first involve the dilution of samples in 10-fold increments
these is in order to disperse the cells present in these samples so that the
ideal number of cells in each sample are between 30- 300 cells or
colonies .the dilution should be made from a sterile solution (or diluent)
which is usually 0.85 % NaCL in water (physiological saline ).
In pour plate method , about 0.1 – 1.0 of the final dilution is transferred
into a sterile petri dish and then overlaid with melted nutrient agar hat
have been cooled to 50oc , the agar is still liquid at these temperature .the
petridish is then gently swirled to mix the bacteria with liquid agar and is
then left to harden . during hardening , the individual cells are fixed in
place and after incubation , they grow to form distinguishable colonies.
In spread plate method , about 0.1 –0.2 ml of the final dilution is
transferred into a plate originally containing the already solidified
nutrient agar medium . the solution is then spread over the surface of the
agar with a sterilized bent glass rod . after evenly spreading the sample
on the solidified agar surface , the late is again incubated for a period of
time so as to allow the colonies to form which can then be counted
By noting how much the sample was diluted and by counting the number
of colonies found to have grown in each dilution , a mathematical
calculation can be done to determine the number of cells likely to have
been present in the original sample . its believed that each colony
observed in the plates came from one single cell and therefore cells
clustered together as one colony are referred to as colony forming unit
(a)Membrane filtration
These method is used when the number of organisms in the sample are
very low . the method therefore concentrates the number of bacteria by
filtratering them using a known volume of liguid through a sterile
membrane filters which have very tiny pores that cannot allow bacteria to
pass through . the filter is then placed on an apropriate agar medium and
then incubated . the number of colonies that grow on the filter indicates
the number of bacteria that were in the filtered solution .
(b) The most probable number (MPN)
These methods applies statistics basing on probability to estimate the
number of cells present in a solution . these is achieved by continuously
diluting the sample until it reaches a point at which subsequent dilution
receives no cell. Usually three sets of three or five tubes containing the
same growth media are prepared . each set receives a measured amount
 SCIENCE LABORATORY TECHNOLOGY
of a sample e.g. water , milk , food etc . these is done in a way such that
the second set receives 10-fold less than the first set and the 3rd set
receives 100-folds less than he 1st set while the 4th set receives 1000-
folds less than the 1st set and so on . i.e. each set is inoculated with an
amount that is 10-folds less than the previous set . These are then
incubated and presence or absence of growth in each test tube is noted .
the results obtained are then compared against an MPN table which gives
statistical estimates of the cell concentration .

B) MEASURING BIOMAS
In these method , the mass of the cells is determined either by measuring
their
turbidity , their total weight or the amount of chemical constituents
produced.
(a) Measuring turbidity
Turbidity is simply cloudiness. Suspension of bacteria in a broth culture
often results in the suspension becoming turbid .
These is due to the scattering of light passing through the liquid by cells .
the amount of light scattered s proportional to the concentration of cells
present in the suspension . A spectrometer is used to measure turbidity .
These instrument
transmits light through the specimen and measures the percentage that
reaches a light detector .These method however requires the medium
to contain relatively high numbers of bacteria in order for the suspension
to be cloudy or turbid .

(ii) Total weight

These method is tidious and time consuming and therefore not used
routinely .but
is useful when measuring growth of filamentous organisms . the method
is done by first using a centrifuge to cause the heavy cells to pull down
and the liquid suspending them to float up and be removed . the weight
of the resulting cells is estimated and these is usually proportional to the
number of cells that were present in the culture media . The dry weight
can be obtained after drying the cells completely for 8-12hrs before
wighing them .
(A) DETECTING BACTERIAL BIOCHEMICAL
CONSTITUENTS
 SCIENCE LABORATORY TECHNOLOGY
Products of microbial growth can be used to estimate the number and test
or confirm their presence in the media .
Some of these products and test include includes
(a) Catalase production .
Some bacteria produce catalase enzymes . The presence of these enzyme
can be detected using hydrogen peroxide H2O2 which is broken down to
produce H2O and O2 . Oxygen production is indicated by production of
bubbles.
(ii) Citrate test
Some bacteria depend the on citrate as the sole carbon source. Citrate
test is done to confirm whether or not the bacteria depends on citrate .
This is indicated by formation of turbidity which is usually indicated by
the characteristic colour change of the medium.
(iii) Gelatinase production
Some bacteria produce gelatinase enzyme which can break down gelatin
to polypeptide and convert it to liquid .Gelatinase test can be used to
confirm such bacteria.
(iv) Oxidase test
These method detects the activity of cytochrome C oxidase that is
produced by some groups of bacteria . these is normally detected using a
specific reaction which normally gives a dark color .
(iv) Urease test
These method is used to detect the enzymatic degradation of urea to
CO2 and NH3 . the media becomes alkaline thus causing the pH indicator
to change its color.
(v) Hydrogen sulfide production test
these method detects H2S that is liberated as a result of degradation of
sulfur containing amino acids. A black precipitate forms due to the
reaction of H2S with an iron salt in the medium.
(vi) Indole test
These method detects the enzymatic degradation or removal of amino
group from trptophan. The product indole that is
produced reacts with a chemical reagent added turning it into a deep
red color.
(vii) Methyl red test
These method detects mixed acids produced . The media becomes red in
colour upon addition of certain indicator when media becomes acidic at
pH below 4.5 .
(viii) Sugar fermentation
 SCIENCE LABORATORY TECHNOLOGY
These method detects the acidity resulting from fermentation of sugar
that have been incorporated into the media .it also detects gas
production .The color of the pH indicator incorporated into the media
changes if acid is produced .an inverted tube (Durham test ) traps any gas
produced .
(ix) Voges –Proskerer test (V-P test)
These test detects acetoine which is an intermediate of fermentation
pathway that leads to production of 2,3 butadiaol .red colour develops
on addition of chemicals that detects acetoine.
NB its advisable not to rely on a single biochemical test for each test you
carry out but instead carry out different simultaneous test which
identifies the organisms faster and more reliably

CHAPTER TWENTY SEVEN

THE BODY FLUIDS

BLOOD

THE BLOOD CELLS

Functions of the Blood - Blood is a liquid tissue that has three major
functions; as a transportation, for regulation, and protection.  Blood
transports materials to and from all the cells of the body  Wastes produced
by the cells carried away in the blood to organs which remove the wastes. 
Blood acts as a regulator.  Blood can absorb heat from warm areas of the
body and releases the heat in cooler areas.  The blood usually maintains a
constant pH and water balance.  Blood also protects the body.  It holds
specialized cells and chemicals that defend the body against diseases.
 SCIENCE LABORATORY TECHNOLOGY
Blood has the ability to clot, preventing the body from loosing large
amounts of blood due to an injury.
The Components of the Blood - Because blood has many functions you
might be able to conclude that the blood is composed of many different
parts.  The liquid part of the blood is called plasma.  Plasma takes up
about 55% of the total volume of the blood.  The remaining 45% of the
blood is made up of red blood cells, white blood cells, and platelets.  An
adult human has between four and six liters of blood in the body.
Plasma - Plasma is the clear liquid portion of the blood.  90% of plasma
is water.  The other 10% contains many types of molecules, including
nutrients, glucose, vitamins, cellularwastes, salts, and proteins.  There are
three major types of proteins which exist in plasma.  These are albumin,
fibrinogen, and globulins.  Each proteins has a specific function to
perform.  The albumin keeps water from leaving the blood and entering
the surrounding cells by osmosis.  It does this by helping to keep the
concentration of the water within the blood the same as the concentration
in the body tissues.  The fibrinogen aids in the clotting of the blood.  Some
globulins transport proteins and other substances from one part of the
body to the next.  Other globulins are known as antibodies, which help to
fight of infection.  Antibodies are proteins that attach to and help destroy
foreign substances in the body.
 Red Blood Cells - These cells are red and carry oxygen and carbon
dioxide.  They are present in huge numbers within the blood.  The human
body conatins 30 trillion red blood cells, or approximately 5 million cells
per cubic millimeter of blood. The red blood cells transport oxygen from
the lungs to the tissues in the body.  They also carry carbon dioxide from
the body tissues to the lungs.  In humans, the matured red blood cells do
not contain a nuclei. Their cytoplasm is filled with an iron-containing
protein called hemoglobin.  Hemoglobin is the substance that gives the
blood its red colour.  Human red blood cells are constructed by bone
marrow and have and a average life span of up to 120 days. New cells are
produced at the same rate red blood cells are destroyed .  This occurs at
pace of about 2 million per second. The old red cells are removed from the
body by the spleen and liver and are then broken down. The iron from the
hemoglobin is then collected and reused.
When a person has an insufficient amount of hemoglobin or too few red
blood cells, this is referred to as anemia.  Both of these conditions lowers
 SCIENCE LABORATORY TECHNOLOGY
the amount of oxygen that can be carried throughout the blood. Anemia
causes the cells not to recieve the proper amount of oxygen.  This is a
heriditary disorder, and is caused by an abnormal form of hemoglobin.

The red blood cell

Platelets - The part of the blood which is

involved in the clotting of blood.  Platelets are formed when bits of
cytoplasm are pinched.  Even though these bits of cytoplasm contain no
nuclei, they surrounded by a membrane.  There about a total of 1.5 trillion
platelets in the blood of an adult human.  

platelets
There are about 300,000 platelets existing in a cubic millimeter of blood.
Their life lasts for about seven days and are produced at about 200 billion
per day.
 Blood Clotting - Unless blood is a free-flowing liquid it will not be able to
circulate easily throughout the body's blood vessels.  However, in being a
 SCIENCE LABORATORY TECHNOLOGY
liquid it could cause a large variety of problems.  If there was an injury
that broke a large blood vessel it could lead to a large loss of blood. This
problem is resolved by the complex mechanism of clotting.  The clots form
a temporary barrier to prevent blood loss until the vessel walls have
healed.                                                                                  
The Clotting Process  - When a blood vessel is injured, platelets begin
to collect near the injury, which forms a barrier known as the platelet plug.
When the platelets come in contact with an injured area, they swell up,
become sticky, and release certain chemicals.
Blood clotting requires many precise reactions to maintain a certain
balance between quick and efficient clot formation.  This balance has to be
kept exact so that your blood will not clot at the wrong time. Prothrombin
and fribrinogen are two proteins that are produced by the liver that are
always present in the plasma of the blood.  The injured tissues and
platelets release prothrombin activator and calcium ions (Ca 2+) to change
prothrombin into the enzyme thrombin.  Then the thrombin splits two
short amino acid chains from each fibrinogen molecule.  The ends of the
fibrinogen then join together, forming threads of fibrin.  Thesefibrin
surround the platelet plug in the damaged area of the blood vessel and
provide the shape for the clot.  Red blood cells are present within the
fibrin which makes the clot appear red.  After this the clot stops the
bleeding, gets smaller, and hardens.
Over time the injury is repaired by
the growth of new cells which will replace
the cells lost because of the injury.
When all the healing has finished an
enzyme called plasmin in activated
and dissolves the fibrin clot.

Blood clotting process

 SCIENCE LABORATORY TECHNOLOGY

Some Clotting Problems - There are many conditions which can cause
the clotting process to be disrupted.  People who have the hereditary
disease haemophilia, lack the essential Factor VIII (Antihaemophilic
Globulin, or AHG) of blood clotting.  These people can recieve certain
injections which will enable ther blood to clot properly.  If you don't have
enough platelets in the blood or lack vitamin K, this will reduce the ability
to clot.
Blood Groups - The ABO grouping and the Rh factor are the most often
used to determine blood type.  The physician Karl Lansteiner determined
that there were four major blood groups among humans.  He designated
them as A, B, AB, and O.  This same system is used today.  It is now a
known fact that blood type is based on a type of glycoprotein present in
the red blood cells.  Type A blood has A-type glycoproteins, type B blood
has B-type glycoproteins, type AB blood has both of these glycoproteins,
and type O blood has neither of them.  The A and B glycoproteins function
as antigens, and they combine specifically with antibody molecules.  When
this kind of reaction occurs, the red blood cells agglutinate (join together).
People who have type O blood are called universal donors.  It can be given
to anyone without fear of agglutination because it does not contain any
antigens that could combine with anti-a or anti-b antibodies  The name
universal recipient is given to a person with type AB blood. AB blood does
not have anti-a or anti-b antibodies that could combine with any antigens.
The Rh factors are another group of antigens found on the surface of red
blood cells.  They are called Rh factors simply because they were first
discovered in rhesus monkeys.  About 85% of  humans are Rh+, which
means they have Rh factors on their red blood cells.  The remaining 15% of
humans are Rh-, which means that they do not contain the Rh
factor.  These Rh factors may present a problem when a mother is Rh- and
the baby is Rh+.  If their blood is to mix and some of the Rh+ blood cells
enter the mother's circulatory system, her immune system will from anti-
Rh antibodies.  In future pregnancies, these anti-Rh antibodies could
enter the babies blood stream.  If this were to happen and the baby was
Rh-,  the antibodies would destroy the babies red blood cells.  This
problem can be eliminated if the mother is given an injection of anti-Rh
antibodies to destroy the baby's Rh+ cells shortly after the birth of each
 SCIENCE LABORATORY TECHNOLOGY
Rh+ child.  This will prevent the mother's immune system from forming
it's own antibodies.
White Blood Cells - There is a variety of colorless blood cells which
make the white blood cells, or known as leukocytes. These white blood
cells are defenders for the body.  They protect the body from bacteria and
viruses, which are disease-causing organisms.  Unlike red blood cells, the
white blood cells contain a nucleus and are larger than the red blood cells.
There are fewer white blood cells than white, but there are still about 60
billion in an adult human body.  The bone marrow and lymphatic tissue
produce approximately 1 million white blood cells every second.  The
white bloods cells are distribute themselves throughout the body by
moving through the circulatory system.  When there is an infection within
the body, the white blood cells collect in the infected area and attack the
foreign organisms.
There are five different types of white blood cells.  The majority of them
function to protect the body in some form.  A portion of the white blood
cells are what are called phagocytic(monocytes & neutrophils).  They
protect the body by fighting the bacterial invaders, and anything which
does not belong in the body.  The lymphocytes take care of the production
of

White blood cells

antibodies and the cells that destroy certain substances and uncommon
cells. Usually, there are 7000 to 10 000 white blood cells present per cubic
millimeter of blood. When an infection of the blood occurs, the numbers
of white blood cells may increase to 30,000 or more per cubic millimeter.
The phagocytic white blood cells eat the bacteria which they encounter.
After these phagocytic cells eat the bacteria some of them die.  This is what
pus is when it forms around an infected area.
white blood cells are the major immunologically active cells of the
immune system ( others are plasma cells , platelets and mast cells )
 SCIENCE LABORATORY TECHNOLOGY
Staining of white blood cells shows two main groups i.e. (I) granulocytes
(ii) agranulocytes
(a) Granulocytes
Granulocytes have lobbed nucleus and are made in the bone marrow but
they differentiate from those hat make the red blood cells
Granulocytes can further be divided into
(a) Eosinophils
(b) Neutrophils
(c ) Basophils
NEUTROPHIL
This granulocyte has very tiny light staining granules (the granules are
very difficult to see). The nucleus is frequently multi-lobed with lobes
connected by thin strands of nuclear material. These cells are capable of
phagocytizing foreign cells, toxins, and viruses.
When taking a Differential WBC Count of normal blood, this type of cell
would be the most numerous. Normally, neutrophils account for 50-70%
of all leukocytes. If the count exceeds this amount, the cause is usually due
to an acute infection such as appendicitis, smallpox or rheumatic fever. If
the count is considerably less, it may be due to a viral infection such as
influenza, hepatitis, or rubella.

EOSINOPHIL
This granulocyte has large granules (A) which are acidophilic and appear
pink (or red) in a stained preparation. This micrograph was color
enhanced to illustrate this feature. The nucleus often has two lobes
connected by a band of nuclear material. (Does it looks like a telephone
receiver?) The granules contain digestive enzymes that are particularly
effective against parasitic worms in their larval form. These cells also
phagocytize antigen - antibody complexes.

These cells account for less than 5% of the WBC's. Increases beyond this
amount may be due to parasitic diseases, bronchial asthma or hay fever.
Eosinopenia may occur when the body is severely stressed.
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BASOPHIL

The basophilic granules in this cell are large, stain deep blue to purple,
and are often so numerous they mask the nucleus. These granules contain
histamines (cause vasodilation) and heparin (anticoagulant).
In a Differential WBC Count we rarely see these as they represent less
than 1% of all leukocytes. If the count showed an abnormally high number
of these cells, hemolytic anemia or chicken pox may be the cause.

Eosinophil Basophils
neutrophils

(b) AGRANULOCYTES

LYMPHOCYTE

The lymphocyte is an agranular cell with very clear cytoplasm which stains
pale blue. Its nucleus is very large for the size of the cell and stains dark
purple. (Notice that the nucleus almost fills the cell leaving a very thin rim
of cytoplasm.) This cell is much smaller than the three granulocytes
(which are all about the same size). These cells play an important role in
our immune response. The T-lymphocytes act against virus infected cells
and tumor cells. The B-lymphocytes produce antibodies.
This is the second most numerous leukocyte, accounting for 25-35% of the
cells counted in a Differential WBC Count. When the number of these cells
exceeds the normal amount, one would suspect infectious mononucleosis
or a chronic infection. Patients with AIDS keep a careful watch on their T-
cell level, an indicator of the AIDS virus' activity.
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MONOCYTE

This cell is the largest of the leukocytes and is agranular. The nucleus is
most often "U" or kidney bean shaped; the cytoplasm is abundant and
light blue (more blue than this micrograph illustrates). These cells leave
the blood stream (diapedesis) to become macrophages. As a monocyte or
macrophage, these cells are phagocytic and defend the body against
viruses and bacteria.These cells account for 3-9% of all leukocytes. In
people with malaria, endocarditis, typhoid fever, and Rocky Mountain
spotted fever, monocytes increase in number.

Lymphocyte Monocyte
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IMMUNOLOGICAL
TECHNIQUES
Immunity is a medical term that describes a state of having sufficient
biological defenses to avoid infection, disease, or other unwanted
biological invasion. Immunity involves both specific and non-specific
components. The non-specific components act either as barriers or as
eliminators of pathogens to stop infection by micro-organisms before they
can cause disease. Other components of the immune system adapt
themselves to each new disease encountered and are able to generate
pathogen-specific immunity
Immunity is the capacity to recognize the intrusion of foreign materials
into the body and to mobilize cell and cell products to help remove that
particular foreign product from the body with greater speed and
effectiveness . i.e. immunity is the ability of the body to defend itself from
infection caused by foreign materials .Cells or tissues which bring about
immunity forms the immune system .the immune systems must be
effective in order to provide the body from infection .  
The general characteristic of an immune system

An immune system should have

(a) memory - these is the ability to remember its first encounter with
such diseases e.g. small pox ,measles etc and prevents such diseases from
occurring again in future
(b) Specificity – these is the capacity to distinguish or discriminate
between antigens even if those antigens are closely related . NB antigens
are molecules which can cause the body to form antibodies. Antibodies
are protein molecules that are synthesized by an animal in response to
the presence of foreign substance known as antigen .
(c) once the immune system is exposed to the same antigen again , it
results to a faster and a grater response (each future response is gets more
efficient)
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(d) immune system distinguishes between self and non self . NB self
means what belongs to the body while none slf means what is foreign . if
immune systems does not make these distinction , it would constantly be
making antibodies against our own parts of our body . however
sometimes the immune system is unable to tolerate the self antigens and
it starts making antibodies against its own self antigens . these
phenomenon is known as auto - immunity
(e) the initial interaction of cells or antibodies with the antigens can
be greatly amplified by a number of secondary mechanisms . such
mechanisms include
 increasing vascular permeability so that the immune cells e.g.
phagocytes can move into tissues effectively to go and engulf the
pathogens
 release the mediator substance from mast cells which are coated with
antibodies
 by activation of complement proteins . these are proteins capable of
destroying foreign materials
 production of lymphokines or cytokines from sensitized lymphocytes
(f) an immune system is under control of immune response genes( Ir
genes) . which controls the ability to discriminate foreign and self
antigens and to remember that the antibodies or cells had earlier own
came across or in contact with that particular antigen and also controls
the ability to improve the immune response by utilizing secondary
mechanisms

Specific immunity
(a)                    Specific immunity is that aspect of your body's defenses
against pathogens (and other foreign material) that acts against specific
molecules, usually requiring that your immune system "learn" the
properties of specific molecules over a number of days or weeks before
mounting an effective response against the foreign material
(b)                    Typically a specific immune response against one pathogen
will be ineffective against a different pathogen, sometimes even a closely
related but still different pathogen
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(c)                    Specific immunity is that aspect of immunity that is primed
when individuals are vaccinated against, for example, pathogens or their
toxins
(d)                    Specific immunity includes humoral immunity and cell-
mediated immunity
(e)                    A number of body organs, tissues, and cell types are
involved in effecting each of these forms of specific immunity
THE IMMUNE SYSTEM
Bacteria, viruses, and any other thing that may cause disease are called
pathogens.  The microorganisms exist almost everywhere in the
environment, which means that you are exposed to these pathogens every
day.  Thankfully, humans have several defences against these
various pathogens.  The defenses keep your body's environment healthy
most of the time.
First-Line Defenses - The body's first line of defense against pathogens
uses mostly physical and chemical barriers such as sweat, skin, tears,
mucus, stomach acid, and so on.  Our skin and other membranes which
line the body passages are fairly effective in keeping most pathogens out of
the body.  Mucus can trap pathogens, which are then washed away or
destroyed by chemicals.  Tears, sweat, and saliva have certain chemicals
which can kill different pathogens.
Second-Line Defenses - If a pathogen is able to get past the body's first
line of defense, and an infection starts, the body can rely on it's second
line of defense.  This will result in what is called an inflammatory
response.  This is a reaction that causes redness, heat, swelling, and pain
in the area of infection.   Redness and heat are due to cappilary dilation
resulting in increased blood flow.  Sweeling is caused by the passage of
plasma from the blood stream and into the damaged tissue.  The pain is
mainly due to the tissue destruction, and to a lesser extent, the swelling.
Third-Line Defenses - Sometimes the second line of defense is still not
enough and the pathogen is then heading for the body's last line of
defense, the immune system.  The immune system will recognize, attack,
destroy, and remembers each foreign substance and pathogen that enters
the body.  It does this by making specialized cells and antibodies that
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makes the pathogens useless.  Unlike the first line and second line defense
the immune system determines between kinds of pathogens. For each type
of pathogen, the immune system produces cells that are specific for that
particular pathogen. 

Nonspecific immunity        
Nonspecific immunity includes those defenses against pathogens, etc.,
that are not specific to each pathogen including such things as physical
barriers, chemical barriers, some cellular defenses, inflammation, fever,
and molecular defenses
Innate immunity (genetic immunity, species immunity)
While specific immunity must be learned (i.e., may be acquired), innate
immunity is even present even before the exposure to a pathogen
Acquired immunity
Acquired immunity contrasts with innate because it requires previous
exposure to a pathogen (or its product) before immunity is acquired by the
host
   Acquired (specific)immunity is the immunity that is responsible for
subsequent exposures to the same pathogens causing less or no disease
(i.e., your becoming "immune" There are two categories of means by
which such immunity may be acquired, artificially and naturally

Naturally acquired immunity (colostrum)

 Naturally acquired immunity is that immunity acquired upon exposure to
a specific pathogen particularly in the course of an infection/disease
Additionally, naturally acquired immunity occurs when an infant obtains
colostrum from mom    "Colostrum is the first fluid secreted by the
mammary glands after childbirth. Although deficient in many nutrients
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found in milk, colostrum contains large quantities of antibodies that cross
the intestinal mucosa and enter the infant's blood.
  The infant is thus naturally immune against many or all of the diseases
that the mother is immune to especially as a consequence of the mother
possessing antibodies against the associated pathogens
Artificially acquired immunity (antiserum, antitoxin) Specific
immunity may also be acquired artificially.Artificially acquired immunity
specifically refers to vaccination (which is an artificial exposure to a
pathogen's antigens, i.e., without infection or, at least, without disease)
and to the transfusion of antibodies from one individual into another
(antiserum or antitoxin, etc.)
Active immunity
Active immunity occurs when an individual's own immune system is
induced to produce a specific immune response against an
antigen/pathogen.   Active immunity can occur either upon infection or
disease (naturally acquired active immunity), or artificially upon
vaccination (artificially acquired active immunity ); note that there is
some ambiguity in the definitions I've used since vaccines can cause
infections so the distinction between artificially and naturally acquired
immunity is really one between how the antigens were acquired, by
natural versus by artificial means  Active immunity can last as long as the
immune system cells, that mediate this immunity , survive within an
individual; this can be for weeks, months, or years
Passive immunity
   Passive immunity results when antibodies are produced by one
individual and then acquired by another. The acquisition of the antibodies
in colostrum by an infant is an example of (naturally acquired) passive
immunity ;the crossing of the placenta by maternal antibodies is another
example of naturally acquired passiveimmunity
Passive immunity may also be artificially acquired, particularly when
antiserum or antibodies produced by one individual are transfused into a
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second individual . In all cases, passive immunity represents the passive
acquisition of an immune response that was actively acquired by another
individual
However, because passiveimmunity involves the transfusion of molecules
rather than the transfusion of immune system cells, passive can last for at
most months since antibodies have a finite life span within the body.
  On the other hand, passive immunity is functional immediately upon
reception, whereas active immunity(ironically) requires time (days,
weeks) before a functional immune response develops

LYMPHOID ORGANS ,TISSUES AND IMMUNE CELLS

Immune system consist of interconnecting network of organs and
tissues . The dominant cells in these organs and tissues are lymphocytes
and therefore these organs and tissues are known as lymphoid .
The immune system consist of a number of lymphoid organs including
(c) thymus glands
(d) lymph nodes
(e) spleen
(f) tonsils
(g) aggregates of lymphoid tissues in noon lymphoid organs e.g. payers
patches in the gut
(h) bone marrow tissues in the long bones
The thymus and the bone marrow are referred to as the central or
primary lymphoid organs or system while the rest are referred to as
peripheral or secondary lymphoid organs . These is because development
and function of the peripheral lymphoid organs depends absolutely on
the cells made by the thymus and the bone marrow
THE PRIMARY (CENTRAL ) LYMPHOID ORGANS
A) THYMUS GLANDS
Thymus gland is a lobed organ located in the thoracic cavity just above
the heart. It consist of many lobes each of which contains a cortex and
medullar .
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Thymus gland begins to function in the embryonic stage but its at its
most active stage at time near or shortly after birth
It however decreases in size after weaning period and will soon stop
functioning . Thymus gland is important in the development of immune
response in an individual . removal of thymus is called thymectomy .
absence of thymus gland in new born leads to premature death due to
lack of lymphocytes in the blood and tissue fluids (lymphopenia ) the
animal may also lack ability to recognize and react to tissue transplant
(does not reject allografts ) but these however is not the case in older
animals these may be due to the reason that the function of the thymus
glands are carried out earlier in the animals life and hence the thymus
becomes less important so long as the remaining parts of the immune
system are functional .
The thymus glands functions by accepting the stem cells from the bone
marrow at the time of birth and ensures completion of the development .
They increase in sizes and develop to mature T – lymphocytes
(thymocytes ) . These thymocytes package themselves in the medullar and
cortex of the thymus glands . Some of the thymocytes mature into
different classes of mature T-cells e.g. helper T-cells , suppressor T-cells
and killer T –cells . Others leave the thymus gland through the blood
stream and migrates into the lymph nodes , spleen , tonsils , payers
patches etc where they form mature T-cells
T-cells are involve both in the cell mediated and humoral immune
system . during the process of maturation , the T-cells becomes
immunocompetent i.e. they gain the ability to respond to specific
antigens , they also gain the ability to distinguish between self and non
self antigens in the body . each T-cells produces small proteins molecules
called lymphokynes (or cytokynes or interleukins ). lymphokynes serve
to make T- cells keep on multiplying , promote inflammation and
stimulates B-cells to start making antibodies
BURSA OF FABRICIOUS
These is another primary lymphoid organ found in birds in addition to
thymus . it is situated near the cloaca . Stem cells enter the bursa of
fabricous where they differentiate into B-cells that are capable of
producing antibodies . Removal of bursa of fabricous is known a
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bursectomy . bursectomised a birds lack B-cells and therefore cannot
produce antibodies but they are able to reject graft .
Mammals including man have no bursa of fabricous yet hey have B –
cells . These means that in mammals the bone marrow serves an
equivalent role as the bursa of fabricous
THE BONE MARROW
Erythrocytes , leucocyte and platelets are produced in the bone marrow
by a process called hemoporesis . the stem cells that give rise to these
blood cells are called hemopoetic stem cells .
Precursors of the lymphocytes originates in the bone marrow but they
latter migrate in the blood stream to the thymus where they differentiate
into T- lymphocytes.
When these stem cells migrate to the spleen or lymphnodes or remain
in the bone marrow , they differentiate into B-lymphocytes . The
formation of lymphoid outside the bone marrow is known as
lymphoporesis .
The blood cells formation depends on the presence of hemopoetic stem
cells in the bone marrow (precursor cells)
Earlier in the 19th and 20th century, blood cells were put into two
categories according to their presumed origin i.e.
(i) Erythrocytes , granular leukocytes and platelets developing
from the bone marrow were called myeloid and their formation was
referred to as myelopoesis .
(ii) Lymphocytes and monocytes were believed to originate in the
lymph nodes , spleen and thymus . They were hence referred to as
lymphoid elements of blood and therefore there formation was called
lymphopoeisis.
But however recent studies have shown that lymphopoetic stem cells
similarly originates from the bone marrow
SECONDARY LYMPHOID ORGANS
(a) lymph nodes
the net work of the lymph vessels in the body forms the lymphatic
system . At some points in the lymphatic system ,are some swellings
referred to as lymph nodes . Lymph nodes are located in some strategic
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places in the body such as in the armpits ,neck regions and groins etc .
Each lymph node receives a flow of lymph through its afferent lymph
vessels.
Each lymph node is a spherical or bean shaped and it is surrounded by a
tough membrane or capsule . Beneath these capsule is a network of
phagocyte lined mesh work called subcapsular sinus . These meshwork
region filters any microorganisms present and phagocytes or engulfs
them . phagocytes present in these region are macrophages .
The lymph nodes have a densely packed lymphocytes and macrophages
organized into spherical nests or cells called follicles. When stimulated by
an antigen , these follicles becomes enlarged and the cells inside multiply
rapidly particularly at the center (germinal center )
An active follicle produces lymphocytes and plasma cells which in turn
produces antibodies . The antibodies are produced as a result of presence
or introduction of any antigen . These explains why armpits lymph nodes
due to a wound on a finger .the wound allows entrance of antigens into
the body which find their way into the lymph node were they are
trapped . In the lymph nodes the follicles becomes very active by
enlarging and producing many lymphocytes and macrophages . The
lymphocytes differentiates into plasma cells which then make antibodies
which shall fight against the antigens introduced . The macrophages
engulf the bacteria at faster rate .all these activities makes the lymph
nodes to swell and become painful
(b) spleen
the spleen have two major functions
(i) it destroys old red blood cells
(ii) it’s a major site for immune response
the spleen function just like the lymph node but the main difference is
that the fluid it deals with is blood and not lymph .the spleen have two
major parts (I) the red pulp and (ii) the white pulp
The red pulp deals with destruction of red blood cells while the white
pulp is where the immune response are generated . The lymphocytes are
concentrated in the white pulp which surrounds the central artery called
periarterial lymph node .
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The T- lymphocytes are found in the central parts of the pulp while B-
lymphocytes are found in the germinal centers of the lymph follicles .
The white pulp is separated from the red pulp by a marginal zone . The
lymphocytes enter and leave the pulp via the capillaries of the central
arteries in he marginal zone . Some mature plasma cells pass from the
marginal zone to the red pulp .
The marginal zone is where the blood lymphocytes ,antigens and tissue
fluids are deposited , there also intense phagocytic activities carried out
by macrophages
Other lymphoid tissues
(i) Gut associated lymphoid tissues (GALT) e.g. payers patches
and appendix .
These tissues contain both T and B independent areas which contain T
and B lymphocytes
(ii) mucosa associated lymphocytes tissues (MALT)
They are found in the gut, urinary tract and respiratory tract . they have
both T and B lymphocytes
Antigen
                  Another way of defining specific immunity is that it is a means
by which a body defends itself against the presence of specific antigens
associated with, for example, pathogens.   Antigens are the protein or
polysaccharide components of pathogens
Antibodies work by interacting with (binding to)specific structures found
on specific antigens
Immunogen
  Synonymous with antigen, an immunogen/antigen is "a substance the
body identifies as foreign and toward which it mounts an immune
response… Most antigens are large protein molecules with complex
structures and molecular weights greater than 10,000 [Daltons]. Some
antigens are polysaccharides, and a few are glycoproteins (carbohydrate
and protein)… Proteins usually have greater antigenic (immunogenic)
strength because they have a more complex structure than
 SCIENCE LABORATORY TECHNOLOGY
polysaccharides." That is, proteins possess many more potential epitopes
than do carbohydrates      "Antigens are found on the surface of viruses
and all cells, including bacteria, other microorganisms, and human cells.
The exact chemical structure of each of a cell's antigens is determined by
genetic information in its DNA. Bacteria can have antigens on capsules,
cell walls, and even flagella. Many microorganisms have several different
antigens somewhere on their surface. Determining how the human body
responds to these different antigenic determinants is important in making
effective vaccines. …antigens on the surfaces of red blood cells determine
blood types, and antigens on other cells determine whether a tissue
transplanted from another person will be rejected."
Epitope (antigenic determinant)
     Complex antigens such as proteins produce more robust immune
responses because each structure/complexity on an antigen can serve as
the site of binding of a different immune system molecule (e.g., an
antibody)
Each of these separate binding areas/structures is called an epitope
Complex antigens possess numerous epitopes and the binding of immune
system molecules (e.g., antibodies) to epitopes can have different effects
depending on the epitope
Hapten
  Haptens are small molecules that can serve as antigens (i.e., display
immunologically recognized epitopes) upon binding to a larger molecule
(e.g., a protein)
    Allergies to penicillin occur because penicillin can serve as a hapten
upon binding to certain body proteins
Antibody (titer)
One of the immune system molecules that bind to the epitopes on
antigens is the antibody
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Antibodies are secreted proteins that are found as soluble proteins in body
fluids (for more on antibodies, see immunoglobulin, below)
One measures the quantity of antibodies in terms of antibody titers (e.g.,
active antibody per unit volume)

Lymphocytes (white blood cells)

Lymphocytes are one category of white blood cells Lymphocytes mediate
specific immunity
We can differentiate lymphocytes into a variety of types including,
particularly, the B lymphocytes (B cells) and the T lymphocytes (T cells)
B lymphocytes (B cells)
   B lymphocytes are the producers of antibodies; that is, they mediate
humoral immunity .B cells are not matured in the thymus
T lymphocytes (T cells)
  T cells mediate cell-mediated immunity.T cells come in a variety of
typeswhich possess different antigens (proteins) on their surfaces and
which have different roles in the immune response
The T in T cell stands for thymus and it is in the thymus that T cells
mature, especially in immature immune systems
Clonal selection hypothesis (self, nonself, tolerance)
The immune system possesses the ability to recognize antigens/epitopes
to which it has never been exposed (nonself)
In addition, the immune system possesses the ability to not recognize
(i.e., not bind to) antigens/epitopes associated with normal body tissues
(self)
The means by which both of these mechanisms occur includes the
selective amplification of those immune system components that
recognize foreign antigens and the selective deletion of those immune
system components that recognize normal body tissues
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Together these mechanisms constitute the clonal selection hypothesis
   "According to this hypothesis, embryos contain many different
lymphocytes, each genetically programmed to recognize a particular
antigen and make antibodies to destroy it. If a lymphocyte encounters and
recognizes that antigen after development is complete, it divides
repeatedly to produce a clone, a group of identical progeny cells that make
the same antibody. If, during embryonic development, it encounters its
programmed antigen as part of a normal host substance (self), the
lymphocyte is somehow destroyed or inactivated. This mechanism
removes lymphocytes that can destroy host tissues and thereby creates
tolerance for self. It also selects for survival [of] lymphocytes that will
protect the host from foreign antigens."
Specificity
The hallmark of specific immunity is the specificity of the immune
response This means that an immune response to specific epitopes will
not be effective against a pathogen lacking these epitopes, even if the
second pathogen is otherwise closely related to the first  Specificity is not
necessarily perfect thus allowing, using the above example, partial
immunity against the second pathogen because the second pathogen
shares some, but not all epitopes with the first pathogen
Memory
  Actively acquired specific immunity possesses memory. This is another
way of saying that an immune response may be primed by exposure to an
antigen, and thereafter with subsequent exposure to the same antigen the
immune response against that antigen occurs much more rapidly
This memory is a function of the circulation of the lymphocytes which
either mediate the specific immune response or can give rise to cells (i.e.,
by dividing) that differentiate into immune-response-mediating cells
Subsequent exposure to an antigen, in sufficient quantity, will also serve
to strengthen subsequent immune responses
Humoral immunity [memory cells, plasma cells]
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That aspect of specific immunity that is mediated by antibodies is termed
humoral immunity
Humoral immunity is particularly effective against toxins (exotoxins),
whole bacteria, and free viruses (i.e., viruses not currently infecting cells)
"Humoral immunity depends first on the ability of B lymphocytes to
recognize specific antigens and second on their ability to initiate responses
that protect the body against foreign agents. The most common response
is the production of antibodies that will inactivate an antigen and lead to
destruction of infectious organisms."
  B cells, which mediate humoral immunity, each produce only a single
kind of antibody (i.e., one structure with the ability to bind to only a single
type/structure of epitope). These antibodies are displayed on the surface
of B cells
  The binding of an antigen to one of these surface B cells induces those
cells either to start producing antibody or to differentiate into cells that
produce antibody
    These antigens may be soluble, found on the surface of pathogens, or
displayed by other immune system cells such as macrophages
   Plasma cells are those B cells that can immediately produce (and
secrete) antibody molecules
Memory cells are those more long-term-stable B cells that can
differentiate into plasma cells
Immunoglobulin (Ig)
Another name for antibody is immunoglobulin. Antibodies come in a
variety of classes (i.e., IgG, IgM, IgA, IgE, and IgD) possessing variations
on the basic antibody structure.These different classes are made by
different body tissues and vary in their properties and uses
By far the most commonly studied is IgG which is the common blood
antibodies
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Antibodies function by binding to antigens and directly inactivating them
(viruses and toxin) they stimulate the attachment of complement
(bacteria)
They directly stimulate lysis (via membrane attack complexes) and
phagocytosis (opsonization) as well as   Stimulating killer T cells
Multivalence (agglutination)
         Note that an antibody is multivalent meaning that a single antibody
molecule can bind to two epitopes (e.g., the upper tips of the "Y" shape of
the antibody molecule)
   Note that each half (left and right) of the molecule are essentially
identical; the two epitopes that a given antibody may bind therefore must
be structurally identical (i.e., a given IgG molecule can bind two otherwise
identical epitopes)
This multivalent nature of antibodies allows antibodies (especially the
highly multivalent IgM) to bind together antigens in a clumping known as
agglutination, which can aid in pathogen phagocytosis
Primary response
  Upon initial exposure to an antigen that a body has never been exposed
to, there is a 10 to 17 day lag before the peak in antibody (particularly IgG)
concentration
Secondary response
  The second (or later) time an antibody is exposed to an antigen there is
only a 2 to 7 day lag before the peak in antibody (particularly IgG)
concentration
This is why your body is able to rapidly defeat many of the pathogens to
which you have been previously exposed
Note that it is only the memory cells from the primary response that
participate in a secondary response
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Cell-mediated immunity
    Cell-mediated immunity, not surprisingly, is mediated by cells rather
than via secreted proteins (i.e., not by antibodies)
     Cell-mediated immunity is particularly active against virus-infected
cells, though also can function against other foreign or otherwise renegade
(e.g., cancer) eukaryotic cells
  Cell-mediated immunity is mediated by cyototoxic T lymphocytes
Cytotoxic T cells (killer T cells)
Cytotoxic T cells act by first recognizing that other body cells are virally
infected, and then killing those cells, thus destroying the viral infection (of
a cell) while destroying the cell
This cell killing by the host immune system is one means by which virus
infections damage hosts and thus cause disease
The means by which cytotoxic T cells recognize that a body cell is virus
infected is as follows:
 Body cells degrade internal cellular proteins on a regular basis, whether
because the protein deteriorates, because the protein was incorrectly
folded, or simply in the course of normal cell metabolism
 Some of these degraded cells are broken up into short peptides which
are then presented on the surface of normal body cells in association with
MHC class I proteins
 If a virus or some other pathogen is infecting a cell, then the pathogen,
too, will produce proteins, and these nonself proteins will also eventually
be presented on the surface of cells in association with MHC class I
proteins
It is the MHC-presented peptide antigens, from intracellularly infecting
pathogens, that cytotoxic T cells recognize; self antigens are not
recognized by T cells because T cells that recognize self antigens are
deleted during T cell maturation
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The MHC class I-associated presentation of the antigens essentially serves
as a means of allowing the immune system to "look" inside of cells, e.g.,
serving as a window onto a cell's metabolism

Vaccination
Vaccination is artificially acquired active immunity.The goal of
vaccination is to prime humoral and cellular immune responses against
pathogens (or their toxins) without simultaneously causing disease
  Typically vaccines are most easily developed against either toxins or
against pathogens that, upon infection, induce lifetime immunity (i.e.,
naturally acquired active immunity) in their hosts
Pathogens for which even infection accompanied by disease does not
result in immunity (particularly diseases that cause gastrointestinal
distress) are not easy to prevent by the application of vaccination (note
that the polio vaccines are seemingly exceptions but instead do not
prevent the gastrointestinal ailment so much as systemic infection by the
virus)
   Note that most vaccines do not prevent infection (i.e., growth of the
pathogen) so much as the disease that results from infection; this is
accomplished either by blocking the effects of pathogen toxins or by
priming the immune system against the pathogen such that infection is
brought under control much more quickly, before full-blown disease
results
   Vaccines do not necessarily confer life-long immunity ; the duration of
immunity typically is dependent on to what degree the vaccination mimics
a natural infection plus to what degree subsequent natural infections are
capable of boosting the immunization
Categories of vaccine type include
a) Live vaccines
b) Whole-killed vaccines
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c)     Toxoid vaccines

d) Recombinant vaccines
Typically vaccines are employed to prevent disease though not as a means
of treating existing infections; exceptional is the rabies vaccines which is
employed, in humans, to prevent the development of disease given
infection
(a) Live vaccine (live-attenuated vaccine)
Live vaccines result in infection but not disease, and confer the most
long-lasting immunity
    Live vaccines typically are attenuated so that while they can still cause
infection, they have a reduced propensity to cause disease
(b) Whole-killed vaccine
      One alternative to a live vaccine is a vaccine consisting of an
inactivated whole pathogen, i.e., a whole-killed vaccine
             An advantage to employing a whole-killed vaccine over a live
vaccine is that the former can be safer
     However, these “dead” vaccines do not result in infection and therefore
do not produce as robust an immune response as live vaccines
(particularly lacking is cytotoxic T cell-mediated immunityagainst
intracellular pathogens), but if boosted by subsequent natural infection by
the same pathogen can produce long-lasting immunity
(c) Toxoid vaccine
                    Toxoid vaccines, against toxins (exotoxins), are not boosted by
subsequent infections (since the amount of toxin produced by natural
infections is not great) and require a sufficient standing antibody titer;
declines in this titer necessitates boosting
(d) Recombinant vaccine
 SCIENCE LABORATORY TECHNOLOGY
  In contrast to a whole vaccine (i.e., either live-attenuated or whole-killed
vaccines), a recombinant vaccine contains only part of a microorganism
(i.e., the vaccine is not whole).
   Such vaccines are typically safer than whole vaccines because they
(ideally) contain only those parts that induce a good immune response
and not those parts that can lead to complications or side effects
    However, recombinant vaccines are also typically less effective than
whole vaccines, especially live-attenuated vaccines, since at best
recombinant vaccines induce an incomplete immune response against a
pathogen (i.e., an immune response against only part of the pathogen and
then, except in the case of DNA vaccines, only inducing humoral
immunity)
Most vaccines are given by hypodermic injection as they are not absorbed
reliably through the gut. Live attenuated Polio and some Typhoid and
Cholera vaccines are given orally in order to produce immunity based in
the bowel.

Booster
  Boosting a vaccine involves revaccination by the same vaccine (or, at
least, against the same organism)
  Booster vaccines are employed for a number of reasons
   To assure that the immune response induced by an at least partially
successful vaccination is boosted to a sufficiently large immune response
to be effective in protecting against disease
  To assure that all recipients of the vaccination display at least some
immune response (e.g., oral polio vaccine does not always lead to
infection, but if given three times over a relatively long period, at least one
of the vaccinations is likely to result in infection and therefore potentially
successful vaccination)
              To replenish the immune response after a long period (e.g., 10
years as is the case for tetanus vaccine)
 SCIENCE LABORATORY TECHNOLOGY

IMMUNOCHEMICAL METHODS
Several invivo and invitro (inside and outside the cell) methods are
available for observing results of combination of antigens and their
specific antibodies . Antibodies reacts with antigens to form precipitation
(if the antigen is a macromolecule ) and agglutinations (if the antigen is
part of the cellular surface of the protein) .the formation of specific
antigens and antibody aggregates usually results in co-precipitation of
certain components of the serum called complements . They are said to
be complement fixed. The extend of complement fixation is directly
related to the antigen antibody combination . If the antigen is on the
cellular surface , it reacts with the antibody in the presence of
complements to form a cytotoxic reaction which result in lysis (breaking)
of the cell.
If crude extracts containing a mixture of antigens are injected in a
mammal, several antibodies that are specific for each antigen are
formed . These formed antibodies can be extracted separately from the
serum by adding each antigen separately (singly).
Immunological methods vary in sensitivity and usefulness, precipitation
and hemagglutination methods are the most common methods used .
But their application vary or depends on the easiness of observation e.g.
precipitin reactions is commonly used because is directly visible and can
be quantified but it however have a disadvantage of being limited to
antigens in aqueous solution.
There are three common methods of measuring the antibody potency
outside (invitro) the living organism
(a) dilution method
thee involves working out the highest dilution of the serum that should
be added to a constant amount of antigen at which detectable reaction
e.g. precipitation , agglutination or lysis occur . The serum dilution
giving this end point (highest dilution) is called titre

(b) optimum proportion method

These involves determining the ratio of antigen and serum at which
flocculation if most rapid and if relative proportions are optimal . In
 SCIENCE LABORATORY TECHNOLOGY
these method, various dilutions of antigens are mixed with constant
volume of serum in series of tubes . The total volume of each tube is being
constant . The tubes in which flocculation first occurs is noted and the
ratio of antigens dilution to the serum dilution is calculated .
(c) quantitative chemical analysis
These method is generally based on the determining the protein nitrogen
content in a precipitated or agglutination product . Quantitative method
is capable of widespread application in the analysis of mixtures
containing immunologically reactive substances . It is highly specific and
permits estimation of a given constituent of a mixture without chemical
fractionation or purification unlike the other analytical chemical
methods . It also requires a small amount of material for analysis, the
method is also capable of considerate precision of + or – 2% -5% under
suitable conditions .
IMMUNODIFUSSION
Immunodifussion is a method involving use of jellified media for
precipitation reaction . In this method , complexes of antigen –antibody
reactions could be analyzed by allowing them to react in a capillary tube
filled with agar. The antibody solution if filled with a warm agar solution ,
which is, then allowed to harden in the tube . When the antigen solution is
added , the reaction of the antigen and the antibody forms a precipitation
zone . The number of these zones is less or equal to the number of the
independent precipitation systems (antigen- antibody reactions) present
in the mixture. A line of precipitation forms where an antigen interacts
with its antibody
IMMUNOELECTROPHORESIS
in this method, the sample to be analyzed is subjected to electrophoresis
in a transparent gel , which is then placed a line of magnetic field by
passing an electric current through the gel. At the end of the
electrophoresis , the component of the analyzed media shall have been
separated and will occupy different positions in the gel depending on
their relative mobidities . therefore an immune serum containing
antibodies is then poured into an elongated trough made in the gel and
parallel to the axis of migration , the antibodies diffuse perpendicularly to
the axis and when antibodies and antigens meet in suitable proportions ,
they form a precipitates in form of arcs
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LABELING OF ANTIGENS AND ANTIBODIES
There are several methods used in labeling antigens and antibodies
(a) Fluorescent immunoassay (FIA)
(b) Radioimmuno assay
(c) Enzyme immunoassay

Fluorescent immune assay

In this method , an UV fluorescent marker dye is attached to the
antibody.
Radio immuno assay
In these method, a radioactive marker e.g. isotope is attached to the
antibody or antigen . This method is highly selective and specific but the
radioactive isotope may decay rapidly. It is also expensive and the assay
must be carried out by a highly trained person
Enzyme immuno assay
In this method , enzyme markers are attached to antibodies or antigens .
These methods have several advantages i.e.
It have no radioactive hazard
It have relatively long shelf life
It is inexpensive and uses readily available equipment’s
The test can be rapid and automated
Multiple simultaneous assays are possible
However the main disadvantage s are
 Plasma constituents in samples may affect enzyme activities
 The assay is relatively complex
 Enzyme activities are difficult to control
 Some reactions are relatively slow
There are two main types of enzyme immuno assay method;
Enzyme linked immunoabsorbent assay (ELISA)
Enzyme multiplied immuno assay technique (EMIT)
(a)Enzyme multiplied immuno assay technique (EMIT)
EMIT is widely used for assaying microammounts of drugs and biological
substances in human fluids
(b) Enzyme linked immunoabsorbent assay (ELISA)
 SCIENCE LABORATORY TECHNOLOGY
ELISA technique is widely applied in medicine and food industry in
detection of pollutants, toxins and microbial and parasitic diseases and
other environmental contaminant. This method is based on the following
assumptions ; that antigens and antibodies can be linked to an insoluble
carrier surface and still retain its activity
Enzyme markers can be attached to an antibody or antigen and will still
retain both its immunological and enzymatic activity
An antigen or an antibody is linked to the inside of a well of a carrier
plate
The sensitized carrier surface binds with the specific antibody or antigen
from the test solution
Enzyme labeled immunoglobulin is attached to the antigen –antibody
complex that is detected by the enzyme label and changes he color of the
added substrate.
The optical density of the final color can be quantified and is proportional
to the amount of antibody or antigen in the original test solution.
N/B. An ideal enzyme link should show
 High enzyme activity at low substrate concentration,
 Should have good stability at the pH required for good antibody –
antigen binding
 It should also provide an inexpensive, accurate and sensitive assay
method .
 It should yield stable enzyme –labeled conjugates
 Available in soluble , purified form at low cost
 Should have no health hazard
 Should not be affected in its activity by sample components
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER TWENTY EIGHT

ENZYMOLOGY
Enzymes are biological molecules that catalyze (i.e., increase the rates of)
chemical reactions.
 SCIENCE LABORATORY TECHNOLOGY
All known enzymes are proteins. They are high molecular weight
compounds made up principally of chains of amino acids linked together
by peptide bonds
Enzymes can be denatured and precipitated with salts, solvents and other
reagents. They have
molecular weights ranging from 10,000 to 2,000,000.

Specificity of Enzymes
One of the properties of enzymes that makes them so important as
diagnostic and research
tools is the specificity they exhibit relative to the reactions they catalyze. A
few enzymes
exhibit absolute specificity; that is, they will catalyze only one particular
reaction. Other
enzymes will be specific for a particular type of chemical bond or
functional group. In
general, there are four distinct types of specificity:
1. Absolute specificity - the enzyme will catalyze only one reaction.
2. Group specificity - the enzyme will act only on molecules that have
specific functional
groups, such as amino, phosphate and methyl groups.
3. Linkage specificity - the enzyme will act on a particular type of
chemical bond regardless of
the rest of the molecular structure.
4. Stereochemical specificity - the enzyme will act on a particular
steric or optical isomer.
Though enzymes exhibit great degrees of specificity, cofactors may serve
many apoenzymes.
For example, nicotinamide adenine dinucleotide (NAD) is a coenzyme for
a great number of
dehydrogenase reactions in which it acts as a hydrogen acceptor. Among
them are the alcohol dehydrogenase, malate dehydrogenase and lactate
dehydrogenase reactions.In enzymatic reactions, the molecules at the
beginning of the process, called substrates, are converted into different
molecules, called products. Almost all chemical reactions in a biological
cell need enzymes in order to occur at rates sufficient for life. Since
enzymes are selective for their substrates and speed up only a few
reactions from among many possibilities, the set of enzymes made in a cell
determines which metabolic pathways occur in that cell.
 SCIENCE LABORATORY TECHNOLOGY

Activation energy
Like all catalysts, enzymes work by lowering the activation energy for a
reaction, thus dramatically increasing the rate of the reaction. As a result,
products are formed faster and reactions reach their equilibrium state
more rapidly. Most enzyme reaction rates are millions of times faster than
those of comparable un-catalyzed reactions. As with all catalysts, enzymes
are not consumed by the reactions they catalyze, nor do they alter the
equilibrium of these reactions. However, enzymes do differ from most
other catalysts in that they are highly specific for their substrates.
Enzymes are known to catalyze about 4,000 biochemical reactions

Enzyme activity can be affected by other molecules. Inhibitors are

molecules that decrease enzyme activity; activators are molecules that
increase activity. Many drugs and poisons are enzyme inhibitors. Activity
is also affected by temperature, pressure, chemical environment (e.g.,
pH), and the concentration of substrate. Some enzymes are used
commercially, for example, in the synthesis of antibiotics. In addition,
some household products use enzymes to speed up biochemical reactions
(e.g., enzymes in biological washing powders break down protein or fat
stains on clothes; enzymes in meat tenderizers break down proteins into
smaller molecules, making the meat easier to chew).

Enzyme nomenclature

An enzyme's name is often derived from its substrate or the chemical

reaction it catalyzes, with the word ending in -ase. Examples are lactase,
alcohol dehydrogenase and DNA polymerase. This may result in different
enzymes, called isozymes, with the same function having the same basic
name. Isoenzymes have a different amino acid sequence and might be
distinguished by their optimal pH, kinetic properties or immunologically.
Isoenzyme and isozyme are homologous proteins. Furthermore, the
normal physiological reaction an enzyme catalyzes may not be the same as
under artificial conditions. This can result in the same enzyme being
identified with two different names. For example, glucose isomerase,
 SCIENCE LABORATORY TECHNOLOGY
which is used industrially to convert glucose into the sweetener fructose, is
a xylose isomerase in vivo (within the body).

The International Union of Biochemistry and Molecular Biology have

developed a nomenclature for enzymes, the EC numbers; each enzyme is
described by a sequence of four numbers preceded by "EC". The first
number broadly classifies the enzyme based on its mechanism.The top-
level classification is

 EC 1 Oxidoreductases: catalyze oxidation/reduction reactions

 EC 2 Transferases: transfer a functional group (e.g. a methyl or
phosphate group)
 EC 3 Hydrolases: catalyze the hydrolysis of various bonds
 EC 4 Lyases: cleave various bonds by means other than
hydrolysis and oxidation
 EC 5 Isomerases: catalyze isomerization changes within a single
molecule
 EC 6 Ligases: join two molecules with covalent bonds.

According to the naming conventions, enzymes are generally

classified into six main family classes and many sub-family
classes.

Structures and mechanisms

Enzymes are in general globular proteins and range from just 62 amino
acid residues in size, for the monomer of 4-oxalocrotonate tautomerase, to
over 2,500 residues in the animal fatty acid synthase. A small number of
RNA-based biological catalysts exist, with the most common being the
ribosome; these are referred to as either RNA-enzymes or ribozymes. The
activities of enzymes are determined by their three-dimensional structure.
However, although structure does determine function, predicting a novel
enzyme's activity just from its structure is a very difficult problem that has
not yet been solved.

Most enzymes are much larger than the substrates they act on, and only a
small portion of the enzyme (around 2–4 amino acids) is directly involved
 SCIENCE LABORATORY TECHNOLOGY
in catalysis. The region that contains these catalytic residues, binds the
substrate, and then carries out the reaction is known as the active site.
Enzymes can also contain sites that bind cofactors, which are needed for
catalysis. Some enzymes also have binding sites for small molecules,
which are often direct or indirect products or substrates of the reaction
catalyzed. This binding can serve to increase or decrease the enzyme's
activity, providing a means for feedback regulation.

Like all proteins, enzymes are long, linear chains of amino acids that fold
to produce a three-dimensional product. Each unique amino acid
sequence produces a specific structure, which has unique properties.
Individual protein chains may sometimes group together to form a protein
complex. Most enzymes can be denatured—that is, unfolded and
inactivated—by heating or chemical denaturants, which disrupt the three-
dimensional structure of the protein. Depending on the enzyme,
denaturation may be reversible or irreversible.

Structures of enzymes with substrates or substrate analogs during a

reaction may be obtained using Time resolved crystallography methods.

Specificity

Enzymes are usually very specific as to which reactions they catalyze and
the substrates that are involved in these reactions. Complementary shape,
charge and hydrophilic/hydrophobic characteristics of enzymes and
substrates are responsible for this specificity. Enzymes can also show
impressive levels of stereospecificity, regioselectivity and
chemoselectivity.

Some of the enzymes showing the highest specificity and accuracy are
involved in the copying and expression of the genome. These enzymes
have "proof-reading" mechanisms. Here, an enzyme such as DNA
polymerase catalyzes a reaction in a first step and then checks that the
product is correct in a second step. This two-step process results in
average error rates of less than 1 error in 100 million reactions in high-
fidelity mammalian polymerases. Similar proofreading mechanisms are
also found in RNA polymerase, aminoacyl tRNA synthetases and
ribosomes.
 SCIENCE LABORATORY TECHNOLOGY
Some enzymes that produce secondary metabolites are described as
promiscuous, as they can act on a relatively broad range of different
substrates. It has been suggested that this broad substrate specificity is
important for the evolution of new biosynthetic pathways.

"Lock and key" model

Enzymes are very specific, this is because both the enzyme and the
substrate possess specific complementary geometric shapes that fit exactly
into one another. This is often referred to as "the lock and key" model.

“Induced fit” model

Induced fit model is a modification to the lock and key model it suggests
that since enzymes are rather flexible structures, the active site is
continuously reshaped by interactions with the substrate as the substrate
interacts with the enzyme. As a result, the substrate does not simply bind
to a rigid active site; the amino acid side-chains that make up the active
site are molded into the precise positions that enable the enzyme to
perform its catalytic function. In some cases, the substrate molecule also
changes shape slightly as it enters the active site. The active site continues
to change until the substrate is completely bound, at which point the final
shape and charge is determined.
 SCIENCE LABORATORY TECHNOLOGY

Mechanisms

Enzymes can act in several ways, all of which lower the activation energy
(Gibbs energy):

 Lowering the activation energy by creating an environment in

which the transition state is stabilized (e.g. straining the shape of a
substrate—by binding the transition-state conformation of the
substrate/product molecules, the enzyme distorts the bound substrate(s)
into their transition state form, thereby reducing the amount of energy
required to complete the transition).
 Lowering the energy of the transition state, but without
distorting the substrate, by creating an environment with the opposite
charge distribution to that of the transition state.
 Providing an alternative pathway. For example, temporarily
reacting with the substrate to form an intermediate ES complex, which
would be impossible in the absence of the enzyme.
 Reducing the reaction entropy change by bringing substrates
together in the correct orientation to react. Considering ΔH ‡ alone
overlooks this effect.
 Increases in temperatures speed up reactions. Thus,
temperature increases help the enzyme function and develop the end
product even faster. However, if heated too much, the enzyme’s shape
deteriorates and the enzyme becomes denatured. Some enzymes like
thermolabile enzymes work best at low temperatures.

It is interesting that this entropic effect involves destabilization of the

ground state, and its contribution to catalysis is relatively small.

Allosteric sites

Allosteric sites are sites on the enzyme that bind to molecules in the
cellular environment. The sites form weak, noncovalent bonds with these
molecules, causing a change in the conformation of the enzyme. This
change in conformation translates to the active site, which then affects the
 SCIENCE LABORATORY TECHNOLOGY
reaction rate of the enzyme. Allosteric interactions can both inhibit and
activate enzymes and are a common way that enzymes are controlled in
the body.

Cofactors and coenzymes

Many enzymes require the presence of other compounds - cofactors -

before their catalytic
activity can be exerted. This entire active complex is referred to as the
holoenzyme; i.e.,
apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic
group or metal-ionactivator) is called the holoenzyme.

Apoenzyme + Cofactor = Holoenzyme

Cofactors

Some enzymes do not need any additional components to show full

activity. However, others require non-protein molecules called cofactors
to be bound for activity. Cofactors can be either inorganic (e.g., metal ions
and iron-sulfur clusters) or organic compounds (e.g., flavin and heme).
Organic cofactors can be either prosthetic groups, which are tightly bound
to an enzyme, or coenzymes, which are released from the enzyme's active
site during the reaction. Coenzymes include NADH, NADPH and
adenosine triphosphate. These molecules transfer chemical groups
between enzymes. Enzymes that require a cofactor but do not have one
bound are called apoenzymes or apoproteins. An apoenzyme together
with its cofactor(s) is called a holoenzyme (this is the active form). Most
cofactors are not covalently attached to an enzyme, but are very tightly
bound. However, organic prosthetic groups can be covalently bound (e.g.,
biotin in the enzyme pyruvate carboxylase). The term "holoenzyme" can
also be applied to enzymes that contain multiple protein subunits, such as
the DNA polymerases; here the holoenzyme is the complete complex
containing all the subunits needed for activity.

Coenzymes
 SCIENCE LABORATORY TECHNOLOGY
Coenzymes are small organic molecules that can be loosely or tightly
bound to an enzyme. Tightly bound coenzymes can be called allosteric
groups. Coenzymes transport chemical groups from one enzyme to
another. Some of these chemicals such as riboflavin, thiamine and folic
acid are vitamins. The chemical groups carried include the hydride ion
(H-) carried by NAD or NADP+, the phosphate group carried by adenosine
triphosphate, the acetyl group carried by coenzyme A, formyl, methenyl or
methyl groups carried by folic acid and the methyl group carried by S-
adenosylmethionine.

A prosthetic group
an organic substance which is dialyzable and thermostable which is firmly
attached to the protein or apoenzyme portion.

Thermodynamics

As all catalysts, enzymes do not alter the position of the chemical

equilibrium of the reaction. Usually, in the presence of an enzyme, the
reaction runs in the same direction as it would without the enzyme, just
more quickly. However, in the absence of the enzyme, other possible
uncatalyzed, "spontaneous" reactions might lead to different products,
because in those conditions this different product is formed faster.
 SCIENCE LABORATORY TECHNOLOGY

Furthermore, enzymes can couple two or more reactions, so that a

thermodynamically favorable reaction can be used to "drive" a
thermodynamically unfavorable one. For example, the hydrolysis of ATP
is often used to drive other chemical reactions.

Enzymes catalyze the forward and backward reactions equally. They do

not alter the equilibrium itself, but only the speed at which it is reached.
For example, carbonic anhydrase catalyzes its reaction in either direction
depending on the concentration of its reactants.

(in tissues; high CO2

concentration)

(in lungs; low CO2

concentration)

Nevertheless, if the equilibrium is greatly displaced in one direction, that

is, in a very exergonic reaction, the reaction is in effect irreversible. Under
these conditions, the enzyme will, in fact, catalyze the reaction only in the
thermodynamically allowed direction.
 SCIENCE LABORATORY TECHNOLOGY
Kinetics

Main article: Enzyme kinetics

Mechanism for a single substrate enzyme catalyzed reaction. The enzyme

(E) binds a substrate (S) and produces a product (P).

Michaelis–Menten kinetics

In biochemistry, Michaelis–Menten kinetics is one of the simplest

and best-known models of enzyme kinetics. It is named after German
biochemist Leonor Michaelis and Canadian physician Maud Menten. The
model takes the form of an equation describing the rate of enzymatic
reactions, by relating reaction rate to , the concentration of a
substrate S.

It involves an enzyme E binding to a substrate S to form a complex ES,

which in turn is converted into a product P and the enzyme. This may be
represented schematically as

where,and denote the rate constants,[4] and the double arrows between S
and ES represent the fact that enzyme-substrate binding is a reversible
process.
 SCIENCE LABORATORY TECHNOLOGY
Under certain assumptions – such as the enzyme concentration being
much less than the substrate concentration – the rate of product
formation is given by

The reaction rate increases with increasing substrate concentration ,

asymptotically approaching its maximum rate , attained when all
enzyme is bound to substrate. It also follows that ,
where is the enzyme concentration. , the turnover number, is
maximum number of substrate molecules converted to product per
enzyme molecule per second.

The Michaelis constant is the substrate concentration at which the

reaction rate is at half-maximum, and is an inverse measure of the
substrate's affinity for the enzyme—as a small indicates high affinity,
meaning that the rate will approach more quickly.[5] The value of
is dependent on both the enzyme and the substrate, as well as
conditions such as temperature and pH.

Factors Affecting Enzyme Activity

Knowledge of basic enzyme kinetic theory is important in enzyme analysis
in order both to understand the basic enzymatic mechanism and to select
a method for enzyme analysis. The conditions selected to measure the
activity of an enzyme would not be the same as those selected to measure
the concentration of its substrate. Several factors affect the rate at which
enzymatic reactions proceed - temperature, pH, enzyme concentration,
substrate concentration, and the presence of any inhibitors or activators.
(i)Enzyme Concentration
 SCIENCE LABORATORY TECHNOLOGY
In order to study the effect of increasing the enzyme concentration upon
the reaction rate, the substrate must be present in an excess amount; i.e.,
the reaction must be independent of the substrate concentration. Any
change in the amount of product formed over a specified periodof time
will be dependent upon the level of enzyme present.
These reactions are said to be "zero order" because the rates are
independent of substrate concentration, and are equal to some constant k.
The formation of product proceeds at a rate
which is linear with time. The addition of more substrate does not serve to
increase the rate. In zero order kinetics, allowing the assay to run for
double time results in double the amount of product.
The amount of enzyme present in a reaction is measured by the activity it
catalyzes. The relationship between activity and concentration is affected
by many factors such as
temperature, pH, etc. An enzyme assay must be designed so that the
observed activity is proportional to the amount of enzyme present in order
that the enzyme concentration is the
only limiting factor. It is satisfied only when the reaction is zero order.
Enzyme activity is generally greatest when substrate concentration is
unlimiting.
As substrate is used up, the enzyme's active sites are no longer
saturated,substrate concentration becomes rate limiting, and the reaction
becomes first order
.

(ii) Substrate Concentration

It has been shown experimentally that if the amount of the enzyme is kept
constant and the substrate concentration is then gradually increased, the
reaction velocity will increase until it reaches a maximum. After this point,
increases in substrate concentration will not increase the velocity It is
theorized that when this maximum velocity had been reached, all of the
available enzyme has been converted to ES, the enzyme substrate
complex.
(iii)Temperature Effects
Like most chemical reactions, the rate of an enzyme-catalyzed reaction
increases as the temperature is raised. A ten degree Centigrade rise in
temperature will increase the activity of
most enzymes by 50 to 100%. Variations in reaction temperature as small
as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the
 SCIENCE LABORATORY TECHNOLOGY
case of enzymatic reactions, this is complicated by the fact that many
enzymes are adversely affected by high temperatures.
Because most animal enzymes rapidly become denatured at temperatures
above 40·C, most enzyme determinations are carried out somewhat below
that temperature.
Over a period of time, enzymes will be deactivated at even moderate
temperatures. Storage of enzymes at 5·C or below is generally the most
suitable. Some enzymes lose their activity when frozen.

(iv)Effects of pH
Enzymes are affected by changes in pH. The most favorable pH value - the
point where the enzyme is most active - is known as the optimum pH.
Extremely high or low pH values generally result in complete loss of
activity for most enzymes. pH is also a factor in the stability of enzymes.
As with activity, for each enzyme
there is also a region of pH optimal stability.
The optimum pH value will vary greatly from one enzyme to another.
In addition to temperature and pH there are other factors, such as ionic
strength, which can
affect the enzymatic reaction. Each of these physical and chemical
parameters must be considered and optimized in order for an enzymatic
reaction to be accurate and reproducible.

(v) Enzyme Inhibition

Enzyme reaction rates can be decreased by various types of enzyme

inhibitors.
 SCIENCE LABORATORY TECHNOLOGY

Competitive inhibitors bind reversibly to the enzyme, preventing the

binding of substrate. On the other hand, binding of substrate prevents
binding of the inhibitor. Substrate and inhibitor compete for the enzyme.

Competitive inhibition

In competitive inhibition, the inhibitor and substrate compete for the

enzyme (i.e., they can not bind at the same time). Often competitive
inhibitors strongly resemble the real substrate of the enzyme. For
example, methotrexate is a competitive inhibitor of the enzyme
dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to
tetrahydrofolate. The similarity between the structures of folic acid and
this drug are shown in the figure to the right bottom. In some cases, the
inhibitor can bind to a site other than the binding-site of the usual
substrate and exert an allosteric effect to change the shape of the usual
binding-site. For example, strychnine acts as an allosteric inhibitor of the
glycine receptor in the mammalian spinal cord and brain stem. Glycine is
a major post-synaptic inhibitory neurotransmitter with a specific receptor
site. Strychnine binds to an alternate site that reduces the affinity of the
 SCIENCE LABORATORY TECHNOLOGY
glycine receptor for glycine, resulting in convulsions due to lessened
inhibition by the glycine. In competitive inhibition the maximal rate of the
reaction is not changed, but higher substrate concentrations are required
to reach a given maximum rate, increasing the apparent Km.

Uncompetitive inhibition

In uncompetitive inhibition, the inhibitor cannot bind to the free enzyme,

only to the ES-complex. The EIS-complex thus formed is enzymatically
inactive. This type of inhibition is rare, but may occur in multimeric
enzymes.

Non-competitive inhibition

Non-competitive inhibitors can bind to the enzyme at the binding site at

the same time as the substrate,but not to the active site. Both the EI and
EIS complexes are enzymatically inactive. Because the inhibitor can not be
driven from the enzyme by higher substrate concentration (in contrast to
competitive inhibition), the apparent Vmax changes. But because the
substrate can still bind to the enzyme, the Km stays the same.

Mixed inhibition

This type of inhibition resembles the non-competitive, except that the

EIS-complex has residual enzymatic activity.This type of inhibitor does
not follow Michaelis-Menten equation.

In many organisms, inhibitors may act as part of a feedback mechanism.

If an enzyme produces too much of one substance in the organism, that
substance may act as an inhibitor for the enzyme at the beginning of the
pathway that produces it, causing production of the substance to slow
down or stop when there is sufficient amount. This is a form of negative
feedback. Enzymes that are subject to this form of regulation are often
multimeric and have allosteric binding sites for regulatory substances.
 SCIENCE LABORATORY TECHNOLOGY

CHAPTER TWENTY EIGHT

TOXICITY TESTING
What is toxicology

Toxicology is the study of how natural or man-made poisons cause

undesirable effects in living organisms.

What is Toxic?

This term relates to poisonous or deadly effects on the body by inhalation

(breathing), ingestion (eating), or absorption, or by direct contact with a
chemical

What is a Toxicant?

A toxicant is any chemical that can injure or kill humans, animals, or

plants; a poison. The term "toxicant" is used when talking about toxic
substances that are produced by or are a by-product of human-made
activities. For example, dioxin (2,3-7,8-tetrachlorodibenzo-p-dioxin
{TCDD}), produced as a by-product of certain chlorinated chemicals, is a
 SCIENCE LABORATORY TECHNOLOGY
toxicant. On the other hand, arsenic, a toxic metal, may occur as a
natural contaminant of groundwater or may contaminate groundwater as
a by-product of industrial activities. If the second case is true, such toxic
substances are referred to as toxicants, rather than toxins.

What is a Toxin?

The term "toxin" usually is used when talking about toxic substances
produced naturally. A toxin is any poisonous substance of microbial
(bacteria or other tiny plants or animals), vegetable, or synthetic chemical
origin that reacts with specific cellular components to kill cells, alter
growth or development, or kill the organism.

What are harmful or adverse effects?

Harmful or adverse effects are those that are damaging to either the
survival or normal function of the individual.

What is Toxicity? The word "toxicity" describes the degree to which a

substance is poisonous or can cause injury. The toxicity depends on a
variety of factors: dose, duration and route of exposure ,shape and
structure of the chemical itself, and individual human factors.

What is a Toxic Symptom?

This term includes any feeling or sign indicating the presence of a poison
in the system.

What are Toxic Effects?

This term refers to the health effects that occur due to exposure to a toxic
substance; also known as a poisonous effect on the body.

What is Selective Toxicity?

 SCIENCE LABORATORY TECHNOLOGY
"Selective toxicity" means that a chemical will produce injury to one kind
of living matter without harming another form of life, even though the two
may exist close together.

How Does Toxicity Develop?

Before toxicity can develop, a substance must come into contact with a
body surface such as skin, eye or mucosa of the digestive or respiratory
tract. The dose of the chemical, or the amount one comes into contact
with, is important when discussing how "toxic" an substance can be.

What is a dose?

The dose is the actual amount of a chemical that enters the body. The dose
received may be due to either acute (short) or chronic (long-term)
exposure. An acute exposure occurs over a very short period of time,
usually 24 hours. Chronic exposures occur over long periods of time such
as weeks, months, or years. The amount of exposure and the type of toxin
will determine the toxic effect.

What is dose-response?

Dose-response is a relationship between exposure and health effect, that

can be established by measuring the response relative to an increasing
dose. This relationship is important in determining the toxicity of a
particular substance . It relies on the concept that a dose, or a time of
exposure (to a chemical, drug, or toxic substance), will cause an effect
(response) on the exposed organism. Usually, the larger or more intense
the dose, the greater the response, or the effect. This is the meaning
behind the statement "the dose makes the poison."

What is the threshold dose?

Given the idea of a dose-response, there should be a dose or exposure level

below which the harmful or adverse effects of a substance are not seen in a
population. That dose is referred to as the ‘threshold dose'. This dose is
also referred to as the no observed adverse effect level (NOAEL), or
 SCIENCE LABORATORY TECHNOLOGY
the no effect level (NEL). These terms are often used by toxicologists
when discussing the relationship between exposure and dose. However,
for substances causing cancer (carcinogens), no safe level of exposure
exists, since any exposure could result in cancer.

What is meant by ‘individual susceptibility?' This term describes

the differences in types of responses to hazardous substances, between
people. Each person is unique, and because of that, there may be great
differences in the response to exposure. Exposure in one person may have
no effect, while a second person may become seriously ill, and a third may
develop cancer.

What is a "sensitive sub-population?

" A sensitive sub-population describes those persons who are more at risk
from illness due to exposure to hazardous substances than the average,
healthy person. These persons usually include the very young, the
chronically ill, and the very old. It may also include pregnant women and
women of childbearing age. Depending on the type of contaminant, other
factors (e.g., age, weight, lifestyle, sex) could be used to describe the
population.

The branches of Toxicology

Toxicology addresses a variety of questions. For example, in agriculture,

toxicology determines the possible health effects from exposure to
pesticides or herbicides, or the effect of animal feed additives, such as
growth factors, on people. Toxicology is also used in laboratory
experiments on animals to establish dose-response relationships.
Toxicology also deals with the way chemicals and waste products affect
the health of an individual.

The field of toxicology can be further divided into the following sub-
disciplines or sub-specialities:
 SCIENCE LABORATORY TECHNOLOGY
a) Environmental Toxicology is concerned with the
study of chemicals that contaminate food, water, soil, or the atmosphere.
It also deals with toxic substances that enter bodies of waters such as
lakes, streams, rivers, and oceans. This sub-discipline addresses the
question of how various plants, animals, and humans are affected by
exposure to toxic substances.
b) Occupational (Industrial) Toxicology is concerned
with health effects from exposure to chemicals in the workplace. This field
grew out of a need to protect workers from toxic substances and to make
their work environment safe. Occupational diseases caused by industrial
chemicals account for an estimated 50,000 to 70,000 deaths, and
350,000 new cases of illness each year in the United States (1).
c) Regulatory Toxicology gathers and evaluates existing
toxicological information to establish concentration-based standards of
"safe" exposure. The standard is the level of a chemical that a person can
be exposed to without any harmful health effects.
d) Food Toxicology is involved in delivering a safe and
edible supply of food to the consumer. During processing, a number of
substances may be added to food to make it look, taste, or smell better.
Fats, oils, sugars, starches and other substances may be added to change
the texture and taste of food. All of these additives are studied to
determine if and at what amount, they may produce adverse effects. A
second area of interest includes food allergies. Almost 30% of the people
have some food allergy. For example, many people have trouble digesting
milk, and are lactose intolerant. In addition, toxic substances such as
pesticides may be applied to a food crop in the field, while lead, arsenic,
and cadmium are naturally present in soil and water, and may be
absorbed by plants. Toxicologists must determine the acceptable daily
intake level for those substances.
e) Clinical Toxicology is concerned with diseases and
illnesses associated with short term or long term exposure to toxic
chemicals. Clinical toxicologists include emergency room physicians who
must be familiar with the symptoms associated with exposure to a wide
variety of toxic substances in order to administer the appropriate
treatment.
f) Descriptive Toxicology is concerned with gathering
toxicological information from animal experimentation. These types of
experiments are used to establish how much of a chemical would cause
 SCIENCE LABORATORY TECHNOLOGY
illness or death. We use information from these studies to set regulatory
exposure limits.
g) Forensic Toxicology is used to help establish cause and
effect relationships between exposure to a drug or chemical and the toxic
or lethal effects that result from that exposure.
h) Analytical toxicology identifies the toxicant through
analysis of body fluids, stomach content, excrement, or skin.
i) Mechanistic Toxicology makes observations on how
toxic substances cause their effects. The effects of exposure can depend on
a number of factors, including the size of the molecule, the specific tissue
type or cellular components affected, whether the substance is easily
dissolved in water or fatty tissues, all of which are important when trying
to determine the way a toxic substance causes harm, and whether effects
seen in animals can be expected in humans.

Classification of Toxic Agents :

Toxic substances are classified into the following:

A. Heavy Metals

Metals differ from other toxic substances in that they are neither created
nor destroyed by humans. Their use by humans plays an important role in
determining their potential for health effects. Their effect on health could
occur through at least two mechanisms: first, by increasing the presence
of heavy metals in air, water, soil, and food, and second, by changing the
structure of the chemical. For example, chromium III can be converted to
or from chromium VI, the more toxic form of the metal.

B. Solvents and Vapors

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Nearly everyone is exposed to solvents. Occupational exposures can range
from the use of "white-out" by administrative personnel, to the use of
chemicals by technicians in a nail salon. When a solvent evaporates, the
vapors may also pose a threat to the exposed population.

C. Radiation and Radioactive Materials

Radiation is the release and propagation of energy in space or through a
material medium in the form of waves, the transfer of heat or light by
waves of energy, or the stream of particles from a nuclear reactor .

D. Pesticides

Pesticide is defines as any substance or mixture of substances intended to

prevent, destroy, repel, or mitigate any pest. Pesticides may also be
described as any physical, chemical, or biological agent that will kill an
undesirable plant or animal pest .

E. Plant Toxins

Different portions of a plant may contain different concentrations of

chemicals. Some chemicals made by plants can be lethal. For example,
taxon, used in chemotherapy to kill cancer cells, is produced by a species
of the yew plant.

F. Animal Toxins

These toxins can result from venomous or poisonous animal releases.

Venomous animals are usually defined as those that are capable of
producing a poison in a highly developed gland or group of cells, and can
deliver that toxin through biting or stinging. Poisonous animals are
generally regarded as those whose tissues, either in part or in their whole,
are toxic.

Subcategories of Toxic Substance Classifications

All of these substances may also be further classified according to their:

 SCIENCE LABORATORY TECHNOLOGY
a) Effect on target organs (liver, kidney, hematopoietic system),
b) Use (pesticide, solvent, food additive),
c) Source of the agent (animal and plant toxins),
d) Effects (cancer mutation, liver injury),
e) Physical state (gas, dust, liquid),
f) Labeling requirements (explosive, flammable, oxidizer),
g) Chemistry (aromatic amine, halogenated hydrocarbon), or
h) Poisoning potential (extremely toxic, very toxic, slightly toxic)

NB; All chemicals (or any chemical) may be poisonous at a given dose and
through a particular route. For example, breathing too much pure oxygen,
drinking excessive amounts of water, or eating too much salt can cause
poisoning or death

Effect of poisons

Poisons cause undesirable effects to living organisms . they can cause one
or more toxic effects which can also be immediate (acute ) or long term
(chronic). Its therefore necessary to understand these effects so that they
are avoided or if they occur , then they can be rationally and successfully
managed . Toxic effects of toxic substances may be classified as follows

(A) local toxic effects

Local toxicity effects are those that occur at the site of first contact
between the biological system and the toxicant e.g. damage of tissues by
corrosive substances e.g. acids and alkalis on the contact points such as
on skin , mouth ,,eyes and respiratory drugs

(B) Systemic effects

These effects always occur away from the site of body contact with the
poison . the poisons are absorbed and distributed away to some other
sensitive sites e.g. in the central nervous system (neurotoxin), the liver
(hepatoxin ) , kidney (renotoxin) and other hemopoetic tissues .
 SCIENCE LABORATORY TECHNOLOGY
(B) Selective toxicity

Selective toxicity of a toxic substance refers to its ability to injure one

tissue , organ or cell without harming another one even if the two organs ,
cells or tissues may be in intimate contact with each other. These
selectivity in effect may be due to to the differences in distribution ,
biotransformation or excretion of the toxicant e.g. carbon tetracloride
(CCL4) is toxic to the liver because the liver have some enzymes that
have greater capacity to metabolize CCL4 to trichloromethyl free radicals
which is very toxic to the liver . also DDT is accumlated in the adipose
tissue but produces no toxic effect here but instead it exerts its toxic
effect in the CNS .

The toxic effect of a given substance is aggravated by the following

(a) the general state of health of the person

(b) general practice and habbits e.g. smoking and alcohol
(c) length of exposure
(d) the level of the dose

( C) Mutagenic effects

A mutagen is a substance that cause damage to the genetic make-up or

DNA of an organism. Exposure to chemical mutagens increases the
likelyhood of DNA damage and these can lead to hereditary problems
such as sickle cell anemia phenyl ketonuria . Radiation’s can also cause
mutagenic effects . Mutagenic effects are often serious to expectant
women than men

(D) Carcinogenic effects

Carcinogenic substances are those substances that have the ability to

induce cancer or tumours to living organisms . Most but not all
carcinogens act directly on the DNA . Carcinogens also have some
characteristic selectivity or specificity in terms of the target organs e.g.
 SCIENCE LABORATORY TECHNOLOGY
some carcinogens may induce blood cancer while others may induce lung
cancer . Carcinogens may be classified as follows

(i) primary carcinogens – they directly cause cancer upon contact

(j) procarcinogens – they are first metabolised and converted into an
active carcinogen first before they can cause cancer
(k) promoters – they stimulate latent cancer cells into characteristic
tumors and cancer

E) Teratogenic effects
Teratogenic effects involve the production of gross structural deformation
or malformation during foetal development . These developmental
abnormalities include skeleton faults e.g. improperly formed limps
vertebral column ,heart malformations , closed segments of the intestinal
tract or nervous imperfection . These hazards normally arise during
early periods of pregnancy i.e. when the embryo begins to form

(F) Reproduction effects

these effects are caused during the time when female and the male
reproductive systems processes such as ovulation and sperm production ,
implatation of the fertilized egg etc are affected by chemical substances

IMPORTANCE OF LABORATORY ANIMALS IN TOXICITY

TESTING

Most biological experiments are done using small animals such as

rats,mice , rabbits and guinea pigs . This is basically because it is ethically
imposible to use human or human specimens in doing this experiments
but it is possible to obtain similar results using laboratory animals . These
animals are easy to maintain in a laboratory environment and have short
life expectancy which enables the effect of a lifetime to be observed in
relatively short life time .
 SCIENCE LABORATORY TECHNOLOGY
Before using any laboratory animals in toxicological test one needs to
consider the following ;

(a) selection of species

(b) The manner of dosing e.g. through mouth. skin or nose etc
(c) the length of time required
(d) varriability in test animals
(e) age of test animal
(f) sex of the test animal

(a) selection of species

Results obtained from toxicological test shall only be considered to be
reliable and accurate if they can be reproduced through other similar
experiments . For one to obtain this reproducible results , its important to
select and use laboratory animals from a single breeding strain that is
genetically pure and the individual selected must be able to respond
uniformly .

(b) manner of dosing

it is also important o specify he manner in which he substance was dosed
. this is because any roué of dosage will affect the outcome of the results
obtained There are several routes for dosing animals either orally ,
through the nose (nasal),respiratory tract or through skin . The final
toxicity values obtained usually differ according to the path of dosing .
The path of dosage chosen should reflect the manner of exposure the
technique should also provide an accurate measure of compounds taken
into the test animal . if the food is dosed , then the amount of food
should be consumed by the animal should be recorded . intraperitonial
injections gives a higher level of accuracy with which the dose level is
known .

(c) The length of time

 SCIENCE LABORATORY TECHNOLOGY
Testing vary with time period. When an animal is dosed one time orally or
by injection or by coating through the skin or when the dosing is done for
a limited period of time. I such a test we determine the level of the
compound that is sufficient to achieve the desired measure of toxicity.

If testing exposes the animal daily for months for the animal’s lifetime,
the study is termed as chronic. chronic testing reveals whether there is
accumulation of either the compound itself or the damage it causes .

The knowledge of acute toxicity is always needed since we may have

excessive contact with any chemical in the surrounding at any time
.while chronic toxicity study is important in evaluating a chemical that
workers contact every day

(c) the variability among test animals

Toxicity of a compound differs from one subject to another. the variation
are explained in terms of the difference in their activities of the key
metabolic enzymes used to chemically change the toxicant , ease of
crossing the barriers into the particular body compartment such as the
brain and the effectiveness of the body system to eliminate the
compound from the body

Differences exist between animals of different species and also within

species. Males sometimes present a greater or less degree of sensitivity to
a particular compound than in identical female of the same species. some
of the enzymes that change a chemical structure of a foreign compound
are produced at a level that depends on levels of hormones such as
estrogen . These results in variations of toxic response. Even though
enzymes convert foreign substances into more readily eliminated
compound, the newly eliminated toxic compound might be more toxic
than the unmetabolized predecessor.

(d) Age of the test animal

New born animals are often more sensitive than adults to almost all
chemical substances .these is due to their body weight and their enzyme
 SCIENCE LABORATORY TECHNOLOGY
system which is still very low .also animals of advanced age show greater
susceptibility to the same compounds .

Types of test
(a) LD 50 test
Many different substances are tested these way including all drugs,
agricultural chemicals, cleaners and cosmetics and food ingredients. LD50
stands for the lethal dose 50%; it refers to the dose of a substance that will
kill half of the test animals. The results are usually expressed as mg /kg
.e.g. if it takes 400mg of a test substance to kill half of all the rabbits
weighing 4 kg then the LD50 is 100mg /kg

A single dose of a test substance is usually passed directly into the

stomach of the test animal via a tube . Different groups of animals are
given increasing doses of the test substance to see which dose will kill half
of them. These animals are observed for 14 days and any that have not
died by then is then killed and dissected. The numbers of death or any
signs of illness should be recorded on daily basis. The signs of toxic
substances to these animals will include abdominal pain, cramps,
convulsion, vomiting, diarrhea, paralysis. Breathing problems and
bleeding. Various species of animals can be used for LD50 test and these
mostly includes small animals like rats , mice , guinea pigs , and even
sometimes other big animals like dogs and rabbits can also be used

LD 50 test are often cruel tests done to animals and it often give no
information on treating human poisoning and therefore its unreliable
way of predicting risks to humans because the results obtained are
altered by many factors which include
(a) species used are often different therefore he LD50 for the same
substance are often different from one species than in another.
Sometimes these differences may be very great or smaller even between
closely related species .

(b) The age and the sex of the animal,

 SCIENCE LABORATORY TECHNOLOGY
(c) the social and the physical environment e.g. temperature and
humidity

It should also be remembered that the lethal dose for human beings may
sometimes be higher or lower than that of the test animals and therefore
LD 50 test for test animals cannot be used to predict for human beings.
Even lipstick which is known to be harmless to humans has been shown to
have adverse effect to test animals. The table bellow shows some LD 50
test

(b) inhalation test

These method is applied when testing for those substances which are
actually volatile or gaseous i.e. which may be inhaled and absorbed
through the lungs . These substances are usually sprayed in the air -tight
animal cage with animals placed inside for about 4 hrs and observed for
any toxic effect

(c) derma test

These method is applied to those substances that can enter the body
through skin. These are often done by first shaving off the animal’s fur
and then the test substance is kept in contact with the skin for about 24
hrs. The animal is then observed for 14 days. The LD 50 dose can there
after be calculated.

NB All the test above is only meant to test for acute effects of drugs. To
find out the chronic or long term effects of drugs , the test animals are fed
smaller doses every day , often for 90 days.

Draize test
The Draize test involves applying 0.5mL or 0.5g of a substance to an
animal's eye or skin for four hours; the test subjects are usually albino
rabbits who are conscious and held immobilized in stocks. The animals
are observed for up to 14 days for signs of erythema and edema on the
skin, and redness, swelling, discharge, ulceration, hemorrhaging,
 SCIENCE LABORATORY TECHNOLOGY
cloudiness, or blindness in the eye. The animals are killed after the test.
Despite two decades of research into alternatives to this test, no non-
animal alternatives have yet been successful,
Alternative methods have however been adopted that can now be used to
predict accurately the toxicity of substances to humans. These methods
uses human cell cultures test. The human cell culture have several
advantages in predicting toxicity which include

(a) they are human cells and therefore they minimize species
differences
(b) they can be taken from the tissue that that particular test
substance is most likely to affect e.g. the skin or the liver
(c) they allow researchers to study how a substance causes damage
to the cells
(d) they avoid causing pain and death t he animal

Others
Other tests include the:

 90-day repeat-dose test, administered by mouth, inhalation, or

skin to 30-80 rats, rabbits, or guinea pigs;
 90-day-repeat-dose test by mouth in non-rodents, in which
dogs are used;
 teratogenicity test, which tests for the effects of a substance on
a fetus, and involves at least 80 mice, rats, hamsters, or rabbits;
 chronic toxicity test, which involves at least 160 rats, who are
given daily doses of the substance for most of their lifespan;
 carcinogenicity test, another lifetime study testing for cancer in
400-500 rodents;
 one- and two-generation reproduction toxicity test, which
involves more than 100 rats or mice;
 test for embryonic genetic damage, which involves 10-60 rats,
hamsters, mice and their offspring;
 toxicokinetic study, which involves eight to ten animals to study
the absorption, metabolism, distribution, and excretion of a substance.
 SCIENCE LABORATORY TECHNOLOGY

PHARMACOLOGY
Pharmacology is the study of drug action. More specifically it is the
study of the interactions that occur between a living organism and
exogenous chemicals that alter normal biochemical function. The field
encompasses drug composition and properties, interactions, toxicology,
therapy, and medical applications and antipathogenic capabilities.
Pharmacology is not synonymous with pharmacy,. Pharmacology deals
with how drugs interact within biological systems to affect function. It is
the study of drugs, of the body's reaction to drugs, the sources of drugs,
their nature, and their properties. In contrast, pharmacy is a medical
science concerned with the safe and effective use of medicines.
Pharmacology as a chemical science is practiced by pharmacologists.
Subdisciplines include

 clinical pharmacology - the medical field of medication effects

on humans
 neuro- and psychopharmacology (effects of medication on
behavior and nervous system functioning),
 pharmacogenetics (clinical testing of genetic variation that
gives rise to differing response to drugs)
 pharmacogenomics (application of genomic technologies to
new drug discovery and further characterization of older drugs)
 pharmacoepidemiology (study of effects of drugs in large
numbers of people)
 toxicology study of harmful effects of drugs
 theoretical pharmacology
 posology - how medicines are dosed
 pharmacognosy - deriving medicines from plants
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THE SCOPE OF PHARMACOLOGY
Pharmacology encompasses a number of aspects including ;

(a) when the drug gets into solution and the site of administration
(b) Absorption of the dissolved drug by a variety of mechanisms i.e.
crossing the cell membrane . the absorption is influenced by the
molecular size of the drug , the lipid solubility , ionization and other
physical and chemical properties .
(c) The distribution of the dissolved drug in or between the blood
plasma proteins . these may be reversibly taken into the red blood cells
or reversibly bound to the red blood cells
(d) the metabolism or biotransformation of drugs in the liver or other
tissues
(e) The excretion or elimination of metabolites in the bile through the
intestines where they can be excreted in feces alternatively , the
metabolites may be reabsorbed back through the portal blood back to
the liver and pass back from the liver to the general circulation and be
carried to other organs or tissues(these circulation is sometimes referred
to as enterohepatic circulation ) , the metabolites may also be excreted
through the kidneys alongside urine, similarly some of the metabolites
may again be reabsorbed by the renal tubules from the urine and
passed back to the blood steam to other organs . other excretory organs
are skin which will excretes the metabolites with sweat and also the
lungs which normally important in excretion of aesthetics etc
The scope of pharmacology is rapidly expanding in many fields of
medicine and veterinary especially in fields such as clinical
pharmacology, neuropharmacology , cardiovascular pharmacology , renal
pharmacology , biochemical pharmacology , molecular pharmacology ,
behavioral pharmacology etc these expanding scope provides better
understanding of therapeutic use of drugs their application . i.e. the
right drug should be given to the right patient at the right time with the
right dose . Drugs should also be safe , available ,affordable and
effective .

When describing the pharmacokinetic properties of a chemical,

pharmacologists are often interested in LADME:
 SCIENCE LABORATORY TECHNOLOGY

 Liberation - disintegration (for solid oral forms {breaking down

into smaller particles}), dispersal and dissolution
 Absorption - How is the medication absorbed (through the skin,
the intestine, the oral mucosa)?
 Distribution - How does it spread through the organism?
 Metabolism - Is the medication converted chemically inside the
body, and into which substances. Are these active? Could they be toxic?
 Excretion - How is the medication eliminated (through the bile,
urine, breath, skin)?

Terms used in pharmacology

Drug –any substance or group of substances which affect living tissue at

molecular level

Bioavailability – amount of drug that is available to the target tissue

after the drug have been administered or the amount of the available drug
in tissues that is enough to produce the desired effect

Medical pharmacology – the study of drugs used for diagnosis

,prevention and treatment of diseases

Posology – the study of dosage , deals with he amount of drugs

necessary to produce the desired effect

Usually recommended dose – the amount of drug that will ordinarily

produce the effect for which it was intended . in addition to the usually
recommended dose ,the usually is indicated for many drugs .these
provides a guide in deciding whether the prescriber should be consulted
about the correctectness of the prescribed dose .

The minimum dose- these is the sallest dose that can give the desired
therapeutically effect

Maximum dose –these the largest dose of a dug that can safely be
administered
 SCIENCE LABORATORY TECHNOLOGY
Toxic dose – the amount of drug that would produce harmful effect.

Lethal dose – the amount of drug that will cause death i.e. LD50 to a
half of the test animals

Single dose – the amount of drug to be taken at one same time

Daily dose – the amount of drugs to be taken in 24hrs . these dose is

usually divided into several individual doses

Maintenance dose – the amount of drug taken to maintain or continue

the desired therapeutically effect . some drugs must be taken on daily
basis in order to maintain the therapeutically effect

Loading dose – thee is the first dose of the drug given to the patient so
as to achieve the drug level quickly . drugs that are given only once or
twice a day may take 2-3 days to reach their maximum effect . to
overcome these delay , a loading dose is given that wil bring about the
maximum desired effect quickly . loading doses are often given to very sick
patients

Od –once daily
Bd – twice daily

Tds- thrice daily

Qd –4 times daily

C/o- clinical observation

H/o – history of

Dx – diagnosis

Rx –treatment

Imp-impression
 SCIENCE LABORATORY TECHNOLOGY

CLASSIFICATION OF DRUGS
Different drugs have different actions and most of them are not easily
interrelated . These makes it not easy to classify them . But however
drugs can be classified depending on ;

(i) their action or effect i.e. according to the biological systems

that they affect
(ii) The basis of their chemistry i.e.
(a) Those that are charged or uncharged according to the existing
environmental pH (electrolytes) –usually most molecules are present
both in ionized and unionized state . the degree of ionization (Ka) often
affects how the molecule will dissolve in the lipid bilayers of cells and
hence their diffusibility . some ionize by losing H+ (acidic groups) while
others ionize by adding the OH- ions (basic groups ) the degree of
ionization is expressed as pKa
(b) Those that are incapable of assuming any charge regardless of the
environmental pH (uncharged nonpolar substances) e.g. digoxin do not
have ionizable groups and therefore they are unaffected by the
environmental pH . they are lipid soluble and so diffuse readily across
tissue boundaries
(c) Those that are permanently charged irrespective of the
environmental pH (charged polar substances ). They carry groups whose
ionization is so strong that they remain charged at all values of the body
pH. They have limited capacity to cross the cell membrane
(iii) according to their chemical structure e.g. those having sulfur in
their chemical structure are collectively known as sulphonamides
(iv) according to the diseases they treat e.g. drugs for malaria
,epilepsy , tuberculosis are grouped by adding the prefix anti – e.g.
antimalaria drugs
(v) according to the organ system on which they have their
pharmacological effects e.g. those drugs acting on the cardiovascular
system , nervous system et c
(vi) according to their generation e.g. some drugs are classified as 1st
, 2 , 3 or 4th generation
nd rd
 SCIENCE LABORATORY TECHNOLOGY
SOURCES OF DRUGS
Most drugs are organic compounds derived from varous sources e.g.

(a) From naturally occurring compounds - they are extracted

from plants e.g. morphine , quinine , digitalis etc and animals e.g.
heparin , insulin , thyroxine etc . they are the oldest type of drugs

(b) From modified natural drugs – these are drugs produced as a

result of modification of naturally occuring drugs .such drugs are made to
become better and effective e.g. semisynthetic insulin and penicilin

(c ) from mineral source – these are obtained from minerals salts e.g.
iron , ,iodine , sulphur etc

(d)from microbial source –these are derived from microorganisms

e.g. antibiotics

(e) from synthetic sources – thee are drugs produced by purely

synthetic . these drugs have many advantages than natural drugs , they
are cheaper and easily available

(f) from genetic engineering - these are the newest and the most
recent drugs

NATURE OF DRUGS

Most of the drugs used nowadays are synthetic and are made from a
mixture of variety of chemicals . plants contain the active principle ,These
is the pharmacologically active substance or ingredient obtained from
plant material e.g. alkaloids , glycosides ,oils , resin and tannins . the
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active ingredients of crude drugs are usually isolated from such plants .
the purpose of isolation are ;

(a) In order to identify the active ingredients

(b) Analyze the pharmacokinetics and pharmacodynamics of the
individual ingredient
(c) To ensure that a precise dosage is used
(d) To seek alternatives on the possibilities of chemical synthesis and
derivatives of the original constituents with improved therapeutic
usefulness
(a) alkaloids
alkaloids are organic nitrogenous substances of plant origin that have
marked pharmacological activities e.g. morphine , codeine ( analgesic) ,
ematine(antiamoebic) , atropine anticholinergic) , quinine (antimalarial ),
quinedine ( cardiac depressant ), vincristine (anticancer ) caffein (CNS
depressant) , pilocarpine(antiglaucoma ) , papaverine (smooth muscle
reluctant ) etc .they have significant pharmacological activities .
Structurally , they posses carbon , hydrogen and nitrogen and oxygen .
They react with acids e.g. hydrochloric , sulfuric , citric and tartaric
acids to form crystalline salts . Free alkaloids are insoluble in water but
their salts are soluble

(b) glycosides
These are compounds which hydrolyze to give rise to one or more sugars
(glycones ) together with other non-sugar compounds (aglycone or
genin). Most glycosides are colourless crystalline compounds but others
like anthracine glycosides are red or orange colored . they are soluble
in water and alcohol but insoluble in other organic solvents e.g. ether
and chloroform . Glycosides are classified on the basis of their sugars e.g.
glucosides , fructosides etc or on the basis of their pharmacological
activities e.g. cardiac glycosides ( e.g. digitalis and strophantus ) exhibit
their action on the heart

(c) Gums
Gums are colloidal exudates of plants which swell or form mucilage in
water . they are used as emulsifying or suspending agents
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(d) Tannin

Tannins or tannic acids are mixtures of esters of garlic acids with

glucose . tannic acid is a stringent and is used as haemostatic

(d) fats oils and waxes

most fats and oils are esters of glycerol with higher long chains fatty
acids . They are either obtained from plants (oils ) or animals (fats ). Fatty
acids may be saturated or unsaturated . They are insoluble in water but
soluble in most organic solvents . Fatty acids are used to treat skin lesions
, wounds , burns ,eczema ,as laxative , liniment , preparation of
ointments , and as suppositories in manufacture of soaps.

Waxes are esters of higher straight chain fatty acids . They may be of
plant origin e.g. carnauba wax or animal origin e.g. bees wax . wax are
difficult to saponify

(e) Resins
Resins are amorphous, brittle , translucent hard solids . They soften and
melts on heating. They are insoluble in water but soluble in organic
solvents e.g. alcohol , ether and chloroform .resin containing drugs
posses purgative and counter irritant actin , resins are used externally as
antiseptics , ointments and plasters . They are employed in the
preparation of emulsions

Majority of drugs lie in the range of MW 100-1000 . Drugs in these range

are large enough to allow selectivity of action and small enough to allow
adequate movement within the various compartments in the body . the
overall shape of the drug molecule is important for the fix of the drug or
to eable the dug to on the receptor molecule.

Drugs may cause any of the following effects

- muscle contraction
- secretions from glands
- change of metabolism in tissues
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- cause or prevent growth or division of cells
- act on the CNS to cause alteration of behavior or to relieve
pain .
Drugs are selective in their action i.e. they act on or affect some tissues
and not others .

some drugs have some side effects , these are undesirable effects of
drugs e.g. morphine is a very powerful pain reliever but is addictive and
causes constipation

NOMENCLATURE OF DRUGS
Drugs can have a variety of names i.e.

(a) chemical names

These is the full chemical name according to by IUPAC names . Such
names are too lengthy cumbersome, complicated and very technical . they
require thorough knowledge of chemistry

(b) approved or official names

They are also known as generic names . it allows easy identification
different products with the same ingredient .Generic names are
internationally accepted common names of drugs but unfortunately ,
some compounds have different approved names in different countries
e.g. adrenaline in the UK is known as epinephrine in US

(c) trade names

These are names that are unique to the particular manufacturer. It’s a
name given to a formulation (medicine) by its manufacturing or
marketing company . it helps in promoting and selling of the drug

DOSAGE FORMS
A dosage form of drug is how the drug or product have been designed
for use by the patient .drugs substances are administered as part of
formulation in combination with one or more none medical agent known
as pharmaceutical ingredient.pharmaceutical ingredient help in
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solubilizing , suspending , thickeninig,diluting ,emulsifying ,stabilizing
,preserving and giving color to the medicinal agent .

Dosage forms are needed for the following reasons

(i) For providing safe and convenient delivery of accurate dosage

(ii) For protection of drugs from harmfull effects of the
environment e.g. oxygen and moisture

(iii) For protection of drugs from the destructive influence of

gastric HCL after oral use

(i) to conceal or disguise the abnoxious taste and smell of a drug

(ii) to provide drugs for topical application e.g. ointment
(iii) to provide drugs for insertion into the body`s orifice
(iv) to provide liquid dosage forms as solutions
(v) to provide preparation of parenteral administration
(vi) to provide various controlled released tablets , capsules and
suspensions for extended drug actions
Dosage forms affects drugs absorption and these is normally result of
the formulation itself and the routes of administration . one drug may be
formulated into multiple dosage forms which results in different
absorption rates and times of onset ,peak and duration of effects

The various dosage forms can be classified into; solid dosage form e.g.
tablets ,pellets , capsules , pills etc , semi- solid dosage they are mainly
meant for topical use e.g. ointments , linements , jellys , lortions etc
form and liquid dosage form meant for injections and syrups

BRANCHES OF PHARMACOLOGY

There are two main branches of pharmacology i.e.

 Pharmacokinetics – these branch is concerned with the

adsorption , distribution ,metabolism (biotransformation ) and excretion
of drugs (ADME). i.e. it deals with what the body does to drugs.
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 Pharmacodynamics - These branch deals with what the drug

does to the body , it studies the biochemical and the physiological effect
of drugs and their mode of action on the body . its also concerned with
the dose effect relationship , factors modifying the drug effects and
dosage and drug toxicity

PHARMACOKINETICS
Pharmacokinetics is a branch of pharmacology dedicated to the
determination of the fate of substances administered externally to a living
organism. Pharmacokinetics is often studied in conjunction with
pharmacodynamics. Pharmacodynamics explores what a drug does to the
body, whereas pharmacokinetics explores what the body does to the drug.
Pharmacokinetics includes the study of the mechanisms of absorption and
distribution of an administered drug, the rate at which a drug action
begins and the duration of the effect, the chemical changes of the
substance in the body (e.g. by enzymes) and the effects and routes of
excretion of the metabolites of the drug.

ADME
Pharmacokinetics is divided into several areas which includes the extent
and rate of Absorption, Distribution, Metabolism and Excretion. This is
commonly referred to as the ADME scheme. However recent
understanding about the drug-body interactions brought about the
inclusion of new term Liberation. Now Pharmacokinetics can be better
described as LADME.

 Liberation is the process of release of drug from the

formulation.
 Absorption is the process of a substance entering the body.
 Distribution is the dispersion or dissemination of substances
throughout the fluids and tissues of the body.
 Metabolism is the irreversible transformation of parent
compounds into daughter metabolites.
 Excretion is the elimination of the substances from the body. In
rare cases, some drugs irreversibly accumulate in a tissue in the body.
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Pharmacokinetics describes how the body affects a specific drug after
administration. Pharmacokinetic properties of drugs may be affected by
elements such as the site of administration and the concentration in which
the drug is administered. These may affect the absorption rate.

A .UPTAKE OR ABSORPTION

One of the most important functions of biomembrane is to be

involved in the exchange of material in the internal and external
membrane, which is very important to the maintenance of normal
activities of life. Drugs are delivered from the parts of the body into the
blood circulation, are then distributed in a variety of organs, tissues,
and go through a series of biomembrane after biotransformation to
discharge from the body. These processes are finished via
transmembrane transport. Transshipment of drugs are in the
following three ways:
1 ． Passive transference: (Also known as downstream
trans-shipment).
The drug infiltrates from the high concentration side opposite to
another. without energy transfer, the transfer speed is directly
proportional to the drug concentration difference between both sides
of the membrane .The greater the concentration gradient is , the easier
the drug diffuses. The transit stops when the drug concentration on
both sides of the membrane reaches equilibrium, generally including
simple diffusion and filtration.
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● Simple diffusion: (Also known as passive diffusion ） .

Most drugs transit through this way, characterized by diffusion from
the higher concentration to the lower concentration, not be affected by
saturation and competitive inhibition. The diffusion rate depends
largely on the concentration gradient on both sides of the membrane
and liposolubility of drugs. The higher the concentration is, the greater
the liposolubility is, and the faster it diffuses. Simple diffusion can be
affected by the spread of drug dissociation degrees, most of the drugs
are weak acid or base, and dissociation and non-dissociation mixed-
exist in solution, non-dissociation is more liposoluble and can through
the biomembrane easily. The number of parts of dissociation and non-
dissociation depends on the size of PKa and the pH value of the
solvent. In the acidic environment, acidic drugs have less dissociation,
high-fat solubility, can be easily absorbed; in the acid environment,
alkaline drugs have more dissociation, low-fat solubility, can not easily
be absorbed. Strong acid or alkali and polar quaternary ammonium
salts are difficult to penetrate the biomembrane.
● Filtration: It’s the common way to transfer through the
channel of water filtering for many small molecules (molecular weight
150 to 200), water-soluble, polar and non-polar material. A variety of
water channels of biological membrane have different diameters, such
as capillary endothelial cell membrane has the large hole, about 4 ~
8nm, and intestinal epithelial cell membrane and the majority
membrane was only 0.4nm. Drug transiting through the water
channel, is very important for the kidney excretion, elimination from
the cerebrospinal fluid and through the liver sinus membrane.
2 ． Facilitated diffusion: (Also known as assistant
diffusion)
Similar with the passive diffusion, drugs spread from the high
concentration to the low concentration, the difference is that it needs a
membrane protein to be involved. Facilitated diffusion can not go
against the concentration gradient, no energy consumption, which is a
passive transfer process through its capacity that has been saturated,
also affected by the metabolic inhibitor. Glucose entering red blood
cells and vitamin B12 being easily absorbed from the intestine are the
way of facilitated diffusion.
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3 ． Active transport (Also known as the adverse current

transfer)
The active transport is a vector-mediated transfer, reversing the
concentration (or potential) gradient. Membranes provide the carrier
for the transshipment, there are enzymes involved in and with energy
consumption, and metabolic inhibitors can block this process. Carriers
have specific affinity to drugs and can combine with them reversally
and generate compounds then permeate from one side of the
membrane to the other side, then release the drug and return in situ,
and continue to transfer to a new transport.
Competitive inhibition occurs when the same carriers transfer two
similar compounds. Strong acid, alkali or most metabolites of the
drugs which are rapidly transmitted to bile and urine possess active
transport mechanism; transshipment of most inorganic ions e.g. Na +,
K+, Cl- and the excretion of penicillin, cephalosporins, as well as
probenecid excreted from kidney belongs to active transport process.
4．Pinocytosis / phagocytosis
Membrane has a certain mobility and plasticity, and can
transform initiatively to intake or release some substances. This
process is called pinocytosis or exocytosis, The intake of solid particles
is known as phagocytosis. Macromolecule drugs (as molecular weight
more than 900) enter cells or permeate into the organization barrier
generally via pinocytic / phagocytosis, such as protein, fat-soluble
vitamin, antigen, tetanus toxin, clostridium botulinum toxin.

5．Ion pair transport

High hydrophilic drugs in the gastrointestinal tract combine with
some endogenous compounds. It can form ion-neutral compound by
binding with organic anion mucoprotein, both lipophilic and
hydrophilic- through the lipid membrane by passive diffusion, which is
called approach of ion transfer

BIOAVAILABILITY AND THERAPEUTIC EFFECTS OF DRUGS

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After a drug is administered , it is absorbed systematically and when the
minimum effective concentration (MEC) is reached at the receptor
site , a pharmacological response is initiated . The time from drug
administration to the MEC is known as the onset time . The
pharmacological activity will be observed as long as the drug
concentration remains above the MEC . As the drug concentration
increase , other receptors may combine with the drug to exert an adverse
response . the concentration of the drug at these level is called the
minimum toxic concentration (MTC ). The drug concentration range
between MEC and MTC is called the therapeutic window

Bioavailability is defined as the fraction of unchanged drug reaching

the systemic circulation following administration of the drug by any
route . it is a measurement of the rate and the extend at which the
absorption occurs . drug products are pharmaceutical equivalent if they
contain same active ingredient and are similar in concentration , dosage
form and routes of administration . they are considered to be
bioequivalent when the rates and extent of bioavailability of active
ingredient in two products are not significantly different

Bioavailabity of drugs vary depending with the route of administration i.e.

the intra-venous injection (100%) > intra-muscular injection > sub-
cuteneous injection > oral administration

Bioavailabity determines the drug blood level . its mostly influenced by

the formulation of that particular drug e.g. tablets or capsules contain
large number of excepient which may affect the disintegration and the
dissolution of the drug hence slowing the amount of the drug reaching
thee blood
Factors affecting absorption and bioavailability of drugs

absorption and bioavailability is influenced by two factors i.e. the

pharmaceutical/physical factors and the pharmacological factors .
pharmaceutical factors include
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(a) the partical size - drugs dissolve more rapidly when its surface
area is increased by decreasing the particle size
(b) salt form - salts of weakly acidic drugs are highly water soluble
and hence enhanced bioavailability
(c) crystal form – amorphous form have faster dissolution rate
compared to the crystalline forms
(d) water of hydration – many drugs combine with water .(hydrated
salts )
(e) nature of recipients or adjuvant contained – excipients
and adjuvant are certain inert substances such as starch lactose etc
which are added to drug as a filling material or as binding agent,wettng
agents etc
 SCIENCE LABORATORY TECHNOLOGY

(c)the concentration

drugs in high concentration are more rapidly absorbed than drugs in

low concentration

(f) Solubility in lipid layer – drug molecules must be in a position

to dissolve through the lipid bilayer of the cell membrane . the cell
membrane also have some tiny pores through which the molecules pass
through . for a drug to be absorbed , it must be in a solution form i.e. it
must be in its unionized lipid soluble form
(g) form of drug – drugs that are given in aqueous solution are more
rapidly absorbed than those given in oily solution or solid form because
they mix radily wih the aqueous absorptive surface

pharmacological factors that influence the absorption and the

bioavailability of drugs include

(a) The absorbing surface area

the surface area over which the drug is exposed is very important in
determining the rate of its absorbtion . drugs are often absorbed fast from
large surface areas such as the pulmonary alveoli and the epithelial
intestinal mucosa (villi)

(b) Vascularity and the blood flow

good circulation of blood at the site of application is very important

(c)presence of food in the stomach – food delays gastric empting

and therefore affects absorption of some drugs . its also important to
note that some food promote gastric emptying and therefore influence
absorption

(a) gastrointestinal diseases – gastrointestinal diseases e.g.

gastroenteritis cause decrease in absorption of drugs
 SCIENCE LABORATORY TECHNOLOGY
(b) fast –pass effect – fast pass effect means the drug degradation
occurring in the liver before the drug enters the systemic circulation
resulting in decreased bioavaiability
(c) drug – drug interaction – drugs can interact with other drugs
taken together . these can also affect bioavailability e.g. liquid paraffin
decreases the bioavailability of vitamin A
(d) pharmacogenetical factors - variation in bioavailability also
exist in human beings due to many genetic reasons .
(e) route of administration
Drugs can be applied locally (topically ) orally and by injection . topical
drug are applied on the skin for local action .the following are some of
the routes . The choice of the best route of administration shall depend on

(a) the chemical nature of the drug e.g. insulin is a protein and
can be destroyed by the stomach enzymes therefore should not be
administered orally
(b) the urgency of the case basing on the condition of the
patient i.e. very sick patient will require the drug to be administered via
injection for faster distribution of drugs
(c) drug interaction
(d) degree of ionization e.g. tubacurine is a muscle relaxant
when administered parentally but will not be absorbed when administered
orally due to its large charge and hence no pharmacological effects
Drugs usually enter the body at the site that is often remote or far away
from the target tissue and are carried by the circulation to interior site of
action . the rate and the efficiency of absorption differs depending on the
route of administration

(I) Topical or local routes – these is where the drugs are applied
directly to the place where it must act . it involves use of ointment ,
creams , lotions , powders , sprays on skin , eyes , throats , mouth,
rectum , vaginal tract , urethra etc

drugs applied on mucous membranes can often be absorbed rapidly

enough to produce actions in the rest of the body . drug absorption
through contact is proportional to the lipid solubilty of the drug ( the
epidemis behaves as a lipid barrier ) absorption across the lipid barrier
 SCIENCE LABORATORY TECHNOLOGY
can be enhanced by suspending the drug in oily vihecle . topical
application is the least effective and the least economical route of
administration i.e. huge quantities of drug are required to get just a small
quantity of it to be absorbed . though the skin is an effective barrier to
almost al substances , it is not a perfect barrier to many substances . the
skin represent the route by which poisoning I man and other animals
occur following exposure to insecticides and other chemicals
(ii) gastrointestinal tract – these can be through ;

(a) the oral mucosa - the mouth can serve as a site for absorption
although many drugs are not capable of penetratig the oral mucosa in
significant amount and others are too irritating to be held in the mouth .
drugs absorbed in the mouth are not exposed to gastric and intestinal
digestive enzymes which could alter their effects by eliminating and
biotransforming the drug
(b) the stomach and the intestine due to its high blood suply and
epithelial surface area . The stomach and the intestines are conducive for
absorption of drugs but however , some drugs can be altered into other
products by the many enzymes and the high pH in the stomach . also the
presence of food and the rate of stomach emptying and the solubility of
the drug in the intestinal fluid may affect the effectiveness of these route
(c) rectal mucosa (suppositories )
rectal administration n is advantages in unconscious patients who are
unable to retain materials given by mouth . also for drugs with
objectionable taste , odor and those that are destroyed by digestive
enzymes rectal route protect the drug from digestive enzymes and the
action of liver which nave a lot of oxidative enzymes which can
metabolize these drugs .these is because blood from the lower part of the
rectum bypasses the liver on its way to the heart . The absorption is
however irregular and incomplete. The drug may also irritate the rectal
mucosa .

Factors that influence the rate of absorption through the GIT

- the permeability of the drug across the mucus membrane

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- the pH of the GIT- these affects the ionization of the drug
(non ionized drugs are more permeable )
- the surface area of the absorption surface eg presence of
villi in the small intestines increases the surface area for absorption of
drugs
- effects of GIT secretions e.g. presence or absence of
proteolitic enzymes which digest the polypeptide drugs e.g. insulin ,
oxytoxin and vasopressin which break into amino acids
- bacterial metabolism of drugs in the GIT – intestinal
commensals i.e. bacteria and other saprophites produce enzymes that
catalyzes a number of reactions including drugs and their drug
metabolites (enterohepatic shunt)

(f) pulmonary endothelium

gases , vapour or aerosols can be inhaled and absorbed through the
alveoli surface of the bronchial mucosa giving rapid access to the
circulation

(d) injection (parenterial administration)

there are several methods which fall under these category i.e.

(g) subcuteneous route

in these method the dug is placed just bellow the epidermis of the skin
i.e. in the subcuteneous layer . these route can be used in administering
irritating drugs . large volume can be painfull due to tissue distortion .
the route also provide for even and slow absorption to obtain a
sustained drug effect

(ii) intra muscular injection

These method involve the injection of an aqueous solution of drug deep
into the muscle . such drugs are rapidly absorbed but irritating
substances or large volumes may be painful

(iii) intra peritoneal

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the peritoneum provides a large absorptive surface with a rapid entry of
drugs into the circulatory system . Intraperitoneal injection is a common
laboratory procedure but not often used clinically because of the dangers
of intestinal injury

(iv) intra-venous
These is where the drug is placed directly into the circulatory system
through the venous blood vessel . There are three type of intravenous
injection

(a) Rapid intra-venous injection – in these method ,blood

concentration is attained immediately . But however these method have
the following limitations ;
 removal of the drug from the system is difficult
 the possibility of transmission of diseases due to contamination is very
high
 Tran version of the drug into the adjacent or sorounding tissues is very
likely
(b) slow intravenous injection (infussion)
In these method , the drug is dripped or slowly made to gain into the
tissues . the level of the drug in the blood increases slowly . these method
is usefull in maintaining the blood level of a drug over a prolonged period
of time .nevertheless , these method also have some disadvantages which
include

 vascular injury
 possibility of transmision of infection
 it requires an expert to administer

(v) intra –arterial injection

these method is occasionaly used to direct high drug concentration into a
specifictarget tissue or organ hence minimizing administration in
general injection . thhese method limits the spead of the drug toother
neighbouring tissues

(vi) intra-thecal injection

 SCIENCE LABORATORY TECHNOLOGY
these method is employed to direct drugs into the CNS by passing the
blood brain barrier and the blood -–cerebrospinal fluid barrier .
antibiotics may be injected into the pleural space or into an abscess
cavity for the treatment of a local pocket of infection surrounded by
fibrous tissues that prevent the antibiotics from getting there via the
blood stream .

B . DISTRIBUTION OF DRUGS

Drug distribution is the process that drugs go through the blood

circulation to reach certain tissues and organs after being absorbed
into blood. Drug concentration in organizations is inequable, often in
dynamic equilibrium. The main factors impacting on drug distribution
are as follows:
Bonding force between drug and plasma protein. Drug possess
two forms in plasma, i.e. binding and free form, often both are in a
dynamic equilibrium. The combination of drugs is not easy to
penetrate the vessel wall, no pharmacological activities, and is not easy
excreted through the kidneys, so that action time is extended. When
the free drugs are distributed, metabolized or excreted and the blood
concentration reduces, the combination of drugs can release free
drugs, thereby delaying the speed of drug’s disappearance from the
plasma, so that prolong the half-life. As a result, the binding between
drug and plasma protein actually is a storage function. Drug and
plasma protein binding rate mainly depends on the chemical structure
of drugs, but very different, such as Sulfametoxydiazine being sulfa, its
protein binding rate is 81% in the dog plasma, while the SD-only 17%.
In addition, the drug and plasma protein binding capacity is limited,
and excessive doses of drugs over saturation will make a significant
increase in drug-free, in some cases even causing poisoning.
Blood flow of local organizations. Liver, kidney, heart, brain
and other organs have large blood flow, it is easy for drug to quickly
reach a higher level through the blood vessel wall.
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Physical and chemical properties of the drug. Such as the

liposolubility, pKa and the molecular weight and so on. Liquid soluble
or water-soluble small molecule drugs permeate membrane easily;
non- lipid -soluble molecules or the dissociation of drug permeate the
membrane hard , thus affect its distribution.
Affinity to the organization. Some drugs have specific affinity to
some organizations, so that in which more drugs can be distributed.
This selective distribution of certain drugs has important clinical
significance, such as selective distribution of iodine in the thyroid, a
concentration higher than that of other organizations, about 10,000
times, it can be used for the treatment of hyperthyroidism; mercury,
arsenic, antimony, and other heavy metals mostly deposit in the liver
and kidneys, offal forums, and the poisoning can cause damage to
these organizations. The organs distributions selected by drugs may
not be the organs act on, such as digitalis selective effect on the heart,
for the strong performance of the heart, but it is mainly distributed in
the liver and skeletal muscle.
Permeability across tissue barriers . Many macromolecular and
high polarity drugs can not penetrate the blood-brain barrier into the
brain, the drug binding with plasma protein can not enter too. Take
the treatment of meningitis as example, SD-to sulfa drugs is the best
choice, because it's plasma protein binding capacity is low. New-born
animals suffering from blood-brain barrier dysplasia or meningitis, the
permeability of blood-brain barrier increase in the number of drugs
into the cerebrospinal fluid. Such as penicillin under normal
circumstances, even if the dose is great it is very difficult to enter the
cerebrospinal fluid, but when the brain is of meningitis, it is easier for
drug to enter the cerebrospinal fluid. Lipid soluble drugs such as the
general anesthetic can enter the fetal blood from maternal blood, in
particular, so attention should be paid to certain drugs through the
placental barrier caused by fetal malformation, poisoning or danger,
the pregnancy animals’ medicine usage should be treated with caution.
C . METABOLISM OR BIOTRANSFORMATION OF DRUGS .

Biotransformation of drugs is the chemical alteration of the drug in the

body i.e. to change the non polar (lipid soluble) compounds to
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polar(water soluble ) compounds so that the metabolites are not
reabsorbed into the renal tubules and are excreted

Metabolism is a wider term which reffer to the total fate of drugs in the
body including absorption , distribution , biotransformation and
excretion leading to lose of their parmacological activities .
metabolismussually produces products which are more water soluble and
easily excreted . the liver is the most important site of biotransformation
of drugs , other sites involved are ; the kidney , plasma , intestines , skin ,
lungs and spleen . biotransformation is mediated through the hepatic
microsomal systems which contain several enzyme systems called
microsomes which are involved in metabolism of many drugs .
Microsomal enzymes facilitates catalyzes most drugs by oxidation
,reduction ,hydrolysis and conjugation reaction .some drugs are however
excreted largely unchanged but majority are metabolised via extremely
complex routes producing a lot of metabolites .

Biotrnsformation may result in inactivation , activation or change in

actiity of the drug .most of the drugs are in active form while
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the metabolites produced are inactive e.g. choloquine is an active drug

but its metabolite i.e. hydrochloroquine is an inactive product . other
drugs are however usually inactive (prodrugs ) but their metabolites
become active after undergoing activation by enzymes to produce new
compouds which are active .

Biotransformation of drugs involves phase 1 and phase II reaction

Phase I reaction (non synthetic )

These phase often serve to introduce relatively reactive groups such as

hydroxyl groups into the drug molecule . these functional group then serve
as the point of attack for conjugation systems which attaches larger
substituents to it .

phase I reaction consist of oxidation , reduction or hydrolysis . the

product formed becomes so reactive and sometimes more toxic than he
parent drug . phase I reactions are catalyzed by mixed function oxidases
(MFO) such as cytochrome P 450 and cytochrome 450 found in the
smooth endoplasmic reticulm of the liver cells . not all drugs are oxidised
by MFO instead some drugs .e.g. alcohols are oxidized by alcohol
dehydrogenases .

Reduction reaction of phase I are much less common than oxidative

reactions e.g. many steroids are administered as ketones e.g. cortisine
and predinosine , and must be first reduced to their coresponding
hydroxyl compounds . hydrolytic reactions occur in many tissues . esters
and amide bonds are also susceptibe to hydrolysis However .not all drugs
go through phase I reactions , instead some go directly to phase 11

Phase II reactions
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If the drug molecule have a suitable handle , i.e. if it is susceptible to
conjugation with a substituent group , then the conjugate is almost
always pharmacologically inert and have less lipid solubility than its
precursor and is therefore easily excreted in the urine or in bile . these
handle ( a chemical group that can accept another chemical group ) e.g.
hdroxyl (OH) ,sulphate ( SO4), methyl (CH3) groups etc makes it to bind
with another group

The choice of a conjugate depends on the nature of the group at which

conjugation takes place , the site where the reaction takes place , the
availabity of the conjugate, species e.g. glutathione is used as a
conjugate in man , whereas ornithin is used in birds , sulphates in dogs
and pigs . conjugation reactions also take place with the aid of enzymes
found in the smooth endoplasmic reticulum of the liver , kidney etc .

FACTORS INFLUENCING BIOTRANSFORMATION

(a ) frequency of exposure to the drug

(a) the chemical properties of he drug – some drugs are enzyme

inducers and some are enzyme inhibitors
(b) dosage - excess or toxic dosage can reduce the amount of enzymes
required for detoxification
(c) route of administration . metabolism is affected by first -pass effect
(d) age the ability to metabolize drugs is deficient in infants compared
to adults
(e) physiological factors – cases of liver or kidney dysfunction limits
the ability to metabolize and excrete drugs
(f) genetical factors
(g) enviromental factors
(h) sex

First –pass effects

some drugs are removed from the portal (liver ) circulation very
efficiently by the liver and metabolised so that the amount reaching the
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systemic circulation is considerably less than the amount absorbed into
the portal vein . these is known as the first- pass effect and is significant
for several clinically important drugs . such drugs may be given
sublingually so as to bypass the portal circulation

EXCRETION OF DRUGS

Excretion is the passage of systematically absorbed drugs or its

metabolites and their final elimination from the body . drugs can be
eliminated from the body by many routes e.g. through ; urine (kidney ),
expired air (lungs) , sweat (skin ) , saliva ,,feaces, milk , skin, hair , nails
etc .

the kidney is the most important organ for excretion of drugs via urine .
knowing the routes of excretion of drugs is important because it helps in
determining the dose and the frequency of drug administration .
clearance is a term used to refer to the measure of the drug elimination .
it is defined as the volume of the plasma cleared of the drug in unit
time . it is expressed

ml/ minute .

Majority of drug excretion follows the first order kinetic whereby the
rate of elimination is directly proportional to the drug concentration . i.e.
a constant fraction of drug is eliminated in a unit time . The rate of
elimination decreases as the concentration of drug available decreases
until a steady state – level is reached when intake equals elimination .

some drugs excretion e.g. alcohol , phenytoin etc however undergo zero
order kinetics whereby a fixed quantity is eliminated per unit. I.e. the
rate of elimination is constant and independent of the mass or
concentration present in circulation

some drugs excretion e.g. asprin , digoxin , warfarin etc however still
undergo mixed order kinetic , these is a dose dependent kinetic whereby
 SCIENCE LABORATORY TECHNOLOGY
smaller doses are handled by first – order kinetics but as the plasma
concentration increases the rate of drug elimination becomes zero order

(a) Renal excretion of drugs and metabolites

Many drugs are excreted through the renal system i.e. through urine .

there are three basic processes that bring about renal excretion i.e.

(a) glumerula filtration

(a) active tubular secretion or absorption

(c) passive diffusion across the tubular epithelium

glumerula filtration

the glumerulli capillaries filters organic substances that are free in

plasma or not bound to plasma into tubules . upto 20% of drugs from

the blood reaching the kidney is removed by glumeruli filtration .

Active tubular secretion

in the proximal renal tubules certain organic anions and cations are
added to the glumeruli filtrate by active tubular secretion . Many organic
acids e.g. penicillin and metabolites are transported by a carrier system
that secretes nuturally occurring substances e.g. uric acid . These carrier
systems are relatively non selective . Both organic ions of similar charges
compete for transport . an example of a drug affecting tubular secretion
is probenicid which was developed to prevent rapid excretion of
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penicilin . Penicillin is secreted in the proximal tubules by acid secreting
systems. Probenicid is also an acid and act competitively by inhibiting
the secretion of penicillin .

Passive diffusion across the tubular epithelium

in the proximal and distal tubules , the non ionized forms of weak acids
and bases undergo net reabsorption . since the tubule cells are less
permeable to ionized electrolytes , passive diffusion is pH dependent i.e .
Reabsorption of drugs can be affected by varying the pH . thus when he
tubular urine is made more alkaline , weak acids shall be excreted more
rapidly in urine

(b)B illiary and feacal excretion of drugs and metabolites

many metabolite of drugs formed in the liver are excreted in the

intestinal tract from the bile . These metabolites may be excreted in the
feaces or reabsorbed into the blood and finally excreted in the urine .

both organic cations and anions are actively transported into the bile
by carrier systems . These carrier systems are non selective and ions of
light charges may compete for transport .

antibiotics such as penicillin and tetracycline are eliminated in active

form in the bile and are used to treat billiary tract infections

(b) excretion by other routes

excretion of drugs into sweat , milk , saliva and tears is quantitatively
unimportant but nevertheless they are considered as routes of drug
elimination . elimination by these route is by diffusion of unionized ,
lipid soluble forms of drugs through the epithelial cells of the glands
and it is pH dependent . milk is more acidic than the plasma and
therefore the basic compounds i.e. drugs may be more slightly
concentrated in milk .
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excretion of drugs through hair and skin is also quantitatively
insignificant but it can still be considered very important and infact its
most used in forensic detection of toxic metals e.g. mercury ,arsenic etc

PHARMACODYNAMICS
Pharmacodynamics is the study of the biochemical and physiological
effects of drugs on the body or on microorganisms or parasites within or
on the body and the mechanisms of drug action and the relationship
between drug concentration and effect.
Pharmacodynamics is often summarized as the study of what a drug
does to the body, whereas pharmacokinetics is the study of what the body
does to a drug.

Effects on the body

The majority of drugs either (a) mimic or inhibit normal
physioloical/biochemical processes or inhibit pathological processes in
animals or (b) inhibit vital processes of endo- or ectoparasites and
microbial organisms.
There are 4 main drug actions:
 depressing
 stimulating
 destroying cells
 replacing substances

Desired activity
The desired activity of a drug is mainly due to one of the following:
 Cellular membrane disruption
 Chemical reaction
 Interaction with enzyme proteins
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 Interaction with structural proteins

 Interaction with carrier proteins
 Interaction with ion channels
 Ligand binding to receptors:
o Hormone receptors
o Neuromodulator receptors
o Neurotransmitter receptors

Undesirable effects
Undesirable effects of a drug include:
 Increased probability of cell mutation (carcinogenic activity)
 A multitude of simultaneous assorted actions which may be
deleterious
 Interaction (additive, multiplicative, or metabolic)
 Induced physiological damage, or abnormal chronic conditions

Therapeutic window/therapeutic index

The therapeutic window is the amount of a medication between the

amount that gives an effect (effective dose) and the amount that gives
more adverse effects than desired effects. This describes the ratio of
desired effect to toxic effect Medication is said to have a narrow or wide
therapeutic index or therapeutic window

. A compound with a narrow therapeutic index (close to one) exerts its

desired effect at a dose close to its toxic dose. A compound with a wide
therapeutic index (greater than five) exerts its desired effect at a dose
substantially below its toxic dose. Those with a narrow margin are more
difficult to dose and administer .medication with a small pharmaceutical
window must be administered with care and control, e.g. by frequently
measuring blood concentration of the drug, since it easily loses effects or
gives adverse effects.
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Fundamental appearance of drug action

The drug action is the initial response between the drug
micromolecules and the cellular macromolecule of the body, the
pharmacological effect is the consequence of the drug action,
displaying changes of the physiological and the biochemical functions
of the body, mainly showing the periaqueductal gray stimulation and
the inhibitory action.
The periaqueductal gray stimulation of drugs is defined that the
physiological and biochemical functions of organs and tissues can be
enhanced in the drug action. For example ， ephedrine and caffeine
which are performance drugs can enhance the excitement of the
central nervous system and body function activities, making the
animal excited. The inhibitory action of drugs is an effect which can
weaken the physiological and biochemical functions of organs and
tissues. For example ， chloral hydrate and barbiturates which are
suppressants weaken the functional activity of the central nervous
system. On the whole, the periaqueductal gray stimulation and the
inhibitory action of the drug do not exist alone; they may exist in
different tissues and organs at the same time. For example, the
caffeine expresses the direct periaqueductal gray stimulation on the
heart, enhancing the myocardial contractile force. by contrast, it shows
the effect of the expansion and looseness in the blood vessel, showing
the inhibitory action. Ephedrin can enhance the myocardial contractile
force, shrink the blood vessel, induce hypertension, showing the
periaqueductal gray stimulation, by contrast, it makes the bronchic
smooth muscle relax，showing the inhibitory action.
Drugs can treat diseases just because they exert the
periaqueductal gray stimulation and inhibitory action, resulting in
adjustment and recovery of the balance which is destructed by the
pathological factors.
Besides the functional drugs showing the inhibitory action and
periaqueductal gray stimulation, some drugs are mainly used in the
pathogen, killing or driving out the invaded micro-organism or
parasite, showing the pharmacologic action by protecting the
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physiologic and biochemical function of the body or recovering the

balance
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Ways of the drug action

● Direct action and indirect action In view of the order of the
drug action, the direct action is defined as the primary action
generated when the drug directly contacts tissues and organs. The
indirect action is defined as the secondary action caused by the direct
action of drugs. for example, digitalis effects the heart directly,
enhancing the myocardial contractile force, decreasing the heart rate,
improving the cardiac function and blood circulation, it shows the
direct action; the direct action results in the increasing of renal the
blood flow，

generating the diuretic effect, making the cutaneous dropsy

induced by the congestive heart failure weakened or eliminated, this
shows the indirect action of the digitalis.
The indirect action is implemented by the anaclasis at times. For
example, the amchlor (a apophlegmatic) has the irritant action which
causes the reflex of the cranial nerve in the gastric mucosa. The reflex
enhances the secretion of the bronchial gland, attenuating the thick
phlegm, making the phlegm expectorate, showing the eliminating
phlegm.
● Local effect and the absorptive action In view of the
position of the drug action ， the local action is defined as the direct
action generated in the position where the drug is effected before it
enters the blood. For example, procaine exerts local anesthesia in the
part where it is given, losing the sensory function of the nerve ending.
The absorptive action is defined as the general action after the drug
enters the blood. For example, after the antipyretic analgesic enters
the blood, it effects on the body temperature regulating center, cutting
down the excitability, enhancing the body heat loss by the perspiration
and anapetia, showing the effect of relieving fever and pain. Given
orally, the barbiturates are absorbed from the intestinal tract, and then
they are distributed in tissues and body fluids, mainly showing the
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depressant effect in the above activated system of the formatio

reticularis, generating hypnic effect.

Selectivity of the drug action

After the drug is absorbed, it generates the non-equicohesive
effect on different tissues. When the majority of drugs are used at the
proper dose, they only show the obvious effect on some tissues and
organs, having the little effect on other ones, even having no effect on
the consecutive cells; we define the situation as the selectivity of the
drug action. For example, digitalis has effect on the cardiac muscle, the
para-adnephrin has effect on the receptor of the adrenal gland.

The therapeutic action and adverse effects of

drugs
When we use drugs to prevent and treat diseases in the clinic,
the drugs may generate many pharmacological or physiological
effects. Some effects are good for the health in the animals,
which are defined as the therapeutic actions of drugs, other effects
are bad for the health, which are defined as adverse effects (including
the side effects and toxic effects). When the majority of drugs treat the
diseases, they have some adverse effects on the body, we define the
phenomenon as the duality of the drug action. Generally, the
therapeutic
actions and adverse effects of drugs can be predicted, and the
advantage and the disadvantage of the used drug can be analyzd
.when the drug treats the disease, we should take the measure to
decrease or eliminate the adverse effects. For example, dogs or cats
have the reflection of the vomitting, when xylazine is given to
them, we should prepare for the prevention. Certainly, some
adverse effects can not be predicted, such as the allergy, i.e. the
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particularity reaction, we should take the measure according to

the reaction of the animal.
1． The therapeutic action
The etiological and the symptomatic treatment are the two
methods of treating the clinical diseases.
● Etiological treatment: we define the method of treating
diseases according to its cause as the etiological treatment. Its purpose
is to eliminate the original pathogenic factors by using the drug. For
example, antibiotics and sulfonamides kill the invaded causative
organisms; the antidote can eliminate the poison in the body, the
anthelminthic drives out the animal parasites that parasitic in the
body. The supplementary treatment whose purpose is added to the
deficient endotrophic or the metabolin and the substitution therapy
also belongs to the etiological treatment.
● Symptomatic treatment: We define the method of treating
diseases according to its symptoms as the symptomatic treatment. It
can not thoroughly cure the disease, but if the symptoms of shock,
cardia failure and convulsion occur, it is necessary to use the effective
symptomatic treatment. It can prevent the aggravation and the
development of the disease. For example, when the body is poisoned
by the phosphate pesticide such as trichlorphone or vapona, the
symptom of being excited of the parasympathetic can be dealt with by
atropine; pressor agents should be used in the crisis blood pressure
caused by shock; pain-killers should be used in the severe pain; and
defervescents should be given for the high fever.
There are different advantages and disadvantages between the
etiological treatment and the symptomatic treatment. We should treat
the disease in the clinic by the two methods in order to achieve quick
recovery of the animals’ health.
2. Adverse effects
When drugs are used at the therapeutical dose, they may exert
adverse effects which are not related to the purpose of the treatment or
do harm to the body which include the side effect, toxic action, allergic
response, secondary reaction and residual effect.
● Side effects: Side effect is defined as the effect that is not related to
the goals of treatment, generally, they are light and easily recuperative.
For example, atropine has many complicated effects, when it plays a
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part in relaxants and antispasmodic drugs of the smooth muscle; its

side effects are shown as inhibiting the secretion of glands and causing
the dry mouth. By contrast, before we use chloral hydrate, atropine is
used to prevent the stimulation which is caused by the chloral hydrate
in the bronchial gland and enhance the glandular secretion; the other
purpose is to prevent the foreign pneumonia, therefore we call this is
the therapeutic action of atropine. At the same time, it plays a part in
relaxing the smooth muscle of the gastrointestinal tract and bladder,
causing intestinal gas and urine retention after the operation, we call
this its side effect. It is inherent to the drug. Being generally
predictable and avoidable. we should try to rectify side effects when we
use drugs in the clinic .For example, in order to eliminate the
excitement of the cerebral cortex caused by ephedrine, barbiturates
can be used simultaneously to overcome the side effects of over-
excitement.
● Toxic action: The majority of drugs have the toxicity, but there are
different Charasteristics with the different drugs. Drugs exert the
pathological changes of the physiological and biochemical functions in
all tissues and organs, toxicity is generally caused by the bigger dose,
the shorter interval time or the longer course of treatment. when we
use the antimicrobial drugs and antiparasite agents in the therapeutic
dose, there are some toxicity in the body, we do not define the effect
which is not related to the purpose of the treatment as the side effect,
defining them as the toxic action. For example, the alficetin at the
therapeutic dose can inhibit the haematopoietic function of the
marrow, generating the aplastic anemia. Some antipyretic analgesics
have the toxicity such as the amidazophen, algopyrin and butalidon.
● Hypersensitive reaction: It is also called the allergy which
belongs to the immune reaction. The drug action tends to be different
for different individuals. Some individuals show the strong sensibility.
The reaction could be different due to the characteristics of the drug
action even when the dose is below the usual dose,
moreover ， different drugs may appear the similar reaction. It is not
the ingredient of the drug inherent effect; the drug antagonists are
ineffective with regard to save it. The symptoms of the hypersensitive
reaction include the breakout, calefy, dribble, disgorging, asthma,
stomachache，alvine flux, anapetia and hypotension. The serious one
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expresses allergic shock. The animal has the phenomenon of the

anhelation, hypoxia, cataphora, convulsion and death. The
hypersensitive reaction is not relevant to the dose, which is not
predictable. The low-grade infection can be treated by antiallergic
drugs such as aleryl, and the allergic shock should be rescued by the
AD or GC.
● Secondary reaction ： The bad consequence caused by the
therapeutic action of drugs is defined as the secondary reaction. For
example, when we chronically use the broad spectrum antibiotics, the
depressant effect occurs with regard to the aeschynomenous strain of
the drug, the relative balance is destroyed between microbial
populations, a lot of insensitive germs or eumycetes are increasing, the
situation can cause the general infection or the virulent enteritis，also
called the dual infection

● Residual effect: When the drug is stopped, and the blood drug
level dropes below the leak point, we define the survival pharmacol
effect as the residual effect. For example, we chronically use the ACH,
after the drug is stoped, the phenomenons of the hypoadrenocorticism
still exist, and the situation can be back to normal in several months

Factors of Affecting the Drug Action

The drug action is manifestation of the interaction between drugs

and body, many factors might interfere with and influence the
procedure. The factors come from the drugs, animals and
environment.
I. Factors of drugs
1. Dosage
The drug action or effect strengthens with the dosage increases
among invariably dosage range, for example, chloral hydrate exerts
mitigate at the low dose, and produces hypnosis at the midst dose, has
anesthesia at the large dose. But for the animals are different, the same
dosage generate the different pharmacology effect or toxicity, for
instance tincture of iodine, it is sterilization at the low concentration
(2%) (as toxicidum), it is stimulatory function at the large
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concentration （ 10% ） (as an irritant drug). So when clinical

medication, it is not applied without the rational dosage. Except for
according to the Chinese veterinary drugs dispensatory, and according
to the physical-chemical property, toxic by-effect, progression
2. Dosage form
Dosage form is an important factor that affects physiological
disposition especially the drug absorption. Generally speaking, gas
dosage form and inject preparation absorb fast, solid dosage form
absorb most slowly. The house pet clinic often utilize inject
preparation, requesting to combine with the immediate effect dosage,
choose drug depot preparation to prolong drug action, decrease the
administration times, and keep the effective concentration of the drugs
in vivo. of disease of the drugs , it can adjust the dosage.
3. Administration route
The different administration route can affect the drug absorption
dose, speed, concentration in vivo and the time of the drug action. It
will bring convenience for clinic, and bring the immediate effect or
slow-release. according to the characteristics of the drug, physiology
and pathology of the animals, we will choose the right administration
route in clinic.
● Oral administration Drugs can be administered via mouth or
infiltrated into the feeds. The method is convenient, and fits for many
animals, and especially it can induce the role of the drug in the
gastrointestinal tract.

But gastrointestinal contents are too much, absorption is not easy,

mis-classification, and is affected and restricted by the digestive
enzyme, pH, so drug absorption is slow. Some drugs that are easily
destroyed by alimentary tract or can irritant gastrointestinal tract are
not for oral use.
● Injection administration The drug action of the intravenous
injection or intravenous drop infusion emerges fast, dosage is exact,
effective concentration is certain, it frequently use acute disease that
requests the exact dosage and will emerge drug action fast, but it can
not be used continuously, frequently, multiple. When using
intramuscular injection, drug absorption is faster than intravenous
injection, and emerge the role of stabilization, method of operation is
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easy. The drugs that irritation is fort can not use intramuscular inject;
the drugs that irritation is light can use deep part intramuscular
injection. The duration of drug action keep long hypodermic inject, the
drugs that irritation is fort, oil suspension and possessing contract
vessel can not use hypodermic injection. The intraperitoneal injection
is used when is not used for oral use and intravenous injection, but
add a bulk of fluid, add essential nutrient substance or a bulk of fluid
replacement, except irritated drugs.
● Per rectum The method that the drugs are dabbling into
rectum deep part, educe local action and absorptive action (for
instance add nourish, relieve fever analgesia).
● Skin and mucosa adminisration The drugs are squashed,
sprinkled, droped above the skin, educe local action (for instance cure
ectoparasite).
● Inhalation administration gaseous and volatilis drugs and
aerosol ingress into body, educe local action (for instance cure
respiratory tract disease) and absorptive action (for instance
anesthesia). The method is convenient; the concentration is easy to be
handled.

4. the drug interaction

If one drug can cure disease, we can not use two kinds or more
than two kinds, and especially two kinds of antibacterials. Two or
more kinds of drugs apply together, which might generate rational
interaction, and even adverse interaction. According to the property
and location of drug interaction, it can divide extra organ interaction
and interaction in vivo. extra organ interaction main display “chemical
incompatibility”, interaction in vivo can divide pharmacokinetics
interaction and pharmacodynamics interaction.
● chemical incompatibility Interaction might occur if two
drugs are mixed, resulting in the drug counteract, hydrolysis,
aerogenesis and reaction, when it may emerge turbid, sedimentation,
aerogenesis and discoloration abnormal phenomena, called chemical
incompatibility. For example mix sodium sulfadiazine with PGI
injection, minute crystal educes in the fluid, because sodium alkaline
sulfadiazine educes at the low pH；for example, when surgery is going
on, if muscle relaxant celocaine apply with narcotic Sod Pent, though
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external appearance is not saw, anectine hydrolysis out-of-service in

the alkaline solution. Consequently, we are discreetly applying two
kinds of drugs together, avoiding chemical incompatibility.
● Pharmacokinetics interaction Two or above kinds of drugs
are used at the same time, one drug may change another drug’s
absorption, distribution, biological transformation or evacuation, and
change drug’s half life period, max concentration and biological
availability.
(1) Absorption: the interaction of absorption process takes
place in the gastrointestinal tract, it embodies several directions: the
interaction of physical and chemical. For example the food filled in
stomach can dilute the drug, affecting absorption; if pH changes, it will
affect decollement and absorption of the drugs; TCs can chelate with
Ca, Fe, Mg, and affect absorption or make the drugs
inactivation.gastrointestinal tract peristaltic movement change. For
instance cholinergic steps up gastric emptying and enterokinesia, it
can make the drugs discard fast, absorption is incompletion.
Anticholine drugs for instance AT decreases gastric emptying velocity
and reduces enterokinesia gradually, and steps down rate of
absorption, degrades max concentration, prolong detention time in the
gastrointestinal tract, increases absorbed dose of the drugs.
composition and function transformation of microbial flora of
intestinal tract. The gastrointestinal tract bacteria play a role in the
metabolism of the drugs. Broad-spectrum antiseptic drugs can change
or kill the microbial flora of gastrointestinal tract so that it can affect
metabolism and absorption of drugs, while antibiotic can decrease the
biological transformation of the DIG gastrointestinal, increasing
absorption. change the biomembranous’ integrality. Some drugs can
contaminate gastrointestinal tract mucosa, and destroy
biomembranous’ integrality and function, and affect absorption or
block active transport process.
（ 2 ） Disposition: the interaction or disposition of drugs is
caused by the competition between drugs for the binding site of
plasma protein, because the binding of plasma protein and drugs is
reversible, and the drugs whose protein chemical force are high can
displace the drugs whose protein chemical force is weaker. If the drugs
that are high protein binding are displaced by others, it can elevate
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concentration of the nonbinding drugs in the blood ，and accordingly

increase the chance of toxic. For example if the protein binding rate of
NSAID (aspirin) is above 90%, and only a little part of drugs are
displaced, it can generate toxic concentration. Otherwise, the drugs
that can affect organ blood flow can affect the distribution of the drugs
in the organ tissue, because, the uptake rate and clearance rate of
drugs is decided by blood flow of the organ. For instance
antiadrenergic drugs AY-64043 can decrease cardiac output, and
accordingly decrease blood flow of the liver, this can degrade clearance
rate of the liver of strong first pass effect drugs (for instance dolicaine),
induce the heighten of the concentration of drugs in the blood.
（3）Biological transformation：the interaction of the drugs
through the biotransformation mainly displays enzyme induction and
inhibition. Many central depressant drugs including sedatives,
Antispasmodic have enzyme induction, for instance phenol barbital
hepatic induce the composition of the micro some enzyme, elevate its
activity, accordingly accelerate transformation of itself and other
drugs, and degrade drug action. But because it can increase production
of the toxic metabolin, toxicity may increase.
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In contrast, some kinds of drugs such as GC inhibits drug enzyme,

steps down metabolism of drugs, elevate concentration of the drugs in
the blood, enhances drug action. The enzyme inhibition of the drug
metabolism can be used for healing, such as AAP is toxicity for cats,
which can be instead of Cimetidine. It can decrease production of the
toxic metabolin
（4）Excretion: the drugs’ interaction mainly have two kinds of
manifestation in the egesting: due to changing the straining of
glomeruli of kidney and compete the initiative externalization of the
kidney tubule, accordingly change the egesting of the drugs in the
urine fluid. Such as Pb and penicillinum use together, because apuria
initiative externalization of proximal convoluted tubule, it can step
down the egesting of penicillinum, elevate plasma concentration, and
prolong half-value period. the drugs that can affect pH of urine fluid
can change degree of dissociation of another drug, accordingly affect
the re absorption in the kidney tubule, such as we alkalinize urine fluid
with sodium bicarbonate, this can accelerate the egesting of salicylate,
acidize urine fluid with ammonium chloride, and thus accelerate the
egesting of alky drugs.
● Reciprocity of pharmacodynamics. Using two more drugs
simultaneously can make the total effect change, due to drug effect or
action mechanism that is different. Effect of two drugs to use
simultaneously is greater than algebraic sum of single effect, which is
called synergism. For example, using Penicillin G and Streptomycin
together may produce synergism. Effect of two drugs to use
simultaneously is equal to algebraic sum of them acting separately,
which is called additive effect. For example, using tetracyclines and
sulfonamides together may produce additive effect. Effect of two drugs
when used simultaneously is less than algebraic sum of them acting
separately, which is called antagonism. For example, using ß-lactam
antibiotics with rapid t bacterial inhibitor etracycline etc. together
could produce antagonism. In clinical synergism is utilized to reinforce
drug effect, such as sulfonamides and trimethoprim TMP to use
together, and antagonism to utilize may decrease adverse effect, for
example,
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Atropine may withstand stimulant symptom of parasympathetic

nerve of organophosphorus pesticide. In addition, adverse effect can
also emerge synergism, for example, nephrolysin of cephalosporins
may increase because of using Gentamicin together. Generally
speaking, the more kinds of drugs are used, the higher incidence of
adverse effect is. So in clinical environment, multiple drugs should be
avoid being used together, especially avoid using combined drugs of
regular dose, because it makes veterinaries lose chances of adjusting
drug dose according to need of animals′ illness.
5. Drug regimen
The key of doing reasonable treatment to diseased animals rests with
choosing drugs and drawing up drug regimen. Once to decide to do
drug treatment to animals′ illness, treatment plans must be made
thoroughly, including selecting drugs (or preparation) first and
ascertaining drug regimen. When several drugs are chose by
veterinaries, they decide to choose drugs according to the process of
pathology of illness、dynamics characteristic of drugs and strength or
weakness of drug effect and so on. When antibacterial drugs are chose
to treat infective illness of animals, doing susceptibility test as far as
possible before using drugs, if narrow-spectrum is capable to be used,
broad-spectrum need not be used.
When choosing functionality drugs, pay attention to frequently
discrepancy of pharmacokinetics of animals′ genera, for example,
when the dog is given with Aspirin of the same dose (10mg/kg) with
the cat, interval of taking drugs of the dog is 12 hours, which should be
48 hours for the cat.
Because the cat lacks glucosiduronate enzyme which makes metabolic
rate of Aspirin lower. Under general circumstances externally applied
agent ought not to be tagged, but to be explained the same as the tag of
ratified drugs in aspects of drug regimen 、 dose 、 period of
treatment、genera and suitability.

Drug regimen includes drug dose 、 spacing interval 、 channel and

period of treatment. Dose is quantity of taking drugs to animals once a
time, veterinary pharmacopeia of China in 2005 no longer marks
 SCIENCE LABORATORY TECHNOLOGY

using dose of drugs, while ascertain dose of taking drugs according to

veterinary drug applied guide. Interval of taking drugs is determined
by pharmacodynamics and pharmacokinetics of drugs. Each drug or
preparation has its specifical continuous time of action. For example,
time of anti-inflammatory action of Dexamethasone is much longer
than Hydrocortisone, so interval of taking drugs of the former is
longer. Drug regimen of drug is mainly restricted by preparation, such
as the tablet、capsules for being taken orally, suspension in injection
only for subcutaneous and Intramuscular injection, not intravenous.

But the choice of drug regimen should also consider the types of
diseases and target drugs, such as Lidocaine in the non-intravenous
drug taken time, which is not valid to control heart ventricle
arrhythmia. Last step of drawing up drug regimen is to determine the
course of treatment, Some diseases by taking a single kind of drugs or
short-term treatment could be reinstated or cured, but many of the
diseases require taking drugs repeatedly in a certain period of time (a
few days, weeks or even longer) in order to achieve therapeutic effect.
Bacterial infection of the disease must have adequate course of
treatment, for example, antibiotics generally requires 2 to 3 days for a
course of treatment, sulfonamides requires 3 to 5 days. Animals can
not stop treatment in body temperature dropping or slightly better
condition, or lead to recurrence of disease or bacteria-induced
resistance to bring subsequent treatment more difficult.
The structure-function relationship of drugs
The chemcial constitution of the drug is closely related to the
pharmacological effect and activity. The specificity of the
pharmacologic action is decided by the specificity of the chemical
reaction, the specificity of the chemical reaction is decided by the
chemical constitution of the drug, and we define the phenomenon
 SCIENCE LABORATORY TECHNOLOGY

as the structure-function relationship. The chemical compound

having the identical or similar elementary structure can associate with
the same acceptor, generating similar or adverse effect. For example,
adnephrin, ephedrine and aleudrin are the adrenomimetics, and
inderal is a antiadrenergic drug. They have the similar chemical
structure. But the similar elementary structure only decides the
characteristics of the drug action, the relative intensity of their effects
is decided by the substituent group of the elementary structure.
Sulfonamides have the paratope amino structure, the effective
intensity of these drugs exerts the apparente difference because other
substituent groups are different. In addition, many drugs having the
same chemcial constitution exist optical isomeride ， the intension of
their pharmacologic actions have the tremendous difference, while the
majority of the levo isomer drugs have the stronger action

The dose-effect relationship of drugs We

define the dosage which generates some reactions as the dose. The
more doses or concentrations are, the stronger is the effect of the drug,
and we define the regular change which is between the dose and the
effect as the dose-effect relationship.
Changes of doses can cause the change of the intension or the
character of the drug effect.
● Ineffective dose: It is defined as the dosage which is too small to
generate any effect.
● Threshold dose（minimal effective dose）：It is defined as the
dosage which can induce the concentration of the drug to reach the
leak point in the body, beginning to generate the drug effect.
● Therapeatic dose: It is defined as the dosage which is used
clinically in order to prevent and treat the disease.
● Maximal dose: The dose limite of the drug is defined as the dose
which is between the minimum toxic dose and the usual dose in the
standard of veterinary drugs. Besides the special requirement, the
maximal dose should be avoided in the clinical medication.
● Minimal toxic dose: The dosage has exceeded the maximal dose
and the body appears signs of intoxication.
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● Lethal dose（toxic dose）: The dose has exceeded the minimum

toxic dose, if the dose is used, the phenomenon of the intoxication or
the death will occur.
● Margin safety: The width which is between the minimal effective
and the maximal dose is defined as margin safety. There is large safety
when the drug is in the large safety range. By contrast, there is small
safety when the drug is in the small safety range.
To estimate the safety or the toxicity of the drug, we generally use
the therapeutic index or the chemitherapeutic index in terms of the
ratio between the LD50 and the ED50, i.e. Therapeutic index = LD50/
ED50, which gives some idea of the margin of safety in use of a drug, by
drawing attention to the importance of the relationship between the
effective and toxic dose. The bigger the therapeutic index is, the
smaller the toxicity is and the higher the relative curative effect is.
There is the clinical sample meaning when it is above three, it can be
applied when it is below seven.

MECHANISM OF DRUG ACTION

Mechanism of drug action is to reveal the nature of drugs, that
researches how drug is choosen to act on the cells and produce the
effect. With different natures of drugs, they also have a wide variety of
mechanisms.

I. Mechanism of the drug receptor

Receptors are the specific bioactive substances which have the
capacity to identify and selectively combine with the biological
macromolecules, which may be composed of protein or lipoprotein, or
it may be part of the nucleic acid or enzymes ,often reside in the cell
membrane. According to the pharmacological experiments, the
occupation theory presume that the drug must be able to show the role
of receptor-binding, which is a combination of chemical, that is,
through
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combination of a variety of chemical bonds (such as Vander Waals's

bond, hydrogen bond, ionic bond, covalent bond, etc.) and the
receptor active groups (such as the sulfhydryl, carboxyl and amino,
etc.). This combination is consistent with the mass action law and it is
reversible, that is, when the concentration of the drugs reduces or
lower, its bands break, this time the role of the drug is to stop.
Combination is only the first step, so that the reaction spread and
speed up.
After the receptor-binding, receptor may be excited, also be
blocked, which mainly depends on the "intrinsic activity" of drugs, that
is, drugs could have a certain strength of the effect after receptor-
binding which is known as agonists. If one drug with affinity of
combination with receptor has no internal activity, it does not only
have a clear effect, but also interfere with gonist or medium to exert
effects because the receptor is taken up and sheltered by the drug, ,
such a drug is known as the antagonist.

II. Non-receptor mechanism of drug action

There are a wide variety of the chemical structures of drugs, the
features of animal body are also ever-changing, it results in that
mechanism of the drug acting on the body is a complex physical,
chemical and biological process. In addition to the receptor
mechanism, there are some non-receptor mechanisms.
● Affecting on the enzymes As the receptor may be an integral
part of the enzyme or enzyme system, the realization of the drug action
is via the receptor effect on the enzyme function, including enzyme
induction, inhibition, activation, as well as the revival etc. Such as the
phenobarbital induces liver microsomal enzyme; caffeine inhibits
phosphodiesterase; adrenergic activates adenylyl cyclase;
Echothiopate Iodide reactivates cholinesterase.
● Changing the physical and chemical conditions Some
drugs act by changing the physical and chemical conditions of
environment around the cells. For example, antacids reduces the
degree of gastric acidity by neutralisation; as considerable
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hyperosmolar mannitol is fast infused into the blood, brain edema can
be eliminated by the effect of hypertonic pressure water absorption.
● Effecting on the ion channels Some drugs can directly act
on Na+, K+, Ca2+ channels on the cell membrane and produce
pharmacological effects, such as procaine to block Na + channels and
have a role of local anesthesia.
● Acting on nucleic acid Many drugs exert their effects by
acting on a link of nucleic acid metabolism, such as many antibiotics
can affect the nucleic acid metabolism of cells.
● Participating in or interfering with the cell metabolism
Some vitamins and microelements may be directly involved in
physiological and biochemical processes of the normal cells so that the
deficiency can be retrieved; sulfa drugs block the folacin metabolism of
bacteria then suppress their growth and reproduction.
● Impacting on the immune function Some drugs act via
influencing the immune function, such as levamisole has the immune-
enhancing effects. ● Effecting on neurotransmitters or the
active substance in the body Any interference or block in the links
of biosynthesis, storage, release or eliminate of neurotransmitters or
auto-active substances in the body can induce significant
pharmacological effects. For example, ephedrine would promote the
release of noradrenaline and play the role of epinephrine; antipyretic
analgesic produce the influence by inhibiting prostaglandin synthesis.
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ENTOMOLOGY
Insects are the most abundant and most diverse group of
organisms on earth. They have maintained a position of
ecological pre-eminence for over 400 million years. While no
single ecological or physiological attribute can account for
this unparalleled success, the insects do have a unique
combination of characteristics which, as a whole, have given
them an unusual survival advantage.  In brief, these attributes
include an exoskeleton, small body size, the ability to fly, a
high reproductive potential, complete metamorphosis, and
adaptability in an ever-changing environment.

Skeleton
an insect's supporting skeleton is located on the outside of its
body.  This exoskeleton is a marvelous structure that not only
gives shape and support to the body's soft tissues, but also
provides protection from attack or injury, minimizes the loss
of body fluids in both arid and freshwater environments, and
assures mechanical advantage to muscles for strength and
agility in movement.  As a "suit of armor", the exoskeleton
can resist both physical and chemical attack.  It is covered by
an impervious layer of wax that prevents desiccation.  Much
of the exoskeleton is fabricated from chitin, a polysaccharide
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that binds with various protein molecules to form a body wall

that may be as flexible and elastic as rubber or as hard and
rigid as some metals.  Freedom of movement is ensured by
membranes and joints in the exoskeleton.  Muscles that attach
directly to the body wall combine maximum strength with
near-optimum mechanical advantage (leverage).  The result is
an ant, for example, that can lift up to 50 times its own body
weight.

Small size
Most species are between 2 and 20 mm (0.1 - 1.0 inch) in
length, although they range in size from giant moths to tiny
parasitic wasps.  Small size is a distinct advantage.  If insects
were as large as cows or elephants, their exoskeleton would
have to be proportionately thicker to support the additional
mass of body tissue.  But a thicker exoskeleton would also be
heavier and more cumbersome.   Even the simplest
movements would require a larger muscle volume and
consume more energy. Beyond this size, the insect's surface
area is just too small for attachment of all the additional
muscle tissue Another advantage of small size is the minimal
resources needed for survival and reproduction.  In some
cases, food requirements are so modest that an insect may
live on a single plant or animal for its entire life and never
exhaust its food supply. small size is a big advantage to
insects that must avoid predation.   They can hide in the
cracks of a rock, beneath the bark of a tree, behind the petal
of a flower, or under a blade of grass.  The exoskeleton is
hard enough for them to burrow between individual grains of
sand, yet flexible enough to let them squeeze through the
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tiniest of cracks.  Small size, together with adaptations in

body shape and coloration, gives many species the ability to
blend so well with their environment that they become
virtually undetectable.

Ability to fly
Insects are the only invertebrates that can fly. Flight gave
these insects a highly effective mode of escape from
predators that roamed the prehistoric landscape.  It was also
an efficient means of transportation, allowing populations to
expand more quickly into new habitats and exploit new
resources

Reproductive Potential

Reproductive success is one of the most significant measures

of an organism's fitness.  In insect populations, females often
produce large numbers of eggs (high fecundity), most of the
eggs hatch (high fertility), and the life cycle is relatively
short (often as little as 2-4 weeks).  Together, these three
characteristics enable insects to produce remarkably large
numbers of offspring.  A typical female lays 100-500 eggs in
her lifetime, but numbers in the thousands are not
uncommon.  The queen of an African termite colony may be
the mother of more than ten million workers during her 20-25
year lifespan.
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Since most insects die before they ever have an opportunity

to reproduce, a high reproductive potential is the species' best
chance for survival.  Many adaptations help maximize this
potential.  Most females, for example, can store sperm for
months or years within the spermatheca, a special region of
the reproductive system.  A single mating can supply a
female with enough sperm to fertilize all the eggs she will
produce in her lifetime.  An unbalanced sex ratio, where
females outnumber males, is another way to maximize
reproductive potential.  Since most insects are not
monogamous, a few males can supply sperm for a large
number of females.  And finally, there are many species (e.g.
aphids, scale insects, thrips, and midges) where males are
entirely absent -- all members of the population are female
and contribute offspring through a process of asexual
reproduction.

Metamorphosis
Most insects undergo significant developmental changes as
they grow from immatures to adults.  These changes,
collectively known as metamorphosis, may involve physical,
biochemical, and/or behavioral alterations that promote
survival, dispersal, and reproduction of the species.

 In the class Insecta, only 9 out of 28 orders undergo

complete metamorphosis, yet these 9 orders represent about
86% of all insect species alive today.  The obvious advantage
to this type of development lies in the compartmentalization
of the life cycle.  Through natural selection, larval form and
function can be optimized for growth and feeding without
compromising adaptations of the adult for dispersal and
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reproduction.  Each stage of the life cycle is entirely free to

adapt to its own ecological role.  In some cases, this means
that immatures and adults may consume different types of
food, exploit different environmental resources, and even
occupy different habitats

Adaptability

A combination of large and diverse populations, high

reproductive potential, and relatively short life cycles, has
equipped most insects with the genetic resources to adapt
quickly in the face of a changing environment.  Their record
of achievement is impressive:  they were among the first
creatures to invade the arid expanses of dry land and exploit
green plants as a source of food, they were the first animals to
use flight as an escape from predators, and they were the first
organisms to develop a complex social hierarchy with
division of labor and cooperative care of the young.  As a
group, they have endured 400 million years of climatological
and geophysical upheaval, including the evaporation of
inland seas, formation of mountain ranges, shifts in
continental plates, onset of ice ages, and the fallout from
cosmic impacts.

Classification of Insects
Taxonomy is the study of the principles of scientific
classification.The animal kingdom is divided in a number of
 SCIENCE LABORATORY TECHNOLOGY

groups called "phyla" (singular: phylum). Examples of phyla

are:

 Protozoa (single-celled animals)

 Porifera (sponges)
 Nemathelminthes (roundworms)
 Mollusca (mollusks, snails, etc.)
 Arthropoda (crayfish, millipedes, centipedes, spiders and
insects)
 Chordata, (fish, amphibians, reptiles, birds, and mammals)

Each phylum is subdivided in classes, for example the class

Hexapoda (= insects). Classes are subdivided into orders, for
example the order Coleoptera (= beetles). Orders are divided
into families, families into genera (singular: genus), and
genera are divided into species (See Table 1). Within the
class Hexapoda there are over 750,000 different species of
insects. 
The scientific name of a species is always a double name (the
genus name, and a specific name). It should be written with a
capital letter in the genus name and either in italics or
underlined. 
Example: Helicoverpa armigera or Helicoverpa armigera
An example of the classification of an insect:
Kingdom – Animal

     Phylum – Arthropoda

          Class -- Hexapoda (= insects) 

               Order -- Lepidoptera (= butterflies and moths)

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                    Family -- Noctuidae (= noctuids) 

                         Genus – Helicoverpa

                              Species -- Helicoverpa armigera (= American bollworm)

The phylum Arthropoda

Some characteristics of the Arthropoda are:

 They have a so called exoskeleton. They do not have

bones, but the hard outer covering supports the muscles.
 The appendages are jointed.
 The body is formed of a number of segments.

Characteristics of the class Hexapoda (Insects)

Some characteristics of insects are:
Body:

 The body is divided into three distinct regions: head,

thorax and abdomen

Head:

 One pair of antennae.

o The antennae are usually used as tactile organs (=
organs pertaining to the sense of touch) or as
olfactory organs (= organs of smell).
 Eyes:
o Most insects possess one pair of compound eyes
and sometimes some simple eyes called "ocelli".
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 Mouthparts.
o There is a big variety in types of mouthparts;
biting, sucking, stinging, licking, etc.

Thorax:

 Three pairs of legs.

o The thorax has three segments. These are called
pro-thorax, meso-thorax and meta-thorax. Each
segment has one pair of legs. The different parts
of the leg are called coxa, trochanter, femur, tibia,
and tarsus.
Note: some insects are legless, or have fewer than
6 legs. Some larvae have leg-like appendages on
the abdomen.
 Often one or two pairs of wings.
o The wings are borne by the second and/or third of
the thoracic segments.
Note: Some insects are wingless.

Abdomen:

 The gonopore (genital opening) is at the posterior end of

the abdomen.
 No appendages used for moving on the abdomen of
adults (except in a few primitive insects).
 Sometimes there are some appendages at the end of the
abdomen.

Classification of Hexapoda (Insects)

The class hexapoda is divided in two subclasses:
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 Apterygota (= primitive wingless insects)

 Pterygota (= winged and secondarily wingless insects)

The subclass Pterygota is divided in two divisions:

 Exopterygota (= insects with a simple metamorphosis,

without pupal stage)
 Endopterygota (= with a complete metamorphosis,
including a pupal stage)

Metamorphosis
After hatching from the egg, an insect grows by a series of
molts. After shedding the old skin they expand into a new
larger one. This molting continues until the adult stage is
reached. At each molt, some externally visible changes occur.
This type of growing is called metamorphosis. The division
of insects into apterygota, exopterygota and endopterygota is
mainly based on differences in the type of metamorphosis.
The apterygota have no metamorphosis. Except for the size,
all larval stages closely resemble the adults (which are
wingless).
The exopterygota undergo a simple metamorphosis. In
molting from egg, via the nymphal stages to an adult, there is
a gradual change in the external appearance. The late
nymphal stages already show the development of wing pads.
But only in the last molt functional wings are developed. The
nymphs usually have the same feeding habits as the adults.
In the endopterygota there is a complete metamorphosis. In
these insects the external (and internal) changes during the
life history are the greatest. The eggs hatch into larvae which
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feed actively during the different instars. The larvae may or

may not have legs. The development of wings is not visible
during the larval stages. After several molts a pupa is formed.
A pupa is an inactive stage, it does not feed and it does not
move. Sometimes the pupa is protected by a cocoon of silk,
or it is found in an earthen cell in the soil. During this pupal
stage big changes take place internally. After the pupal stage,
a highly active winged adult appears. Often, the larvae and
the adults live in different types of habitat and use different
types of food.

Orders of insects
Apterygota
Order Thysanura  Bristletails

Order Diplura  Diplurans (Two-pronged Bristletails)

Order Protura  Proturans

Order Collembola  Springtails

Exopterygota
Order Ephemeroptera  Mayflies

Order Odonata  Dragonflies and Damselflies

Order Orthoptera * Grasshoppers, Locusts and Crickets

Order Dictyoptera  Cockroaches and Mantids

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Order Grylloblattodea  Rock crawlers

Order Phasmida  Stick insects and Leaf insects

Order Dermaptera  Earwigs

Order Isoptera * Termites

Order Embioptera  Web-spinners

Order Plecoptera  Stoneflies

Order Zoraptera  Zorapterans

Order Psocoptera  Psocopterans (Psocids, Booklice)

Order Mallophaga  Chewing lice (Biting lice)

Order Anoplura 
Sucking lice
(= Siphunculata)

Order Thysanoptera * Thrips

Order Hemiptera  

   suborder Heteroptera * Bugs

   suborder Homoptera * Cicadas, Hoppers, Psyllids, Whiteflies,

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Aphids, and Scale insects

Endopterygota
Order Neuroptera  Alderflies, Dobsonflies, Fishflies, Snakeflies,
Lacewings, Antlions, and Owlflies

Order Coleoptera * Beetles

Order Strepsiptera  Twisted-winged parasites (Stylopids)

Order Mecoptera  Scorpionflies

Order Trichoptera  Caddisflies

Order Lepidoptera * Butterflies and Moths

Order Diptera * True Flies

Order
Fleas
Siphonaptera 

Order Hymenoptera Sawflies, Ichneumons, Chalcids, Ants,

* Wasps, and Bees
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INSECT EXTERNAL ANATOMY

The Exoskeleton
An insect's exoskeleton (integument) serves not only as a
protective covering over the body, but also as a surface for
muscle attachment, a water-tight barrier against desiccation,
and a sensory interface with the environment. It is a multi-
layered structure with four functional regions: epicuticle,
procuticle, epidermis, and basement membrane.

The epidermis is primarily a secretory tissue formed by a

single layer of epithelial cells. It is responsible for producing
at least part of the basement membrane as well as all of the
overlying layers of cuticle. The basement membrane is a
supportive bilayer of amorphous mucopolysaccharides (basal
lamina) and collagen fibers (reticular layer). The membrane
serves as a backing for the epidermal cells and effectively
separates the hemocoel (insect's main body cavity) from the
integument.
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The procuticle lies immediately above the epidermis. It

contains microfibers of chitin surrounded by a matrix of
protein that varies in composition from insect to insect and
even from place to place within the body of a single insect.

As the procuticle forms, it is laid down in thin lamellae with

chitin microfibers oriented at a slightly different angle in each
subsequent layer. In some parts of the body, procuticle
stratifies into a hard, outer exocuticle and a soft, inner
endocuticle.

Differentiation of exocuticle involves a chemical process

(called sclerotization) that occurs shortly after each molt.
During sclerotization, individual protein molecules are linked
together by quinone compounds. These reactions "solidify"
the protein matrix, creating rigid "plates" of exoskeleton
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known as sclerites. Quinone cross-linkages do not form in

parts of the exoskeleton where resilin (an elastic protein) is
present in high concentrations. These areas are membranes
-- they remain soft and flexible because they never develop a
well-differentiated exocuticle.

The epicuticle is the outermost part of the cuticle. Its function

is to reduce water loss and block the invasion of foreign
matter. The innermost layer of epicuticle is often called the
cuticulin layer, a stratum composed of lipoproteins and
chains of fatty acids embedded in a protein-polyphenol
complex. An oriented monolayer of wax molecules lies just
above the cuticulin layer; it serves as the chief barrier to
movement of water into or out of the insect's body. In many
insects a cement layer covers the wax and protects it from
abrasion.

The Head

In most insects, the head capsule is a sturdy compartment that

houses the brain, a mouth opening, mouthparts used for ingestion
of food, and major sense organs (including antennae, compound
eyes, and ocelli). The surface of the head is divided into regions
(sclerites) by a pattern of shallow grooves (sutures).

Antennae
The antennae are a pair of sense organs located near the
front of an insect's head capsule.  Although commonly called
"feelers", the antennae are much more than just tactile
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receptors.  They are usually covered with olfactory receptors

that can detect odor molecules in the air (the sense of smell). 
Many insects also use their antennae as humidity sensors, to
detect changes in the concentration of water vapor. 
Mosquitoes detect sounds with their antennae, and many flies
use theirs to gauge air speed while they are in flight.

Although antennae vary widely in shape and function, all of

them can be divided into three basic parts:

1. scape -- the basal segment that articulates with the

head capsule
2. pedicel -- the second antennal segment
3. flagellum -- all the remaining "segments"
(individually called flagellomeres)
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Types of Antennae:
Name Appearance Example(s)

Setaceous --
Dragonflies
bristle-like

Ground beetles
Filiform -- thread-
and
like
Cockroaches
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Moniliform --
Termites
bead-like

Serrate --
Click beetles
sawtoothed

Clavate --
Carrion beetles
gradually clubbed
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Capitate --
Butterflies
abruptly clubbed

Lamellate -- nested
Scarab beetles
plates

Fire-colored
beetles
Pectinate -- comb-
and
like
Male glow-
worms

Plumose -- brush-
Mosquitoes
like
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Geniculate -- Weevils and

elbowed Ants

Aristate -- pouch-
like with lateral House flies
bristle

Thorax
The second (middle) tagma of an insect's body is called the
thorax.  This region is almost exclusively adapted for
locomotion -- it contains three pairs of walking legs and, in
many adult insects, one or two pairs of wings.
Structurally, the thorax is composed of three body segments: 
prothorax, mesothorax, and metathorax. These segments are
joined together rigidly to form a "box" that houses the
musculature for the legs and wings. Each segment has a
dorsal sclerite, the notum (pronotum, mesonotum, and
metanotum) which may be further subdivided into an anterior
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scutum and a posterior scutellum. The ventral sclerite of

each segment is the sternum (prosternum, mesosternum, and
metasternum).The side of each segment is called the pleuron
- it is usually divided by a pleural suture into at least two
sclerites:; an anterior episternum and a posterior epimeron.

Legs
Most insects
have three
pairs of walking legs -- one pair on each thoracic segment.  
Each leg contains five structural components (segments) that
articulate with one another by means of hinge joints:

1. Coxa
2. Trochanter
3. Femur
4. Tibia
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5. Tarsus

The term pretarsus refers to the terminal segment of the

tarsus and any other structures attached to it, including:

ungues -- a pair of claws

arolium -- a lobe or adhesive pad between the claws
empodium -- a large bristle (or lobe) between the claws
pulvilli -- a pair of adhesive pads

Leg Adapations and Modifications:

 

Characteristic Appearance Example(s)

Ground
Cursorial --
beetles
adapted for
and
running
Cockroaches
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Raptorial --
adapted for Praying
catching and mantids
holding prey

Natatorial -- Diving bugs

adapted for and
swimming Water beetles

Fossorial --
adapted for Mole crickets
digging in soil

Saltatorial --
adapted for Grasshoppers
jumping
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Wings
Insects are the only invertebrates that can fly.  Their wings
develop as evaginations of the exoskeleton during
morphogenesis but they become fully functional only during
the adult stage of an insect's life cycle.  The wings may be
membranous, parchment-like, heavily sclerotized, fringed
with long hairs, or covered with scales.  Most insects have
two pairs of wings -- one pair on the mesothorax and one pair
on the metathorax (never on the prothorax).  Wings serve not
only as organs of flight, but also may be adapted variously as
protective covers (Coleoptera and Dermaptera), thermal
collectors (Lepidoptera), gyroscopic stabilizers (Diptera),
sound producers (Orthoptera), or visual cues for species
recognition and sexual contact (Lepidoptera).

In most cases, a characteristic network of veins runs

throughout the wing tissue.  These veins are extensions of the
body's circulatory system.  They are filled with hemolymph
and contain a tracheal tube and a nerve.  In membranous
wings, the veins provide strength and reinforcement during
flight.  Wing shape, texture, and venation are quite distinctive
among the insect taxa and therefore highly useful as aides for
identification.
 SCIENCE LABORATORY TECHNOLOGY

Wing adapations and modifications:

Characteristic Appearance Order(s)

Elytra -- hard,
sclerotized front
Coleoptera
wings that serve as
and
protective covers for
Dermaptera
membranous hind
wings

Hemelytra -- front
wings that are
leathery or
Hemiptera:
parchment-like at the
Heteroptera
base and
membranous near
the tip
Tegmina -- front
Orthoptera,
wings that are
Blattodea,
completely leathery
and
or parchment-like in
Mantodea
texture
 SCIENCE LABORATORY TECHNOLOGY

Halteres -- small,
club-like hind wings
that serve as
Diptera
gyroscopic
stabilizers during
flight

Fringed wings --
slender front and Thysanopter
hind wings with long a
fringes of hair

Hairy wings -- front

and hind wings Trichoptera
clothed with setae

Scaly wings -- front

and hind wings
covered with Lepidoptera
flattened setae
(scales)
 SCIENCE LABORATORY TECHNOLOGY

Hamuli -- tiny
hooks on hind wing Hymenopter
that hold front and a
hind wings together

Frenulum -- Bristle
near base of hind
wing that holds front Lepidoptera
and hind wings
together

Abdomen
An insect's abdomen is the third functional region (tagma) of
its body; the abdomen is located just behind the thorax.   In
most insects, the junction between thorax and abdomen is
 SCIENCE LABORATORY TECHNOLOGY

broad, but
in some
groups,
the
junction
is very
narrow

(petiolate) giving the appearance of a "wasp-waist".

Each segment of the abdomen consists of a dorsal sclerite, the

tergum, and a ventral sclerite, the sternum, joined to one
another laterally by a pleural membrane.   The front margins
of each segment often "telecope" inside the sclerites of the
preceding sement, allowing the abdomen to expand and
contract in response to the actions of skeletal muscles.

In many adult insects, there is a spiracle (opening to the

respiratory system) near the pleural membrane on each side
of the first eight abdominal segments.   Some spiracles may
be permanently closed, but still represented by a dimple in
the sclerite.

At the very back of the abdomen, the anus (rear opening of

the digestive system) is nestled between three protective
sclerites:   a dorsal epiproct and a pair of lateral paraprocts.  
A pair of sensory organs, the cerci, may be located near the
anterior margin of the paraprocts.   These structures are
tactile (touch) receptors.   They are usually regarded as a
 SCIENCE LABORATORY TECHNOLOGY

"primitive" trait because they are absent in the hemipteroid

and holometabolous orders.

The insect's
genital opening
lies just below
the anus:   it is
surrounded by
specialized
sclerites that
form the
external genitalia.   In females, paired
appendages of the eighth and ninth abdominal segment fit
together to form an egg-laying mechanism called the
ovipositor.   These appendages consist of four valvifers (basal
sclerites with muscle attachments) and six valvulae (apical
sclerites which guide the egg as it emerges from the female's
body).   In males, the genital opening is usually enclosed in a
tube-like aedeagus which enters the female's body during
copulation (like a penis).   The external genitalia may also
include other sclerites (e.g. subgenital plate, claspers, styli,
etc.) that facilitate mating or egg-laying.   The structure of
these genital sclerites differs from species to species to the
extent that it usually prevents inter-species hybridization and
also serves as a valuable identification tool for insect
taxonomists.

 Egg Structure
SCIENCE LABORATORY TECHNOLOGY

In most insects, life begins as an independent egg.

This type of reproduction is known as ovipary. Each
egg is manufactured within the female's genital
system and is eventually released from her body
through an ovipositor, a tube-like, saw-like, or blade-
like component of her external genitalia. Production
of eggs by the female's body is called öogenesis and
the egg-laying process is known as oviposition. Each
insect species produces eggs that are genetically
unique and often physically distinctive as well --
spherical, ovate, conical, sausage-shaped, barrel-
shaped, or torpedo-shaped. Yet regardless of size or
shape, each egg is composed of only a single living
cell -- the female gamete.

 Morphogenesis
SCIENCE LABORATORY TECHNOLOGY

 Once an insect hatches from the egg it is usually able

to survive on its own, but it is small, wingless, and
sexually immature. Its primary role in life is to eat and
grow. If it survives, it will periodically outgrow and
replace its exoskeleton (a process known as molting).
In many species, there are other physical changes that
also occur as the insect gets older (growth of wings
and development of external genitalia, for example).
Collectively, all changes that involve growth, molting,
and maturation are known as morphogenesis.
  


Molting
 The molting process is triggered by hormones
released when an insect's growth reaches the physical
limits of its exoskeleton. Each molt represents the end
of one growth stage (instar) and the beginning of
another (Figure 1). In some insect species the number
of instars is constant (typically from 3 to 15), but in
others it may vary in response to temperature, food
availability, or other environmental factors. An insect
is known as an imago (adult) when it becomes
sexually mature. At this point, molting stops and
SCIENCE LABORATORY TECHNOLOGY

energy for growth is channeled into production of

eggs or sperm.
 An insect cannot survive without the support and
protection of its exoskeleton, so a new, larger
replacement must be constructed inside the old one --
much like putting an overcoat under a sweater! The
molting process begins when epidermal cells respond
to hormonal changes by increasing their rate of
protein synthesis. This quickly leads to apolysis --
physical separation of the epidermis from the old
endocuticle. Epidermal cells fill the resulting gap with
an inactive molting fluid and then secrete a special
lipoprotein (the cuticulin layer) that insulates and
protects them from the molting fluid's digestive
action. This cuticulin layer becomes part of the new
exoskeleton's epicuticle.
 Summary of Molting
SCIENCE LABORATORY
Step 1: Apolysis -- separationTECHNOLOGY
of old exoskeleton
from epidermis
Step formation
 After 2: Secretion of inactive
of the cuticulinmolting fluid byfluid
layer, molting
epidermis
Step 3: Production of cuticulin layer for new
exoskeleton
Step 4: Activation of molting fluid
Step 5: Digestion and absorption of old
endocuticle
Step 6: Epidermis secretes new procuticle
Step 7: Ecdysis -- shedding the old exo- and
epicuticle
Step 8: Expansion of new integument
Step 9: Tanning -- sclerotization of new
exocuticle
becomes activated and chemically "digests" the
endocuticle of the old exoskeleton. Break-down
products (amino acids and chitin microfibrils) pass
through the cuticulin layer where they are recycled by
the epidermal cells and secreted under the cuticulin
layer as new procuticle (soft and wrinkled). Pore
canals within the procuticle allow movement of lipids
and proteins toward the new epicuticle where wax and
cement layers form.
 When the new exoskeleton is ready, muscular
contractions and intake of air cause the insect's body
to swell until the old exoskeleton splits open along
lines of weakness (ecdysial sutures). The insect sheds
its old exoskeleton (ecdysis) and continues to fully
expand the new one. Over the next few hours,
sclerites will harden and darken as quinone cross-
linkages form within the exocuticle. This process
SCIENCE LABORATORY TECHNOLOGY

(called sclerotization or tanning) gives the

exoskeleton its final texture and appearance.
 An insect that is actively constructing new
exoskeleton is said to be in a pharate condition.
During the days or weeks of this process there may be
very little evidence of change. Ecdysis, however,
occurs quickly (in minutes to hours). A newly molted
insect is soft and largely unpigmented (white or
ivory). It is said to be in a teneral condition until the
process of tanning is completed (usually a day or
two).
 Metamorphosis
 Each time an insect molts, it gets a little larger. It may
also change physically in other ways -- depending on
its type of metamorphosis: ametabola, hemimetabola,
or holometabola.


 Ametabolous insects undergo little or no structural

change as they grow older. Immatures are called
young; they are physically similar to adults in every
way except size and sexual maturity. Other than size,
SCIENCE LABORATORY TECHNOLOGY

there is no external manifestation of their age or

reproductive state.


 Hemimetabolous insects exhibit gradual changes in

body form during morphogenesis. Immatures are
called nymphs or, if aquatic, naiads. Maturation of
wings, external genitalia, and other adult structures
occurs in small steps from molt to molt. Wings may
be completely absent during the first instar, appear in
the second or third instar as short wing buds, and
grow with each molt until they are fully developed
and functional in the adult stage. Developmental
changes that occur during gradual metamorphosis are
usually visible externally as the insect grows, but
adults retain the same organs and appendages as
nymphs (eyes, legs, mouthparts, etc.).
SCIENCE LABORATORY TECHNOLOGY

 Holometabolous insects have immature forms

(larvae) that are very different from adults. Larvae
are "feeding machines", adapted mostly for
consuming food and growing in size. They become
larger at each molt but do not acquire any adult-like
characteristics. When fully grown, larvae molt to an
immobile pupal stage and undergo a complete
transformation. Larval organs and appendages are
broken down (digested internally) and replaced with
new adult structures that grow from imaginal discs,
clusters of undifferentiated (embryonic) tissue that
form during embryogenesis but remain dormant
throughout the larval instars. The adult stage, which
usually bears wings, is mainly adapted for dispersal
and reproduction.
 Most larvae can be grouped into one of five
categories based on physical appearance:
SCIENCE LABORATORY TECHNOLOGY

Comm
Descrip Exam
Appearance Larval Type on
tion ples
Name
Body
cylindri
cal with
short
thoracic Moths
Caterpi legs and and
Eruciform
llar 2-10 butterfl
pairs of ies
fleshy
abdomin
al
prolegs
Elongat
ed,
flattened
body
with
promine
Lady
nt
Campodeifor Crawle beetle,
antenna
m r lacewi
e and/or
ng
cerci.
Thoraci
c legs
adapted
for
running
SCIENCE LABORATORY TECHNOLOGY

Body
robust
and
"C"-
shaped
June
with no
Scarabaeifor White beetle,
abdomin
m grub dung
al
beetle
prolegs
and
short
thoracic
legs
Body
long,
smooth,
and
cylindri
Click
cal with
Wirew beetle,
Elateriform hard
orm Flour
exoskele
beetle
ton and
very
short
thoracic
legs
Vermiform Maggo Body House
t fleshy, fly,
worm- flesh
like. No fly
SCIENCE LABORATORY TECHNOLOGY

head
capsule
or
walking
legs

 Pupae can be grouped into one of three

categories based on physical appearance:

Com
Pupal Descrip Examp
Appearance mon
Type tion les
Name
Obtect Chrys Develop Butterf
alis ing lies
appenda and
ges moths
(antenn
ae,
wings,
legs,
etc.)
held
tightly
against
the
body by
a shell-
like
casing.
Often
SCIENCE LABORATORY TECHNOLOGY

found
enclose
d within
a silken
cocoon.
All
develop
ing
Beetles
appenda
Exarat ,
None ges free
e Lacewi
and
ngs
visible
external
ly
Body
encased
within
the hard
exoskel
Coarct Pupari
eton of Flies
ate um
the
next-to-
last
larval
instar
 SCIENCE LABORATORY TECHNOLOGY

INSECT INTERNAL ANATOMY

Digestive and Excretory Systems

An insect uses its digestive system to extract nutrients and
other substances from the food it consumes.   Most of this
food is ingested in the form of macromolecules and other
complex substances (such as proteins, polysaccharides, fats,
nucleic acids, etc.) which must be broken down by catabolic
reactions into smaller molecules (i.e. amino acids, simple
sugars, etc.) before being used by cells of the body for
energy, growth, or reproduction.   This break-down process is
known as digestion.

All insects have a complete digestive system.   This means

that food processing occurs within a tube-like enclosure, the
alimentary canal, running lengthwise through the body from
mouth to anus. Ingested food usually travels in only one
direction.   This arrangement differs from an incomplete
digestive system (found in certain lower invertebrates like
hydra and starfish) where a single opening to a pouch-like
 SCIENCE LABORATORY TECHNOLOGY

cavity serves as both mouth and anus.   Most biologists

regard a complete digestive system as an evolutionary
improvement over an incomplete digestive system because it
permits functional specialization -- different parts of the
system may be specially adapted for various functions of
food digestion, nutrient absorption, and waste excretion.   In
most insects, the alimentary canal is subdivided into three
functional regions:   foregut (stomodeum), midgut
(mesenteron), and hindgut (proctodeum).
In addition to the alimentary canal, insects also have paired
salivary glands and salivary reservoirs. These structures
usually reside in the thorax (adjacent to the foregut).  
Salivary ducts lead from the glands to the reservoirs and then
forward, through the head, to an opening (the salivarium)
behind the hypopharynx.   Movements of the mouthparts help
mix saliva with food in the buccal cavity.

 Circulatory System
Insects, like all other arthropods, have an open circulatory
system which differs in both structure and function from the
closed circulatory system found in humans and other
vertebrates.   In a closed system, blood is always contained
within vessels (arteries, veins, capillaries, or the heart itself).  
In an open system, blood (usually called hemolymph) spends
much of its time flowing freely within body cavities where it
makes direct contact with all internal tissues and organs.
The circulatory system is responsible for movement of
nutrients, salts, hormones, and metabolic wastes throughout
the insect's body.   In addition, it plays several critical roles in
defense:   it seals off wounds through a clotting reaction, it
 SCIENCE LABORATORY TECHNOLOGY

encapsulates and destroys internal parasites or other invaders,

and in some species, it produces (or sequesters) distasteful
compounds that provide a degree of protection against
predators.   The hydraulic (liquid) properties of blood are
important as well.   Hydrostatic pressure generated internally
by muscle contraction is used to facilitate hatching, molting,
expansion of body and wings after molting, physical
movements (especially in soft-bodied larvae), reproduction
(e.g. insemination and oviposition), and evagination of
certain types of exocrine glands.   In some insects, the blood
aids in thermoregulation:   it can help cool the body by
conducting excess heat away from active flight muscles or it
can warm the body by collecting and circulating heat
absorbed while basking in the sun.
A dorsal vessel is the major structural component of an
insect's circulatory system.   This tube runs longitudinally
through the thorax and abdomen, along the inside of the
dorsal body wall.   In most insects, it is a fragile,
membranous structure that collects hemolymph in the
abdomen and conducts it forward to the head.
 SCIENCE LABORATORY TECHNOLOGY

In the abdomen, the dorsal vessel is called the heart.   It is

divided

segmentally into chambers that are separated by valves (ostia)

to ensure one-way flow of hemolymph.   A pair of alary
muscles are attached laterally to the walls of each chamber.  
Peristaltic contractions of the these muscles force the
hemolymph forward from chamber to chamber.   During each
diastolic phase (relaxation), the ostia open to allow inflow of
hemolymph from the body cavity.   The heart's contraction
rate varies considerably from species to species -- typically in
the range of 30 to 200 beats per minute.   The rate tends to
fall as ambient temperature drops and rise as temperature (or
the insect's level of activity) increases.
In front of the heart, the dorsal vessel lacks valves or
musculature.   It is a simple tube (called the aorta) which
continues forward to the head and empties near the brain.  
Hemolymph bathes the organs and muscles of the head as it
emerges from the aorta, and then haphazardly percolates back
over the alimentary canal and through the body until it
reaches the abdomen and re-enters the heart.
To facilitate circulation of hemolymph, the body cavity is
divided into three compartments (called blood sinuses) by
two thin sheets of muscle and/or membrane known as the
dorsal and ventral diaphragms.   The dorsal diaphragm is
 SCIENCE LABORATORY TECHNOLOGY

formed by alary muscles of the heart and related structures; it

separates the pericardial sinus from the perivisceral sinus.  
The ventral diaphragm usually covers the nerve cord; it
separates the perivisceral sinus from the perineural sinus.
In some insects, pulsatile organs are located near the base of
the wings or legs.   These muscular "pumps" do not usually
contract on a regular basis, but they act in conjunction with
certain body movements to force hemolymph out into the
extremities.
About 90% of insect hemolymph is plasma:   a watery fluid
-- usually clear, but sometimes greenish or yellowish in color.
Compared to vertebrate blood, it contains relatively high
concentrations of amino acids, proteins, sugars, and inorganic
ions.   Overwintering insects often sequester enough ribulose,
trehalose, or glycerol in the plasma to prevent it from
freezing during the coldest winters.   The remaining 10% of
hemolymph volume is made up of various cell types
(collectively known as hemocytes); they are involved in the
clotting reaction, phagocytosis, and/or encapsulation of
foreign bodies.   The density of insect hemocytes can
fluctuate from less than 25,000 to more than 100,000 per
cubic millimeter, but this is significantly fewer than the 5
million red blood cells, 300,000 platelets, and 7000 white
blood cells found in the same volume of human blood.   With
the exception of a few aquatic midges, insect hemolymph
does NOT contain hemoglobin (or red blood cells).   Oxygen
is delivered by the tracheal system, not the circulatory
system.
 SCIENCE LABORATORY TECHNOLOGY

Respiratory System
All insects are aerobic organisms -- they must obtain oxygen
(O2) from their environment in order to survive.   They use
the same metabolic reactions as other animals (glycolysis,
Kreb's cycle, and the electron transport system) to convert
nutrients (e.g. sugars) into the chemical bond energy of ATP.
During the final step of this process, oxygen atoms react with
hydrogen ions to produce water, releasing energy that is
captured in a phosphate bond of ATP.
The respiratory system is responsible for delivering sufficient
oxygen to all cells of the body and for removing carbon
dioxide (CO2) that is produced as a waste product of cellular
respiration.   The respiratory system of insects (and many
other arthropods) is separate from the circulatory system.   It
is a complex network of tubes (called a tracheal system) that
delivers oxygen-containing air to every cell of the body.

Air enters the insect's body through valve-like openings in the

exoskeleton.   These openings (called spiracles) are located
laterally along the thorax and abdomen of most insects --
usually one pair of spiracles per body segment.   Air flow is
regulated by small muscles that operate one or two flap-like
valves within each spiracle -- contracting to close the
 SCIENCE LABORATORY TECHNOLOGY

spiracle, or relaxing to open it.

After passing through a

spiracle, air enters a
longitudinal tracheal trunk,
eventually diffusing
throughout a complex,
branching network of
tracheal tubes that
subdivides into smaller and
smaller diameters and
reaches every part of the
body.   At the end of each tracheal branch, a special cell (the
tracheole) provides a thin, moist interface for the exchange
of gasses between atmospheric air and a living cell.   Oxygen
in the tracheal tube first dissolves in the liquid of the
tracheole and then diffuses into the cytoplasm of an adjacent
cell.   At the same time, carbon dioxide, produced as a waste
product of cellular respiration, diffuses out of the cell and,
eventually, out of the body through the tracheal system.
Each tracheal tube
develops as an
invagination of the
ectoderm during
embryonic
development.   To
prevent its collapse
under pressure, a
thin, reinforcing
"wire" of cuticle (the
taenidia) winds
 SCIENCE LABORATORY TECHNOLOGY

spirally through the membranous wall.   This design (similar

in structure to a heater hose on an automobile or an exhaust
duct on a clothes dryer) gives tracheal tubes the ability to flex
and stretch without developing kinks that might restrict air
flow.

The absence of taenidia in certain parts of the tracheal system

allows the formation of collapsible air sacs, balloon-like
structures that may store a reserve of air.   In dry terrestrial
environments, this temporary air supply allows an insect to
conserve water by closing its spiracles during periods of high
evaporative stress.   Aquatic insects consume the stored air
while under water or use it to regulate buoyancy.   During a
molt, air sacs fill and enlarge as the insect breaks free of the
old exoskeleton and expands a new one.   Between molts, the
air sacs provide room for new growth -- shrinking in volume
as they are compressed by expansion of internal organs.

Small insects rely almost exclusively on passive diffusion and

physical activity for the movement of gasses within the
tracheal system.   However, larger insects may require active
ventilation of the tracheal system (especially when active or
under heat stress).   They accomplish this by opening some
spiracles and closing others while using abdominal muscles
to alternately expand and contract body volume.   Although
these pulsating movements flush air from one end of the body
to the other through the longitudinal tracheal trunks, diffusion
is still important for distributing oxygen to individual cells
through the network of smaller tracheal tubes.   In fact, the
rate of gas diffusion is regarded as one of the main limiting
factors (along with weight of the exoskeleton) that prevents
 SCIENCE LABORATORY TECHNOLOGY

real insects from growing as large as the ones we see in

horror movies!

Reproductive System
The reproductive organs of insects are similar in structure and
function to those of vertebrates:  a male's testes produce
sperm and a female's ovaries produce eggs (ova).  Both types
of gametes are haploid and unicellular, but eggs are usually
much larger in volume than sperm.
Most (but not all) insect species are bisexual and biparental --
meaning that one egg from a female and one sperm from a
male fuse (syngamy) to produce a diploid zygote.  There are,
however, some species that are able to reproduce by
parthenogenesis, a form of asexual reproduction in which
new individuals develop from an unfertilized egg (virgin
birth).  Some of these species alternate between sexual and
asexual reproduction (not all generations produce males),
while others are exclusively parthenogenetic (no males ever
occur).
Sexual reproduction might well be the most important
"adaptation" ever acquired by living organisms.  It provides a
mechanism for shuffling and recombining genetic
information from two parents to create new ("hybrid")
genotypes that can be tested in the fire of natural selection. 
Only phenotypes that withstand the "heat" can participate in
the next round of reproduction.

External vs. Internal Fertilization

As long as primitive arthropods lived in the water, their
sperm could simply swim from the male's body to the
 SCIENCE LABORATORY TECHNOLOGY

female's body where fertilization could occur.  But in order to

adopt a terrestrial lifestyle, animals that engaged in such
external fertilization had to protect their sperm from
desiccation.  The solution, still used today by myriapods and
insects, was to encapsulate large numbers of sperm within a
water-tight lipoprotein shell secreted by the male's accessory
glands.  These "packages" of sperm are known as
spermatophores.  In myriapods and primitive hexapods (e.g.
Collembola), males leave spermatophores on the ground
where they may be found and picked up by a passing female. 
Silverfish and bristletails have more elaborate courtship
activities in which the male leads his mate to a freshly
deposited spermatophore.
Today, all of the more "advanced" insects exhibit internal
fertilization -- males deposit their sperm inside a female's
body during an act of copulation.  This novel adaptation,
which appeared soon after insects diverged from their
myriapod-like ancestors, presumably ensured that more
sperm found their way to a receptive female.  But the genetic
programming for spermatophore production still persists in
most modern insects.  After a male deposits his
spermatophore inside a female's reproductive system, she
digests the lipo-protein coat and uses it as a source of
additional nutrition for her eggs.  In some cases, the quality
(or quantity) of this nuptial gift may even determine whether
a female accepts or rejects the male's gametes.
 SCIENCE LABORATORY TECHNOLOGY

Male Reproductive System

The male's reproductive system contains a pair of testes,

usually located near the back of the abdomen.   Each testis is
subdivided into functional units (called follicles) where
sperm are actually produced.   A typical testis may contain
hundreds of follicles, generally aligned parallel to one
another.   Near the distal end of each follicle, there are a
group of germ cells (spermatogonia) that divide by mitosis
and increase in size to form spermatocytes.   These
spermatocytes migrate toward the basal end of the follicle,
pushed along by continued cell division of the
spermatogonia. Each spermatocyte undergoes meiosis:   this
yields four haploid spermatids which develop into mature
spermatozoa as they progress further along through the
follicle.
Mature sperm pass out of the testes through short ducts (vasa
efferentia) and collect in storage chambers (seminal vesicles)
that are usually little more than enlarged sections of the vasa.
Similar ducts (vasa deferentia) lead away from the seminal
 SCIENCE LABORATORY TECHNOLOGY

vesicles, join one another near the midline of the body, and
form a single ejaculatory duct that leads out of the body
through the male's copulatory organ (called an aedeagus).
One or more pairs of accessory glands are usually associated
with the male's reproductive system.   These are secretory
organs that connect to the reproductive system by means of
short ducts -- some may attach near the testes or seminal
vesicles, others may be associated with the ejaculatory duct.  
The glands have two major functions:

1. Manufacture of seminal fluid, a liquid medium that

sustains and nourishes mature sperm while they are in the
male's genital system.
2. Production of spermatophores, pouch-like structures
(mostly protein) that encase the sperm and protect them
as they are delivered to the female's body during
copulation.

Female Reproductive System

The female's reproductive system contains a pair of ovaries.  
When the insect is actively reproducing, these organs swell
with developing eggs and may nearly fill the abdomen.   Each
ovary is subdivided into functional units (called ovarioles)
where the eggs are actually produced.   A typical ovary may
contain dozens of ovarioles, generally aligned parallel to one
another.   Near the distal end of each ovariole, there are a
group of germ cells (oogonia) that divide by mitosis and
 SCIENCE LABORATORY TECHNOLOGY

increase in size to form oocytes. During active oogenesis,

new oocytes are produced on a regular schedule within each
ovariole.   These oocytes migrate toward the basal end of the
ovariole, pushed along by continued cell division of the
oogonia.   Each oocyte undergoes meiosis:   this yields four
cells -- one egg and three polar bodies.   The polar bodies
may disintegrate or they may accompany the egg as nurse
cells.
As developing eggs move down the ovariole,
they grow in size by absorbing yolk (supplied
by adjacent nurse cells or accessory cells).  
Thus, each ovariole contains a linear series of
eggs in progressive stages of maturation, giving
the appearance of a "chain of beads" where
each bead is larger than the one behind it.   By
the time an egg reaches the base (calyx) of the
ovariole it has reached full size -- often growing
up to 100,000 times larger than the original
oocyte.
Mature eggs leave the ovaries through short lateral oviducts.  
Near the midline of the body, these lateral oviducts join to
form a common oviduct which opens into a genital chamber
called the bursa copulatrix.   Female accessory glands (one or
more pairs) supply lubricants for the reproductive system and
secrete a protein-rich egg shell (chorion) that surrounds the
entire egg.   These glands are usually connected by small
ducts to the common oviduct or the bursa copulatrix.
During copulation, the male deposits his spermatophore in
the bursa copulatrix.   Peristaltic contractions force the
spermatophore into the female's spermatheca, a pouch-like
chamber reserved for storage of sperm.   A spermathecal
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gland produces enzymes (for digesting the protein coat of the

spermatophore) and nutrients (for sustaining the sperm while
they are in storage).   Sperm may live in the spermatheca for
weeks, months, or even years!
During ovulation, each egg passes across the opening to the
spermatheca and stimulates release of a few sperm onto the
egg's surface.   These sperm swim through the micropyle (a
special opening in the egg shell) and get inside the egg.  
Fertilization occurs as soon as one sperm's nucleus fuses
with the egg cell's nucleus.   Oviposition (egg laying) usually
follows closely after fertilization.   Once these processes are
complete, the egg is ready to begin embryonic development.

The Nervous System

An insect's nervous system is a network of specialized cells
(called neurons) that serve as an "information highway"
within the body.  These cells generate electrical impulses
(action potientials) that travel as waves of depolarization
along the cell's membrane.  Every neuron has a nerve cell
body (where the nucleus is found) and filament-like processes
(dendrites, axons, or collaterals) that propagate the action
potential.  Signal transmission is always unidirectional --
moving toward the nerve cell body along a dendrite or a
collateral and away from the nerve cell body along an axon.
Neurons are usually divided into three categories, depending
on their function within the nervous system:

1. Afferent (sensory) neurons -- these bipolar or multipolar

cells have dendrites that are associated with sense organs
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or receptors.  They always carry information toward the

central nervous system.
2. Efferent (motor) neurons -- unipolar cells that conduct
signals away from the central nervous system and
stimulate responses in muscles and glands.
3. Internuncial (association) neurons -- unipolar cells (often
with several collaterals and/or branching axons) that
conduct signals within the central nervous system.

Individual nerve cells connect with one another through

special junctions, called synapses.  When a nerve impulse
reaches the synapse, it releases a chemical messenger
(neurotransmitter substance) that diffuses across the
synapse and triggers a new impulse in the dendrite(s) of one
or more connecting neurons.  Acetylcholine, 5-
hydroxytryptamine, dopamine, and noradrenaline are
examples of neurotransmitters found in both vertebrate and
invertebrate nervous systems.
Nerve cells are typically found grouped in bundles.  A nerve
is simply a bundle of dendrites or axons that serve the same
part of the body.  A ganglion is a dense cluster of
interconnected neurons that process sensory information or
control motor outputs.
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The Central Nervous System

Like
most

Side view of body showing relative position of

circulatory (yellow), digestive (green), and nervous
(blue) systems.

other arthropods, insects have a relatively simple central

nervous system with a dorsal brain linked to a ventral nerve
cord that consists of paired segmental ganglia running along
the ventral midline of the thorax and abdomen.  Ganglia
within each segment are linked to one another by a short
medial nerve (commissure) and also joined by intersegmental
connectives to ganglia in adjacent body segments.  In general,
the central nervous system is rather ladder-like in
appearance:  commissures are the rungs of the ladder and
intersegmental connectives are the rails.  In more "advanced"
insect orders there is a tendency for individual ganglia to
combine (both laterally and longitudinally) into larger ganglia
that serve multiple body segments.
An insect's brain is a complex of six fused ganglia (three
pairs) located dorsally within the head capsule.  Each part of
the brain controls (innervates) a limited spectrum of activities
in the insect's body:
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 Protocerebrum:  The first pair of ganglia are largely

associated with vision; they innervate the compound eyes
and ocelli.
 Deutocerebrum:  The second pair of ganglia process
sensory information collected by the antennae.
 Tritocerebrum:  The third pair of ganglia innervate the
labrum and integrate sensory inputs from proto- and
deutocerebrums.  They also link the brain with the rest of
the ventral nerve cord and the stomodaeal nervous
system (see below) that controls the internal organs.  The
commissure for the tritocerebrum loops around the
digestive system, suggesting that these ganglia were
originally located behind the mouth and migrated forward
(around the esophagus) during evolution.

Located ventrally in the head capsule (just below the brain

and esophagus) is another complex of fused ganglia (jointly
called the subesophageal ganglion).  Embryologists believe
this structure contains neural elements from the three
primitive body segments that merged with the head to form
mouthparts.  In modern insects, the subesophageal ganglion
innervates not only mandibles, maxillae, and labium, but also
the hypopharynx, salivary glands, and neck muscles.  A pair
of circumesophageal connectives loop around the digestive
system to link the brain and subesophageal complex together.
In the thorax, three pairs of thoracic ganglia (sometimes
fused) control locomotion by innervating the legs and wings. 
Thoracic muscles and sensory receptors are also associated
with these ganglia.  Similarly, abdominal ganglia control
movements of abdominal muscles.  Spiracles in both the
thorax and abdomen are controlled by a pair of lateral nerves
that arise from each segmental ganglion (or by a median
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ventral nerve that branches to each side).  A pair of terminal

abdominal ganglia (usually fused to form a large caudal
ganglion) innervate the anus, internal and external genitalia,
and sensory receptors (such as cerci) located on the insect's
back end.
 

The Stomodaeal Nervous System

An insect's internal organs are largely innervated by a
stomodaeal (or stomatogastric) nervous system.  A pair of
frontal nerves arising near the base of the tritocerebrum link
the brain with a frontal ganglion (unpaired) on the anterior
wall of the esophagus.  This ganglion innervates the pharynx
and muscles associated with swallowing.  A recurrent nerve
along the anterio-dorsal surface of the foregut connects the
frontal ganglion with a hypocerebral ganglion that innervates
the heart, corpora cardiaca, and portions of the foregut. 
Gastric nerves arising from the hypocerebral ganglion run
posteriorly to ingluvial ganglia (paired) in the abdomen that
innervate the hind gut.
In comparison to vertebrates, an insect's nervous system is far
more de-centralized.  Most overt behavior (e.g. feeding,
locomotion, mating, etc.) is integrated and controlled by
segmental ganglia instead of the brain.  In some cases, the
brain may stimulate or inhibit activity in segmental ganglia
but these signals are not essential for survival.  Indeed, a
headless insect may survive for days or weeks (until it dies of
starvation or dehydration) as long as the neck is sealed to
prevent loss of blood!
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Insect Senses
All insects have sense organs that allow them to see, smell,
taste, hear, and touch their environment.   Since these are the
same five senses we humans experience, it is tempting to
conclude that insects see what we see, hear what we hear,
smell what we smell, etc.   But experimental evidence has
shown that an insect's sensory capabilities are very different
(both qualitatively and quantitatively) from those of humans
and other vertebrates.
All sense organs (receptors) act as transducers -- converting
light energy, chemical energy, or mechanical energy from the
environment into electrical energy of nerve impulses in
sensory neurons.   Signals generated by insect sensory
receptors travel to the brain or ventral nerve cord where they
stimulate appropriate behavioral responses:   finding
resources (e.g. food, mate, etc.), avoiding danger, or reacting
to changes in the environment.   All sensory receptors are
derived from embryonic ectoderm and are integral parts of
the insect's exoskeleton.   They can be grouped into one of
three categories, depending on function:
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Category Function Examples

Tactile receptors
Detect movements, vibrations, or other
echanoreceptors Proprioceptors
mechanical disturbances
Sound receptors

Taste buds on
Detect the presence of chemical substances in palps
hemoreceptors the air (smell) or on substrates (taste)
Antennal sensilla

Detect the presence and quality of incident Compound eyes

hotoreceptors light (electromagnetic radiation) Ocelli
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Mechanoreceptors

Insect mechanoreceptors can be found almost anywhere on

the surface of an insect's body.   They may act as tactile
receptors, detecting movement of objects in the environment,
or they may provide proprioceptive cues (sensory input
about the position or orientation of the body and its
appendages).   These receptors are innervated by one or more
sensory neurons that fire in response to stretching, bending,
compression, vibration, or other mechanical disturbance.  
Some mechanoreceptors produce a phasic response when
stimulated -- that is, they fire once when activated and again
when deactivated.   Other receptors generate a tonic
response, firing repeatedly as long as a stimulus persists.  
Neural processing centers in the brain or segmental ganglia
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interpret the combinations of tonic and phasic signals sent

from nearby receptors.
Trichoform sensilla are probably the simplest
mechanoreceptors.   These are tactile hairs (setae) that are
innervated by a sensory neuron.   Dendrites of the neuron
attach near the base of the hair and generate a nerve impulse
whenever they detect movement.   Hair beds (clusters of
tactile setae) are often found behind the head, on the legs, or
near joints where they respond to movements of the body.
Campaniform sensilla are flattened oval discs that usually
serve as flex receptors in the exoskeleton.   They respond
whenever mechanical stress causes the exoskeleton to bend.  
Campaniform sensilla are found throughout the body --
especially on the legs, near the base of the wings, and along
sutures where two sclerites of the exoskeleton meet.
Stretch receptors are multi-polar neurons that usually
accompany muscle or connective tissue.   They are
commonly embedded in intersegmental membranes and in
the muscular walls of the digestive system.   Some insects
may stop feeding when the gut expands enough to stimulate
stretch receptors in the crop.   In others, oviposition may
begin when mature eggs stimulate stretch receptors in the
reproductive system.
Pressure receptors provide sensory information about an
aquatic insect's depth in the water.   These receptors are
usually associated with a cushion of air against the body or
within the tracheal system.   Increasing water pressure
deflects hair-like processes within the receptor and stimulates
tonic and phasic impulses.
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Chordotonal organs include several types of

mechanoreceptors in which one or more bipolar neurons
bridge a gap between two internal surfaces of the
exoskeleton. Each neuron is usually accompanied by two
other cells which form a sheath (the scolopale cell) and a
point of attachment (the cap cell).   Together, these three cells
create a unit (called a scolopidia) that may occur singly or in
groups.   Common types of chordotonal organs include:
Subgenual organs -- located in the legs of many insects,
these receptors contain relatively few scolopidia yet they
appear to be very sensitive to substrate vibrations.   Insects
may lack specialized sound receptors, yet they can still "hear"
vibrations transmitted through the substrate.
Tympanal organs -- lie beneath a drum-like membrane (the
tympanum) where they respond to sound vibrations.   These
"ears" may be located on the thorax (in some Hemiptera), on
the abdomen (in grasshoppers, cicadas, and some moths), or
on the front tibia (in crickets and katydids).
Johnston's organs -- found within the pedicel of each
antenna.   In some insects, they function as a proprioceptors,
supplying information on position or orientation of the
antennae.   In mosquitoes and midges, they respond to certain
frequencies of airborne sound by detecting resonant
vibrations in antennal hairs.   (Shorter hairs near the tip of the
antennae resonate to higher frequencies than longer hairs near
the base).
 
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Chemoreceptors
Insects have the ability to sense various chemical substances
in their environment.   When these chemicals are present in
gaseous form (at relatively low concentrations), they may be
detected as odors (smells) by olfactory receptors.   When
they are in solid or liquid form (usually at higher
concentrations) they are perceived as tastes by gustatory
receptors.   In general, the sense of taste involves direct
contact with a substrate (contact chemoreception) whereas
olfaction usually implies detection of compounds in gaseous
or airborne form (remote chemoreception).
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Taste
Gustatory receptors are commonly described as
thick-walled hairs, pegs, or pits where the
dendrites of several (usually up to five) sensory
neurons are exposed to the environment through
a single opening (pore) in the cuticle.   Each
neuron appears to respond to a different range of
compounds (e.g. sugar, salt, water, protein, acid,
etc.).   Taste receptors are most abundant on the
mouthparts, but may also be found on the
antennae, tarsi, and genitalia (especially near the
tip of the female's ovipositor).

Smell
Olfactory receptors are usually thin-walled pegs,
cones, or plates with numerous pores through
which airborne molecules diffuse.   Dendrites of
sensory neurons branch profusely within these
pores and may respond to very low
concentrations of detectable compounds (e.g. sex
pheromones).   Some receptors respond to a
wide range of substances while others are highly
specific.   Olfactory receptors are most abundant
on the antennae, but may also be associated with
the mouthparts or external genitalia.
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Common chemical sense

High concentrations of irritant compounds (e.g. ammonia,
chlorine, acids, essential oils, etc.) simulate avoidance
reactions and cleaning behavior.   Insects can detect these
compounds even when all known chemoreceptors have been
covered or destroyed.   The irritants evidently trigger a
generalized response from other types of sensory neurons.

Photoreceptors
Compound Eyes

A pair of compound eyes are the

principle visual organs of most
insects; they are found in nearly all
adults and in many immatures of
ametabolous and hemimetabolous
orders.  As the name suggests,
compound eyes are composed of
many similar, closely-packed
facets (called ommatidia) which are the structural and
functional units of vision.  The number of ommatidia varies
considerably from species to species:  some worker ants have
fewer than six while some dragonflies may have more than
25,000.
Externally,each ommatidium is marked by a convex
thickening of transparent cuticle, the corneal lens.  Beneath
the lens, there is often a crystalline cone secreted by a pair of
semper cells.  Together, the lens and the crystalline cone form
 SCIENCE LABORATORY TECHNOLOGY

a dioptric apparatus that refracts incoming

light down into a receptor region containing
visual pigment.
The light-sensitive part of an ommatidium
is called the rhabdom.  It is a rod-like
structure, secreted by an array of 6-8
specialized neurons (retinula cells), and
centered on the optical axis just below the
crystalline cone.  The rhabdom contains an array of closely
packed microtubules where light-sensitive pigments (e.g.
rhodopsin, etc.) are stored.  These pigments absorb certain
wavelengths of incident light and generate nerve impulses
through a photochemical process similar to that of
vertebrates.
Most diurnal insects have pigment cells surrounding each
ommatidium.  These cells limit a facet's field of view by
absorbing light that enters through adjacent corneas.  Each
facet points toward a slightly different part of the visual
field.  In composite, they render a mosaic-like impression of
the environment.  Nocturnal and crepuscular insects have
pigment cells that do not completely isolate each facet.  Their
ommatidia are stimulated by light from larger fields of view. 
This produces a brighter but theoretically less distinct mosaic
image.
Since insects cannot form a true (i.e. focused) image of the
environment, their visual acuity is relatively poor compared
to that of vertebrates.  On the other hand, their ability to sense
movement, by tracking objects from ommatidium to
ommatidium, is superior to most other animals.  Temporal
resolution of flicker is as high as as 200 images/second in
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some bees and flies (in humans, still images blur into
constant motion at about 30 images/second).
Unlike humans, most insects can distinguish between
polarized light (coming directly from the sun) and
unpolarized light (reflected from water vapor and other
particles in the atmosphere).  This ability allows them to
detect the sun's position in the sky, even on cloudy or
overcast days, and use it as an orientation cue.
Compared to humans, insects have a range of spectral
sensitivity that is shifted toward shorter wavelengths (higher
frequencies).  Thus, insects can "see" light in the ultraviolet
range that is invisible to humans.  On the other hand, insects
cannot detect wavelengths at the red end of the spectrum that
are visible to humans.  True color vision, however, involves
more than just a wide range of spectral sensitivity.  Most
insects have only a limited ability to discriminate different
colors of light, but a few (especially bees and butterflies)
have "true" color vision.
 
 

Ocelli -- Simple eyes

Two types of "simple eyes" can be found in the class Insecta:

dorsal ocelli and lateral ocelli (=stemmata).   Although both
types of ocelli are similar in structure, they are believed to
have separate phylogenetic and embryological origins.
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Dorsal Ocellus Dorsal ocelli are commonly

found in adults and in the
immature stages (nymphs) of
many hemimetabolous
species.  They are not
independent visual organs
and never occur in species
that lack compound eyes. 
Whenever present, dorsal
ocelli appear as two or three
small, convex swellings on
the dorsal or facial regions of
the head.  They differ from
compound eyes in having
only a single corneal lens
covering an array of several dozen rhabdom-like sensory
rods.  These simple eyes do not form an image or perceive
objects in the environment, but they are sensitive to a wide
range of wavelengths, react to the polarization of light, and
respond quickly to changes in light intensity.  No exact
function has been clearly established, but many physiologists
believe they act as an "iris
mechanism" -- adjusting the Lateral Ocellus
sensitivity of the compound
eyes to different levels of
light intensity.
Lateral ocelli (=stemmata)
are the sole visual organs of
holometabolous larvae and
certain adults (e.g.
Collembola, Thysanura,
Siphonaptera, and
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Strepsiptera).  Stemmata always occur laterally on the head,

and vary in number from one to six on each side. 
Structurally, they are similar to dorsal ocelli but often have a
crystalline cone under the cornea and fewer sensory rods. 
Larvae use these simple eyes to sense light intensity, detect
outlines of nearby objects, and even track the movements of
predators or prey.  Covering several ocelli on each side of the
head seems to impair form vision, so the brain must be able
to construct a coarse mosaic of nearby objects from the visual
fields of adjacent ocelli.

Extra-ocular Photoreception
Some (perhaps most) insects respond to changes in light
intensity even when all known photoreceptive structures are
rendered inoperative.  This dermal light sense has been
attributed to the response of individual neurons in the brain
and/or ventral nerve cord.  There is also convincing evidence
that some insects can perceive infrared radiation (heat),
although specific receptors for this ability have not been
described.

The Endocrine System

A hormone is a chemical signal sent from cells in one part of
an organism to cells in another part (or parts) of the same
individual.   They are often regarded as chemical messengers.
Although typically produced in very small quantities,
hormones may cause profound changes in their target cells.  
Their effect may be stimulatory or inhibitory.   In some cases,
a single hormone may have multiple targets and cause
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different effects in each target.   There are at least four

categories of hormone-producing cells in an insect's body:

1. Endocrine glands -- secretory structures adapted

exclusively for producing hormones and releasing them
into the circulatory system.
2. Neurohemal organs -- similar to glands, but they store
their secretory product in a special chamber until
stimulated to release it by a signal from the nervous
system (or another hormone).
3. Neurosecretory cells -- specialized nerve cells (neurons)
that respond to stimulation by producing and secreting
specific chemical messengers.   Functionally, they serve as
a link between the nervous system and the endocrine
system
4. Internal organs -- hormone-producing cells are associated
with numerous organs of the body, including the ovaries
and testes, the fat body, and parts of the digestive system.

Together, these hormone-secreting structures form an

endocrine system that helps maintain homeostasis,
coordinate behavior, and regulate growth, development, and
other physiological activities.
In insects, the largest and most obvious endocrine glands are
found in the prothorax, just behind the head.   These
prothoracic glands manufacture ecdysteroids, a group of
closely-related steroid hormones (including ecdysone) that
stimulate synthesis of chitin and protein in epidermal cells
and trigger a cascade of physiological events that culminates
in molting.   For this reason, the ecdysteroids are often called
"molting hormones".   Once an insect reaches the adult stage,
 SCIENCE LABORATORY TECHNOLOGY

its prothoracic glands atrophy (wither away) and it will never

molt again.
Prothoracic glands produce and release ecdysteroids only
after they have been stimulated by another chemical
messenger, prothoracicotropic hormone (PTTH for short).  
This compound is a peptide hormone secreted by the corpora
cardiaca, a pair of neurohemal organs located on the walls of
the aorta just behind the brain.   The corpora cardiaca release
their store of PTTH only after they receive a signal from
neurosecretory cells in the brain.   In a sense, they act as
signal
amplifiers --
sending out a
big pulse of
hormone to
the body in
response to a
small
message
from the
brain.
The corpora allata, another pair of neurohemal organs, lie just
behind the corpora cardiaca.   They manufacture juvenile
hormone (JH for short), a compound that inhibits
development of adult characteristics during the immature
stages and promotes sexual maturity during the adult stage.  
Neurosecretory cells in the brain regulate activity of the
corpora allata -- stimulating them to produce JH during larval
or nymphal instars, inhibiting them during the transition to
adulthood, and reactivating them once the adult is ready for
reproduction.   The chemical structure of juvenile hormone is
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rather unusual:   it is a sesquiterpene compound -- more

similar to defensive chemicals found in pine trees than to any
other animal hormone.
The neurosecretory cells are found in clusters, both medially
and laterally in the insect's brain.   Axons from these cells can
be traced along tiny nerves that run to the corpora cardiaca
and corpora allata.   The cells produce and secrete brain
hormone, a low-molecular-weight peptide that appears to be
the same as (or very similar to) prothoracicotropic hormone
(PTTH) manufactured by the corpora cardiaca.   Insect
physiologists suspect that brain hormone is bound to a larger
carrier protein while it is inside the neurosecretory cell, and
some believe that each cluster of cells may produce as many
as three different brain hormones (or hormone-carrier
combinations).   Large numbers of neurosecretory cells also
occur in the ventral ganglia of the nerve cord, but their
function is unknown.
Many other tissues and organs of the body also produce
hormones.   Ovaries and testes, for example, produce gonadal
hormones that have been shown to coordinate courtship and
mating behaviors.   Ventral ganglia in the nervous system
produce one compound (eclosion hormone) that helps an
insect shed its old exoskeleton and another compound
(bursicon) that causes hardening and tanning of the new one.  
There are still other hormones that control the level of sugar
dissolved in the blood, adjust salt and water balance, and
regulate protein metabolism.
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Hormonal Control of Molting &

Metamorphosis
When an immature insect has grown sufficiently to require a
larger exoskeleton, sensory input from the body activates
certain neurosecretory cells in the brain.   These neurons
respond by secreting brain hormone which triggers the
corpora cardiaca to release their store of prothoracicotropic
hormone (PTTH) into the circulatory system.   This sudden
"pulse" of PTTH stimulates the prothoracic glands to
secrete molting hormone (ecdysteroids).

Molting hormone affects many cells throughout the body, but

its principle function is to stimulate a series of physiological
events (collectively known as apolysis) that lead to synthesis
of a new exoskeleton.   During this process, the new
exoskeleton forms as a soft, wrinkled layer underneath the
hard parts (exocuticle plus epicuticle) of the old exoskeleton.
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The duration of apolysis ranges from days to weeks,

depending on the species and its characteristic growth rate.  
Once new exoskeleton has formed, the insect is ready to shed
what's left of its old exoskeleton.   At this stage, the insect is
said to be pharate, meaning that the body is covered by two
layers of exoskeleton.
As long as ecdysteroid levels remain above a critical
threshold in the hemolymph, other endocrine structures
remain inactive (inhibited). But toward the end of apolysis,
ecdysteroid concentration falls, and neurosecretory cells in
the ventral ganglia begin secreting eclosion hormone. This
hormone triggers ecdysis, the physical process of shedding
the old exoskeleton. In addition, a rising concentration of
eclosion hormone stimulates other neurosecretory cells in the
ventral ganglia to secrete bursicon, a hormone that causes
hardening and darkening of the integument (tanning) due to
the formation of quinone cross-linkages in the exocuticle
(sclerotization).
In immature insects, juvenile hormone is secreted by the
corpora allata prior to each molt.   This hormone inhibits the
genes that promote development of adult characteristics (e.g.
wings, reproductive organs, and external genitalia), causing
the insect to remain "immature" (nymph or larva).   The
corpora allata become atrophied (shrink) during the last larval
or nymphal instar and stop producing juvenile hormone.  
This releases inhibition on development of adult structures
and causes the insect to molt into an adult (hemimetabolous)
or a pupa (holometabolous).
At the approach of sexual maturity in the adult stage, brain
neurosecretory cells release a brain hormone that
"reactivates" the corpora allata, stimulating renewed
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production of juvenile hormone.   In adult females, juvenile

hormone stimulates production of yolk for the eggs.   In adult
males, it stimulates the accessory glands to produce proteins
needed for seminal fluid and the case of the spermatophore.  
In the absence of normal juvenile hormone production, the
adult remains sexually sterile.

INSECT`S ECOLOGY
Insect ecology is the branch of entomology that focuses on
the interrelationships between insects and their
environment.  The term "environment" encompasses both the
abiotic world (non-living things like climate and geology) as
well as the biotic world (all living organisms including
plants, animals, microorganisms, etc.).  All of these
components interact within a framework called the biocenose
(a natural community).
Communities are groups of organisms (populations) that
maintain persistent associations with each other.  The
members of a typical community include plants, animals, and
other organisms that are biologically interdependent through
predation, parasitism, and symbiosis.  The structure of a
biotic community is largely characterized by the trophic
(feeding) relationships among its member species.  These
relationships are often represented simplistically as a food
chain.  Each link in the food chain represents a trophic level
encompassing either producers or consumers.
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In most communities, green plants are the dominant

producers.  They represent the first link in a typical food
chain.  Plants capture kinetic energy from sunlight and,
through the process of photosynthesis, manufacture organic
molecules (e.g. simple sugars) from carbon dioxide and
water.  The captured energy is "stored" in the chemical bonds
of these molecules.  Some of the stored energy is used by
plants for their own survival and growth, some is lost as heat,
and some passes on to consumers when the plant is eaten, or
to decomposers when the plant dies.
Primary consumers occupy the second link of a food chain. 
These animals, often called herbivores, survive by feeding
exclusively on plants or plant products.  The third link
includes primary carnivores, secondary consumers that live
as predators or parasites of herbivores.  Any remaining links
in the food chain are occupied by secondary or tertiary
carnivores (predators or parasites of other carnivores).  Since
energy becomes limiting at the uppermost trophic levels,
there are seldom more than four or five links in a terrestrial
food chain.
 
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Food Webs

Very few animals have a diet that is restricted to only a single

food source, so the concept of a linear food chain is
extremely simplistic.  In reality, trophic relationships within a
community are more like a food web in which dozens of
plant species support a wide variety of herbivores which in
turn are consumed by numerous predators and parasites.  If
one species within a food chain becomes scarce (perhaps due
to bad weather or over-exploitation), there will be serious
repercussions on all other species in the chain.  But in a
complex food web, changes in individual populations are
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likely to have a smaller impact because they are buffered by

the availability of an alternative prey or host species.

Insect Herbivores
Animals that feed on plant tissues or plant products are often
called herbivores.  This term applies not only to insects that
injure a plant by chewing leaves or sucking sap but also to
more benign species who only collect pollen, nectar, or plant
resins.  Entomologists frequently use the noun
"phytophagy" and the adjective "phytophagous" when
referring to any of these nutritional strategies.  Both words
are derived from Greek roots:  "phyton" meaning plant and
"phagein" the verb to eat or devour.
Phytophagous insects generally use visual or olfactory (odor)
cues to locate a host plant.  Visual cues may be as simple as
the vertical silhouette of a tree or the contrast of white
flowers against a dark background of foliage.  Some insects
are strongly attracted to certain shapes or colors which they
evidently associate with "food".  Red spheres, for example,
attract adult apple maggots, white pans of water attract
aphids, and bright yellow sticky traps attract leafhoppers. 
Odor cues are plant volatiles such as the saponins in alfalfa,
the mustard oils in crucifers, or the terpenes in conifers. 
Sometimes these attractants are primary plant compounds
such as sugars (e.g. glucose), nucleotides (e.g. adenine), or
amino acids (e.g. alanine) that a plant needs for its own
survival and growth.  But in other cases, the attractants are
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secondary plant compounds that have no nutritional value

to either the plant or the insect.  These substances may be
manufactured by the plant as a chemical defense against
herbivores but they unwittingly serve as token feeding
stimulants to a select group of specially adapted species. 
Milkweed plants, for example, produce cardenolides that
deter feeding by most phytophagous insects.  These
chemicals, however, attract monarch butterflies, oleander
aphids, milkweed beetles, and a few other species that have
the ability to digest or detoxify the compounds.
Insect herbivores often
have a cyclical pattern
of feeding behavior. 
After an initial phase of
attraction to the host
plant, appropriate tactile
(touch) and olfactory
(odor) cues trigger the
impulse to take a first
bite.  Additional
gustatory (taste) stimuli must be present in order for
continued feeding to occur.  After a bout of feeding is
complete, the insect may leave the host plant to engage in
other activites.
Since many plants conduct chemical warfare against insect
herbivores by manufacturing repellents or deterrents, it is
common for insects to be rather narrow and specialized in
their choice of host plant.  A monophagous insect restricts
itself to a single host species -- it is a consummate specialist,
adapting its behavior and physiology to a single nutritional
resource.  Some of these insects must rely on intestinal
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symbionts to supply essential dietary components that are not

supplied by their host (see Insect Nutrition).  Oligophagous
insects have a slightly broader host range -- often adopting
any plant within a close circle of related genera or the
members of a single taxonomic family.  These insects are less
likely to starve if a preferred host plant is unavailable.  A few
insects are polyphagous.  These species are equipped with
"broad-spectrum" detoxification enzymes that can overcome
a wide range of plant defenses.  It can be metabolically
"expensive" to produce these enzymes, but on the other hand,
there is no shortage of available food!
Some of the more polyphagous insects (like grasshoppers and
armyworms) will consume every part of their host plant.  But
most insect herbivores are more selective:  they specialize as
leaf chewers, sap suckers, stem borers, root pruners, gall
makers, leaf miners, collectors of pollen or nectar, etc.  Each
of these feeding strategies represents a separate ecological
niche and all of the species that feed on the same plant in the
same way are known as members of a feeding guild.  Within
a feeding guild, all species compete directly with each other
for exactly the same resource.  Between members of different
guilds, competition is usually less direct and less severe.  As
a result, there is strong selective pressure limiting the number
of species within each guild.  Direct competitors usually are
not closely related to each other (phylogenetically) and their
association tends to be relatively recent in origin and short-
lived in duration compared to more symbiotic (mutualistic)
interactions.  Natural selection tends to favor adaptations that
minimize competition between species within a feeding guild.
Herbivory has had both positive and negative impacts on
plants over evolutionary time.  Flowering plants (the
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Angiosperms) have certainly benefited by attracting insect

herbivores and exploiting them as pollinators.  These plants
often provide nectar (or other nutritional "rewards") to their
insect accomplices.  Bright colors, distinctive odors,
geometrical patterns, and in some cases even subterfuge are
tactics used by plants to attract specific pollinators and
maintain their interest from blossom to blossom (pollinator
fidelity).  On the other hand, insects are also vectors of plant
diseases.  Aphids and leafhoppers (Hemiptera: Homoptera)
are notorious for spreading plant viruses and mycoplasmas as
they feed.  Bark beetles (Coleoptera) invade the woody
tissues of living trees, inoculating them with fungal
pathogens that weaken and eventually kill the tree.  Bacteria,
protozoa, and nematode pathogens are also carried from plant
to plant by insect herbivores.  Pathogens may be carried
externally on an insect's feet, mouthparts, or ovipositors, or
internally in the salivary glands, digestive tract, or
reproductive system.  Some plant diseases like fire blight (a
bacterium) and mummy berry (a fungus) are collected and
spread by insect pollinators that are attracted to sticky-sweet
exudates produced by the infected plants.

Insect Carnivores

Carnivores eat meat!   Some insect carnivores catch and kill

other insects (or non-insect arthropods) as food, some
parasitize the bodies of other animals, and some feed by
sucking blood.   Zoophagy is a term for all these feeding
strategies.   It is derived from the Greek words "zoion"
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meaning animal and "phagein" the verb to eat or devour.

Predators are zoophagous insects that kill and eat numerous
prey individuals in the course of their growth and
development.   They are generally larger than their prey and
must often immobilize or overpower it before feeding.  

Insect Decomposers
The dead bodies of plants and animals are a rich source of
organic matter that provides nutrition for many insects called
saprophages (from the Greek words "sapros" meaning rotten
and "phagein" the verb to eat or devour.  Insects adapted to
this lifestyle are an essential part of the biosphere because
they help recycle dead organic matter.
Within the ranks of saprophagous insects, entomologists
recognize several major groups:

 those that feed on dead or dying plant tissues

 those that feed on dead animals (carrion), and
 those that feed on the excrement (feces) of other animals.

The dead plant feeders include a wide variety of soil- and

wood-dwelling species that shred leaves or tunnel in woody
tissues.  They accelerate decay by increasing the surface area
exposed to weathering and the action of other decomposers. 
They are largely responsible for creating a layer of humus
that often covers the soil.  This layer serves as an incubator
for the fungi, bacteria, and other microorganisms that release
carbon, nitrogen, and mineral elements for uptake by living
plants.  Carrion feeders include numerous beetles, fly larvae
(maggots), wasps, ants, mites, and others.  Each species
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colonizes the dead body for only a limited period of time but,
as a group, they rapidly consume and/or bury the decaying
flesh.  Blow flies, usually the first to arrive on a carcass, are
also the first to complete development and depart.  Other
species follow over time in a relatively predictable sequence
as the body decomposes.  This change in the species
composition of saprophages is called faunal succession.  It
provides a reliable way to determine the time elapsed since
death and has become a useful tool for police, medical
examiners, and other practicioners of forensic entomology.

Many species of manure flies and dung beetles are attracted

to the odor of animal excrement.  Adults lay their eggs on
fresh feces and larvae feed on the organic matter in these
waste products.  Many dung-feeders exhibit distinct
preferences for particular types of manure:  the species
associated with horse manure, for example, may be quite
different from those found on the same farm in cattle
manure.  One group of dung beetles, called tumblebugs,
form the excrement into a small ball and roll it into a hole
that was previously dug in the soil.  They lay an egg on the
ball of dung and cover it with soil to serve as a nursery for
their larvae.

In addition to their role as decomposers, some saprophagic

insects also serve as pollinators for plants like skunk cabbage
and wild ginger.  These plants produce drab colored, foul
smelling flowers that attract the attention of blow flies or
carrion beetles.  The insects crawl around in the flowers
looking for food and unwittingly pick up pollen.  Finding
nothing to eat, the insects leave and continue to forage for
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food, perhaps visiting another blossom and transferring

pollen.

Survival Strategies
Despite their small size and apparent vulnerability, insects are
not entirely at the mercy of the elements.  They are equipped
with high reproductive rates and numerous behavioral and
physiological adaptations that assure them a fair fight in the
struggle for survival.  The following sections describe a
number of common adaptations that help insects survive
adversity or adapt to their environment.

Migration
Day-to-day activities of insects often involve trivial
movements associated with feeding, mating, or oviposition. 
These movements are "trivial" only in the sense that they are
short-range, random in direction, and do not result in much
dispersal of the population.  At some time in their life cycle,
however, many (perhaps most) of these insects will engage in
a period of directional movement that carries them beyond
the range of their local habitat.  This long-distance
movement, called migration, is a survival strategy with at
least six potential advantages:

 escape from natural enemies

 find more favorable growing conditions
 reduce competition or relieve overcrowding
 locate new (or unoccupied) habitats
 disperse to alternate host plants
 reassort the gene pool to minimize inbreeding
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Although most insects migrate by flying, a few species

(chinch bugs, Mormon crickets, and armyworms, for
example) travel on the ground.  Migration by flight is often
aided by prevailing winds.  Once airborne, small insects may
be lifted by thermal convection and carried hundreds of miles
on frontal air masses.  Even wingless individuals may be
carried aloft by "ballooning" on silk threads or blowing off
tall vegetation.  Larger insects, like dragonflies and monarch
butterflies, may control the direction of their migratory
flights, but most smaller insects are carried wherever the
wind blows them.
Migration is usually a distinct phase in the life cycle, almost
always occurring before the onset of reproductive maturity. 
Migrants are innately programmed to move; they are not
distracted by food or mates.  Once the migratory urge is
satisfied, the insect is generally in a physiological state to
continue development or commence reproduction.  Migration
can be a very risky venture:  in some species more than 90%
of a population may die in transit.  Despite such high
mortality rates, the reproductive success of individuals who
survive the trek apparently makes the gamble worthwhile for
the species as a whole.

Diapause
The life cycle of many insect species may include a hormone-
induced period of "dormancy" called diapause.  Like
hibernation in mammals, diapause is characterized by a
reduction in oxygen consumption, metabolic rate, and
physical activity.  Feeding and growth are generally
interrupted while the individual subsists on stored food
reserves.  Diapause typically occurs during the egg stage in
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some species, during a nymphal or larval instar in other

species, or during the pupal stage in still other species.  Even
adults may enter a "reproductive diapause" which causes a
significant delay in the onset of sexual maturity. 
In temperate climates, many species enter diapause in the fall
as an overwintering adaptation.  Other species, however, have
a summer diapause that helps them survive the dryness and/or
heat.  In either case, the onset of diapause is triggered by an
environmental cue that precedes the adverse weather
conditions (short daylengths in fall, for example).  Diapause
continues, even under apparently favorable conditions, until it
is "broken" (terminated) by other environmental cues, such as
long day lengths or exposure to a substantial period of low
temperature. 
Diapause is not always correlated with adverse environmental
conditions.  It can also regulate development within a
population to ensure optimal timing of emergence or
temporal synchrony with environmental resources.  Female
rabbit fleas, for example, have an obligate adult diapause that
is broken only by feeding on the blood of a pregnant host
rabbit.  By the time the baby rabbits are old enough to be
weaned, the flea's offspring will be mature and ready to
accompany the rabbits when they leave the nest.  In this
ecological relationship, diapause is an adaptation that keeps
the flea population from exceeding the carrying capacity of
its host.

Cold-hardiness
Since insects are poikilotherms (cold-blooded animals), their
body temperature is usually similar to that of the air (or
water) around them.  Species that live in cold mountain
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streams (like mayfly naiads) or on the surface of ice and

snow (like grylloblattids) are adapted for activity at low
temperatures.  Most other insects, however, slow down as the
temperature falls. 
They reach a dormant state, called torpor or quiescence,
when they get very cold.  Physiologically, many insects
prepare for winter weather by producing "antifreeze"
compounds (such as glycerol, sorbitol, or trehalose) in their
hemolymph and body tissues.  High concentrations of these
compounds can increase cold-tolerance by lowering the
freezing point of body fluids and preventing the formation of
ice crystals that would cause internal injury.  In species that
manage to survive in arctic and alpine environments, the
overwintering stage may undergo extensive dehydration --
any ice crystals that do form will be too small to cause
cellular damage.  During the long Antarctic winter, larvae of
Belgica antarctica, a wingless midge, become literally frozen
in place until the arrival of a spring thaw.
Unlike diapause (see above), a period of quiescence lasts only
as long as the weather is cold.  When temperatures rise,
quiescent insects resume normal activity -- at least until the
next cold front arrives!  This explains why there may be a
great deal of insect activity on warm, sunny days in the
middle of winter.

Parthenogenesis
Sexual reproduction involves the union of a female's egg (1n)
with a male's sperm (1n) to form a diploid zygote (2n). 
Although sexual reproduction is the paradigm in most insects,
there are many common species that are able to reproduce
asexually (without mating), through a process known as
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parthenogenesis.  In these species, females are able to

produce viable offspring without a contribution of sperm
from a male. 
One type of parthenogenesis (called arrhenotoky) occurs in
all members of the order Hymenoptera (ants, bees, and
wasps) and also in a few species of thrips and scale insects. 
In these insects, all females are diploid (2n) and all males are
haploid (1n).  Mated females have voluntary control over the
release of stored sperm so they can opt to lay either a
fertilized egg (female) or an unfertilized egg (male).  This
adaptation, which allows a female to regulate the sex of her
offspring, is undoubtedly an important factor in the evolution
of colony structure for social ants, bees, and wasps.
Another type of parthenogenesis (called thelytoky) is found
in many aphids, scale insects, some cockroaches and stick
insects, and a few weevils.  In these insects, females produce
diploid (2n) eggs that develop directly into female offspring
having exactly the same genetic make-up as their mother. 
Since each daughter subsequently has the ability to clone
herself through parthenogenesis, this form of reproduction
has the potential to produce a large number of offspring in a
short period of time.  Parthenogenesis is clearly an advantage
for species that live in a stable environment and exploit an
abundant food resource.  The lack of genetic variability
within the population, however, can be a disadvantage if
(when) environmental conditions change.  For this reason,
many parthenogenetic species have evolved a seasonal cycle
that includes at least one generation of sexual reproduction at
the end of several generations of asexual reproduction.
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Polymorphism
Just as each human has a unique physical appearance, there
are also individual differences among the members of a
single species in the insect world.  Females are often larger
than males, and one sex may have distinctive colors or
markings to attract the opposite sex.  But there are also many
examples of species with two or more colors, shapes, or sizes
where the differences are competely unrelated to gender
characteristics.  Each of these "versions" is called a morph,
and therefore, species that exhibit this "split-personality" are
said to be polymorphic.
In social insects, polymorphism is often associated with
division of labor in the nest.  Among ants, for example, large
individuals with big mandibles usually serve as soldiers or
foragers, while smaller individuals concentrate on care of the
young or other housekeeping tasks.  In honey bees, the
workers have wax glands, stings, and pollen baskets that are
not present in queens or drones.  This type of specialization
among individuals is an adaptation that gives social insects
the ability to utilize their resources more efficiently.
In non-social species, polymorphism may be related to
habitat diversity.  England's peppered moth, Biston betularia,
is a well-known example of such a species.  The light-colored
morph of this moth is hard to find in the daytime when it rests
against a background of lichens growing on the bark of trees. 
A dark-colored morph is easy to see against the lichen, but
hard to spot against the dark background of bare bark. 
Depending on the background, the less-visible morph is the
one most likely to survive bird predation.  During the
industrial revolution, the dark morph predominated because
air pollution from London's factories killed much of the
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lichen on trees in surrounding forests.  Now that air pollution

has been reduced, the lichen are able to survive and the
moth's light-colored morph is most abundant.
In Africa, the desert locust, Schistocerca gregaria, has two
morphs that differ both in physical appearance and behavior. 
Under low population densities, these grasshoppers develop
into adults that are largely green in color, have relatively
short wings, and show little or no tendency to migrate.  Under
crowded conditions, however, these grasshoppers develop
into brownish adults with longer wings.  These individuals
eventually form huge swarms containing millions of
individuals that migrate over hundreds of miles.  Crowding
affects the balance of neurotransmitters, especially serotonin,
and triggers a shift in development to the migratory form.
Different morphs may also be associated with different
generations throughout the year.  The seasonal cycle of many
parthenogenetic aphids includes several generations of
wingless (apterous) individuals followed by a generation of
winged (alate) migrants.  This alternation of generations
provides a mechanism for dispersal from one habitat to
another as environmental conditions and host plant quality
change throughout the year.  The rosy apple aphid, Anuraphis
rosea, is typical of many such species.  In early spring it
reproduces asexually, completing several wingless
generations on apple trees, its primary host.  As the apple
foliage matures and becomes less desirable, an alate
generation develops (partheogenetically) and flies to narrow-
leaved plantain, the secondary host.  Several more wingless
generations develop asexually on plantain until, in late
summer or early fall, another alate generation develops and
flies back to the primary host.  This generation gives birth to
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a sexual generation (both males and females) that will mate

and lay overwintering eggs on the apple trees.

Insect Defenses
For many insects, a quick escape by running or flying is the
primary mode of defense.  A cockroach, for example, has
mechanoreceptive hairs (setae) on the cerci that are sensitive
enough to detect the change in air pressure that precedes a
fast moving object (like your foot).  Nerve impulses from
these receptors travel through giant neurons to thoracic
ganglia at speeds up to 3 meters per second, triggering an
evasive response by the legs in less than 50 milliseconds. 
House flies have a similar reaction time when you try to swat
them.  They leap into the air and begin flapping their wings
30-50 milliseconds after sensing a threat.
Tiger moths (family Arctiidae) can detect ultrasonic
echolocation by bats.  At low intensity, they fly away from
the bat, but if the bat's call increases to a certain threshold
they quickly drop from the air in an evasive, looping dive. 
Other alarm reactions may be less dramatic, but just as
effective:  Madagascar cockroaches hiss when disturbed;
cuckoo wasps curl up into hard, rigid balls; tortoise beetles
have strong adhesive pads on their tarsi and hold themselves
tight and flat against a leaf or stem.  Other insects simply
"play dead" (thanatosis) -- they release their grip on the
substrate and fall to the ground where they are hard to find as
long as they remain motionless.
An insect's hard exoskeleton may serve as an effective
defense against some predators and parasites.  Large weevils
are notorious for their hard bodies - as you may discover for
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yourself the first time you bend an insect pin trying to push it
through the thorax.  Most diving beetles are hard, slick, and
streamlined; even if you can catch them, they will often
squirm out of your grip.
Spines, bristles, and hairs may be effective mechanical
deterrents against predators and parasites.  A mouthful of hair
can be an unpleasant experience for a predator and parasitic
flies or wasps may have a hard time getting close enough to
the insect's body to lay their eggs.  Some caterpillars
incorporate body hairs into the silk of their cocoon as an
additional defense against predation.
Some insects have a "fracture line" in each appendage (often
between the trochanter and the femur) that allows a leg to
break off easily if it is caught in the grasp of a predator.  This
phenomenon, called autotomy, is most common in crane
flies, walkingsticks, grasshoppers, and other long-legged
insects.  In most cases, sacrificing a limb in this manner
creates only a minor disability.  In fact, walkingsticks
(especially young nymphs) may regenerate all or part of a
missing appendage over the course of several molts.

Chemical Defenses
Many insects are equipped to wage chemical warfare against
their enemies.  In some cases, they manufacture their own
toxic or distasteful compounds.  In other cases, the chemicals
are acquired from host plants and sequestered in the
hemolymph or body tissues.  When threatened or disturbed,
the noxious compounds may be released onto the surface of
the body as a glandular ooze, into the air as a repellent
volatile, or aimed as a spray directly at the offending target.
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Defensive chemicals typically work in one of four ways:

1. Repellency -- a foul smell or a bad taste is often enough to

discourage a potential predator.  Stink bugs, for example,
have specialized exocrine glands located in the thorax or
abdomen that produce foul-smelling hydrocarbons.  These
chemicals accumulate in a small reservoir adjacent to the
gland and are released onto the body surface only as
needed.  The larvae of certain swallowtail butterflies have
eversible glands, called osmeteria, located just behind the
head.  When a caterpillar is disturbed, it rears up, everts
the osmeteria to release a repellent volatile, and waves its
body back and forth to ward off intruders.
2. Induce cleaning -- irritant compounds often induce
cleaning behavior by a predator, giving the prey time to
escape.  Some blister beetles (family Meloidae) produce
cantharidin, a strong irritant and blistering agent that
circulates in their hemolymph.  Droplets of this blood ooze
from the beetle's leg joints when it is disturbed or
threatened -- an adaptation known as reflex bleeding. 
Irritant sprays are produced by some termites,
cockroaches, earwigs, stick insects, and beetles.  The
notorious bombardier beetles store chemical precursors
for an explosive reaction mixture in specialized glands. 
When threatened, these precursors are mixed together to
produce a forceful discharge of boiling hot benzoquinone
and water vapor (steam).
3. Adhesion -- sticky compounds that harden like glue to
incapacitate an attacker.  Several species of cockroach
guard their backsides with a slimy anal secretion that
quickly cripples any worker ants that launch an attack. 
Similarly, members of the soldier caste in nasute termites
have nozzle-like heads equipped with a defensive gland
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that can shoot a cocktail of defensive chemicals at

intruders.  The compounds, which are both irritating and
immobilizing, have been shown to be highly effective
against ants, spiders, centipedes, and other predatory
arthropods.
4. Cause pain or discomfort -- Saddleback caterpillars, larvae
of the io moth, and various other Lepidopteran larvae
have hollow body hairs that contain a painful irritant. 
Simply brushing against these urticating hairs will cause
them to break and release their contents onto your skin. 
The consequence is an intense burning sensation that may
last for several hours.  Many ants, bees, and wasps (the
aculeate Hymenoptera) deliver venom to their enemies by
means of a formidable stinger (modified ovipositor).  The
venom is a complex mixture of proteins and amino acids
that not only induces intense pain but may also trigger an
allergic reaction in the victim.

Protective Coloration
Biologists recognize that there is usually an underlying
rationale for the great diversity of shapes and colors found in
the insect world.  We may not know why a particular species
has parallel ridges on the pronotum or black spots on the
wings, but we can be reasonably certain that this shape or
color has contributed in some way, however small, to the
overall fitness of the species.  It is obvious that at least some
of the colors and patterns serve a defensive function by
offering a degree of protection from predators and parasites. 
These patterns, collectively known as protective coloration,
fall into four broad categories:

1. Crypsis
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Insects that blend in with their surroundings often manage to

escape detection by predators and parasites.  This tactic,
called cryptic coloration, involves not only matching the
colors of the background but also disrupting the outline of the
body, eliminating reflective highlights from smooth body
surfaces, and avoiding sudden movements that might betray
location.  Obviously, this tactic loses much of its
effectiveness if an insect moves from one type of habitat to
another.  Well-camouflaged insects usually stay close to
home or make only short trips and return quickly to the
shelter of their protective cover.  Many ground-dwelling
grasshoppers and katydids, for example, have colors of
mottled gray and brown that help them "disappear" against a
background of dried leaves or gravel.  On the other hand,
closely related species that live in foliage are usually a shade
of green that matches the surrounding leaves.  The larvae of
some lacewings improve their camouflage by attaching bits
of moss or lichen from their environment onto the dorsal side
of their body.

2. Mimesis

Some insects "hide in plain sight" by resembling other

objects in the environment.  A thorn could really be a
treehopper; a small twig might be a walkingstick, an assassin
bug, or the caterpillar of a geometrid moth; and sometimes a
dead leaf turns out to be a katydid, a moth, or even a
butterfly.  This "mimicry" of natural objects is often known
as mimesis.  It goes far beyond imitation of plant parts:
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o Some swallowtail larvae resemble bird droppings,

others have false eyespots on the thorax that create a
convincing imitation of a snake's head.
o The likeness of a caterpillar can be found on the
outer edge of many lepidopteran wings, perhaps serving
to fool predatory birds that may peck at the wing margin
instead of the butterfly's body.
o Many butterflies and moths have eyespots on the
wings that emulate the face of an owl or some other large
animal.
o Slug caterpillars and hag moth larvae look like hair
balls or small furry mammals.
3. Warning Colors

Insects that have an active means of defense (like a sting or a

repellent spray) frequently display bright colors or
contrasting patterns that tend to attract attention.  These
visually conspicuous insects illustrate aposematic
coloration, a term derived from the Greek words apo- (from
a distance) and sema (a sign or signal) -- meaning "a signal
from afar".  A predator quickly learns to associate the
distinctive coloration with an "unpleasant" outcome, and one
such encounter is usually enough to insure avoidance of that
prey in the future.  A few individuals will die as sacrifices,
but for the species as a whole, it pays to advertise!

4. Mimicry

If a distinctive visual appearance is sufficient to protect an

unpalatable insect from predation, then it stands to reason that
other insects might also avoid predation by adopting a similar
appearance.  This ploy, essentially a form of "false
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advertising", was first recognized and described by Henry W.

Bates in 1861.  Today, it is commonly known as Batesian
mimicry.  Viceroy butterflies (mostly palatable to birds) are
largely protected from predation because they resemble
monarch butterflies (very distasteful).  Many species of bee
flies, flower flies, robber flies, and clear-winged moths are
similarly protected because they mimic the appearance (and
often the behavior) of stinging bees and wasps.  Batesian
mimicry is usually a successful strategy as long as the model
and mimic are found in the same location, the mimic's
population size is smaller than that of the model, and
predators associate the model's appearance with an
unpleasant effect.
In 1879, Fritz Müller recognized that two or more distasteful
species often share the same aposematic color patterns. 
Many species of wasps, for example, have alternating bands
of black and yellow on the abdomen.  This defensive tactic,
commonly known as Müllerian mimicry, benefits all
members of the group because it spreads the liability for
"educating the predator" over more than one species.  In fact,
as the number of species in a Müllerian complex increases,
there is a greater selective advantage for each individual
species.

5. Mimicry has been carried to extremes in some tropical

Lepidoptera where both related and unrelated species
resemble each other in size, shape, color, and wing pattern. 
Collectively, these butterflies (and sometimes moths) form
mimicry rings that may include both palatable and
unpalatable species.  In South America, for example,
longwing butterflies (Family Nymphalidae) form a mimicry
 SCIENCE LABORATORY TECHNOLOGY

ring that includes at least twelve different species (including

one moth).

Although natural selection favors individuals in a population

with the best camouflage or mimicry, it also favors the
predator or parasite with the best prey-finding acumen.  As a
result of these competing interests, coevolution between
predator and prey populations inevitably leads to an ongoing
escalation of offensive and defensive measures -- a scenario
that Leigh Van Valen of Chicago University describes as an
evolutionary "arms race". 
In order to survive in the arms race, both predator and prey
must constantly evolve in response to the other's changes. 
Failure to "keep up" concedes a competitive advantage to the
opponent and may lead to extinction. 
The idea that perpetual change is necessary just to maintain
the status quo has been coined the Red Queen's Hypothesis. 
This name refers to a scene from the stories of Alice in
Wonderland by Lewis Carroll.  In Through the Looking
Glass, Alice meets a chess piece, the Red Queen.  After
running hard to follow the Queen, Alice discovers that she
has not moved from where she started.  Asked about this
paradox, the Red Queen replies, "Here, you see, it takes all
the running you can do to keep in the same place."

Population Dynamics
A population is a group of individuals (all members of a
single species) who live together in the same habitat and are
 SCIENCE LABORATORY TECHNOLOGY

likely to interbreed.  Each population has a unique physical

distribution in time and space.  It may contain individuals of
different ages and its size (density) is likely to change over
time, growing or shrinking according to the reproductive
success of its members.  The study of population dynamics
focuses on these changes -- how, when, and why they occur. 
In entomology, a good understanding of population dynamics
is useful for interpreting survey data, predicting pest
outbreaks, and evaluating the effectiveness of control tactics.
Birth (natality), death (mortality), immigration, and
emigration are the four primary ecological events that
influence the size (density) of a population.  This relationship
can be expressed in a simple equation:

Change in
=   (Births + Immigration) - (Deaths +
Population
Emigration)
Density

All other factors (both biotic and abiotic) exert their impact
on population density by influencing one (or more) of the
variables on the right-hand side of the above equation.  Such
factors, known as secondary ecological events, may affect
the frequency, extent, magnitude, or duration of a primary
ecological event.  Cold winter temperatures, for example,
could increase mortality and reduce population density.  On
the other hand, low predation rates in the summer might
increase natality and allow the population to grow.  Most
secondary ecological events act as "population regulating
factors".  Whenever they limit a population from reaching its
maximum reproductive potential, they are regarded as
"environmental resistance".
 SCIENCE LABORATORY TECHNOLOGY

Secondary ecological events can be divided into two broad

categories:  density-independent factors and density-
dependent factors.

 Density-Independent Factors include events or

conditions, often weather- or climate-related, that affect
all individuals equally, regardless of the overall population
density.  A hard freeze, for example, will kill the same high
percentage of the potato leafhoppers in a farmer's peanut
field -- no matter if the population contains a few hundred
or a few million individuals.  In another species, high
temperatures and/or low humidity might have a similar,
non-selective impact on mortality.  Favorable climatic
conditions can have a positive effect on population
density just as much as unfavorable conditions can have a
negative effect.  Larvae of Japanese beetles, for example,
thrive in years when ample summer rainfall keeps soil
conditions moist.  Other density-independent events
might include wildfires, hurricanes, or hail storms.  For an
aquatic species, a low concentration of dissolved oxygen
or a flash flood after heavy rainfall would qualify as
density-independent events because a small population
would suffer the same percent mortality as a large
population.
 Density-Dependent Factors include events or conditions
that change in severity as a population's size increases or
decreases.  Common examples of density-dependent
factors include predation, parasitism, and disease (one
species exploiting another).  A large, dense population, for
example, is usually more susceptible to the spread of
parasites or contagious disease than a small, sparse
population.  Predators often adapt to changes in the
density of their prey populations by migrating into areas
 SCIENCE LABORATORY TECHNOLOGY

of high prey density (numerical response) or by focusing

their attention primarily on the most abundant prey
species (behavioral response).  As a result, large and small
populations tend to suffer different rates of predation. 
Competition for limited resources is also density-
dependent -- each individual's share of the "pie"
decreases as a population grows numerically.  In a small
population, members may face competition mostly from
individuals of other species who use the same resources
(interspecific competition).  In large populations,
however, competition may also come from other
members of the same species (intraspecific competition). 
In either case, competition undermines survival and
reproduction.  Any physical trait or behavioral adaptation
that reduces or eliminates competition is likely to be
favored by natural selection.

    Intraspecific     Interspecific
Competition     Competition    
Contest Competition:   In Each species occupies a
situations where resources unique ecological niche
(food, space, etc.) are fairly within its community.   The
stable over time, intraspecific niche is a Gestalt-like concept
competition may take the encompassing all of the biotic
form of "contests" in which and abiotic parameters that
individuals lay claim to a determine where a population
"territory" and defend it from lives (its "habitat") as well as
all intruders.   Each territory the role it plays within the
generally provides enough food web (its "profession").  
resources for the owner's Interspecific competition
survival and reproduction; occurs whenever the niche
 SCIENCE LABORATORY TECHNOLOGY

failure to "win" a territory can parameters of two (or more)

be a competitive different species overlap.  
disadvantage.   Since only the The more the overlap, the
strongest (most "fit") greater the competition.
individuals are likely to hold a Interspecific competition
territory, they have the best usually leads to one of three
chance to pass on their genes possible evolutionary
to the next generation. outcomes:
Scramble Competition:   In
situations where resources are 1. Competitive
temporary or transient, there is exclusion -- one
little or no advantage to species is
defending a territory.   Insects competitively
that compete for these types of superior and drives
resources (blow flies on a
the other species to
corpse, for example)
extinction.
"scramble" for access.   The
first arrivals encounter the 2. Range restriction --
best conditions for survival each species is
and reproduction (first come, confined to a subset
first served).   Latecomers of the range where it
encounter a depleted resource is able to out-
that may no longer support compete the other
growth and development. species.
3. Competitive
displacement -- the
two species evolve in
divergent directions,
adapting to different
resources or
specializing in other
ways that allow them
to co-exist with little
or no direct
 SCIENCE LABORATORY TECHNOLOGY

competition.

Density-dependent emigration (movement away from

crowded conditions) is another important regulator of
population size.  It not only reduces overcrowding in the
home range, but it also increases the likelihood of
establishing new populations elsewhere.  In the long term,
emigration benefits the individuals who remain behind as
well as the pioneers who find new places to live.
Cooperative interactions may also give populations a
competitive advantage, allowing them to reduce mortality,
use resources more efficiently, or accomplish tasks that could
not be performed by solitary individuals.  Intraspecific
cooperation has certainly contributed to the evolutionary
success of all social insects (ants, bees, wasps, and termites). 
These species outnumber all other animals in many terrestrial
habitats and, despite their small size, they usually play
dominant roles in community ecology, both as consumers and
as decomposers.  Cooperative interactions between different
species (i.e. mutualism and commensalism) are also common
in the insect world.  These symbiotic relationships occur not
only between different insect species (e.g. ants and aphids)
but also between insects and microorganisms, between
insects and vertebrates, and between insects and plants.
 

Population Growth
When food is abundant and growing conditions are favorable,
a population has the potential to increase in number from
 SCIENCE LABORATORY TECHNOLOGY

generation to generation -- just as money in a bank savings

account accrues interest over time.  The population's intrinsic
growth rate ("r") is similar to the bank account's interest rate
-- it is a measure of how quickly the increase occurs.  Growth
is said to be geometric when each generation's increase is a
constant percentage of the total population size.  Geometric
growth is also known as exponential growth because the

larger the population gets, the faster it grows.  With a 5%

growth rate, for example, a population of 50 beetles would
grow by only 31 individuals in 10 generations whereas a
population of 10,000 would grow by 6,289 during the same
amount of time.  The "J-shaped" curve in Figure 1 represents
the typical form of an exponential growth curve.
Obviously, exponential growth cannot continue indefinitely
in a resource-limited environment.  Eventually a population
 SCIENCE LABORATORY TECHNOLOGY

becomes so large that it runs out of free space, outgrows its

food supply, or exhausts other assets.  The upper limit on
population density is called the environmental carrying
capacity   (usually represented by the symbol "K").  As
population density approaches the carrying capacity,
competition becomes more intense, mortality increases, the
birth rate drops, and any one of the following alternatives is
possible:

 The population may level out and stabilize below the

carrying capacity.  This pattern is known as a logistic or
sigmoid (S-shape) growth curve.
 The population may briefly overshoot the carrying
capacity and then crash, resulting in repeated cycles of
"boom" and "bust".
 The population may oscillate around (or below) the
carrying capacity.

In reality, all of these models are gross over-simplifications. 

Natural populations respond to a wide range of environmental
conditions that are rarely constant over time.  Some species
have complex life cycles requiring different resources or
conditions at each stage of development.  Others show
distinct temporal or seasonal variability in their response to
environmental conditions.
r- and K-selection.   Some insects are ecological
opportunists.  They exploit disturbed or unstable
environments, take full advantage of transient resources,
produce large numbers of offspring in short periods of time,
and rapidly disperse into new habitats when conditions turn
unfavorable.  This life history strategy, often called "r-
selection," is typically found in species that have a short life
 SCIENCE LABORATORY TECHNOLOGY

cycle, small body size, and high mobility (ex. house flies). 
Most individuals in "r-selected" populations die before
reaching sexual maturity so a high reproductive potential is
essential for the species to avoid extinction.  These insects
often play a major role as colonizers in the early stages of
ecological succession -- they are also likely to be regarded as
pests if their colonial empire spreads into farms, ranches, or
human habitations!
On the other hand, life expectancy is usually longer for
species that live in stable habitats (like mature grasslands or
climax forests).  More of these individuals reach sexual
maturity and populations tend to stabilize near the
environmental carrying capacity.  Under these conditions,
often called "K-selection," there is no particular advantage to
having large numbers of offpring.  Selection pressures focus
on intraspecific competition and efficient use of resources. 
"K-selected" species often have longer life cycles, larger
body size, and relatively low growth rates.
Ecologists recognize that r- and K-selection are opposite ends
in a broad spectrum of life history strategies.  Most species
fall somewhere in the middle of the range with a blend of "r-
selected" and "K-selected" characteristics.  Ants and termites,
for example, produce large numbers of small, expendable
offspring but they have long-lived colonies that are highly
competitive in stable environments.
 SCIENCE LABORATORY TECHNOLOGY

Insects as Pests

Most children are fascinated by the living organisms that

share their environment; they love to watch an ant groom
itself, a bee gather nectar, or a spider build its web.  But this
interest is seldom encouraged in our busy society.  Adult
attitudes toward insects (and related arthropods) are generally
negative -- the ant is a nuisance, the bee might sting, and the
spider... Through these attitudes we send our children a clear,
subconscious message that insects (and most other parts of
the natural environment) are unsafe, unclean, and
unappealing.  Most children adopt this viewpoint before they
leave elementary school; they grow up, like their parents,
convinced that the "only good bug is a dead bug".
The mass media continually reinforces this belief with reports
about killer bees, giant grasshoppers, poisonous spiders, and
crops destroyed by marauding bands of insects.  This cultural
 SCIENCE LABORATORY TECHNOLOGY

indoctrination has produced a society that seems to be

increasingly consumed by efforts to eliminate insects from all
facets of daily life.  Pest control has become big business. 
Nearly 75 million pounds of broad-spectrum insecticides are
manufactured and sold each year for use in American homes
and gardens.  Annual revenues from insecticide sales to
homeowners exceed 450 million dollars.
Aside from ladybugs (which are considered "cute") and
butterflies (they're "pretty"), most insects are regarded with a
certain degree of fear or revulsion.  A few, like honey bees
and praying mantids, may be recognized as "useful", but all
others are just "pests".
A "pest" is defined as any organism that causes annoyance or
injury to human beings, human possessions, or human
interests.  The injury may be physical (bites and stings),
medical (causing illness or disease), or economic (monetary
loss to goods or property).  Injury may arise directly from the
pest itself, or may develop indirectly as a result of the actions
or behavior of the pest.
Despite public perceptions, there are relatively few insects
that really fit the above definition of a pest.  Admittedly, a
few species (very few) appear to have "no redeeming social
value" (pubic lice and bed bugs spring quickly to mind).  But
most insects we encounter in our daily lives are either benign
or beneficial.  Out of the 800,000 - 1,000,000 species of
insects that have been described so far, not more than 1,000
(about 1/10 of 1%) can be regarded as serious pests, and less
than 10,000 (about 1%) are even occasional or sporadic pests.
In reality, many of the insects we label as pests are essential
components of our natural ecosystem -- we can't seem to live
with them, but neither could we live without them!  Termites,
 SCIENCE LABORATORY TECHNOLOGY

for example, are serious pests when they move into the floor
joists of a home.  An infestation may cost hundreds of dollars
to eradicate, and if ignored, could eventually destroy the
house.  But in a forest ecosystem, these termites are
beneficial.  They are a vital component of the biogeochemical
cycle that releases nutrients from dead plant material. 
Without termites and other decomposers living on the forest
floor, organic molecules would be locked away in piles of
dead wood, unavailable to living organisms until released by
fire or erosion.
In general, the species we regard as pests usually affect us in
one or more of the following ways:

 They are an annoyance or nuisance

 They endanger human health or safety
 They threaten the welfare of useful plants or domestic
animals
 They damage stored products or structural materials

These categories are not mutually exclusive.  Many of our

most serious pests (cockroaches, for example) could be listed
in several of these categories.

Nuisance and Aesthetic Pests

Aesthetic pests cause no damage and inflict no injury.  They

may be annoying, unsightly, or disruptive (like chirping
crickets that keep you awake at night, or dance flies that
swarm around your head in the spring), but their presence
will cause no injury or financial liability.  An insect is most
likely to be regarded as an aesthetic pest when it
 SCIENCE LABORATORY TECHNOLOGY

inadvertently invades a home or business establishment.  The

human occupants of these environments are seldom tolerant
of other species -- even the most benign visitors are promptly
executed (or forcibly evicted).  Boxelder bugs (Boisea
trivittata) are typical victims of such intolerance.  In autumn,
homeowners may become upset when they discover
aggregations of these red and black insects on brick walls,
foundations, or concrete steps near a residence.  The bugs
may be numerous and occasionally annoying, but they do not
bite or sting, they pose no threat to the house or its contents,
and they will eventually move elsewhere to find
overwintering quarters.  Still, a large can of insecticide is the
homeowner's typical response.
Buzzing or swarming insects are often more than just
nuisance pests.  "Fly worry" is recognized by poultry and
livestock producers as a serious threat to their livelihood. 
Large fly populations (even non-biting flies) can decrease
milk production in a herd of dairy cattle, depress egg laying
in a poultry house, or reduce daily weight gain of feeder
pigs.  Economic losses associated with nuisance pests can
range from negligible to severe.  At least a dozen species of
bird lice (Mallophaga) are considered economic pests by
poultry producers.  Although these lice feed only on feathers
and dry skin, they cause enough irritation to induce
sleeplessness, loss of appetite, and poor health.  Lice
populations in excess of 35,000 individuals have been
collected from the body of a single chicken.
To some extent, a fear of insects is quite normal.  You may
react instinctively to brush away an annoying fly or jump
back at the sudden appearance of a spider.  But some people
are so afraid of insects that it can affect their ability to
 SCIENCE LABORATORY TECHNOLOGY

function normally in society. These people suffer from

entomophobia, an irrational and often debilitating fear of
insects.  People with severe entomophobia are frequently
confined to their homes; they are unable to enjoy gardening,
walking in the woods, or visiting the beach because they
dread an encounter with an insect.  Psychiatrists estimate that
1-2% of the adult U.S. population is affected by some degree
of entomophobia that limits their activity or interferes with
their quality of life.
Occasionally, a fear or loathing of insects is compounded by
the victim's own imagination.  Some people convince
themselves that their body has become "infested with
parasites" and go to great lengths to rid themselves of the
imaginary vermin.  This type of paranoia, known medically
as delusionary parasitosis, is generally regarded as a form of
obsessive/compulsive behavior.  It is common enough to be
recognized as a psychological disorder by the American
Medical Association.  One case study, reported in 1957,
documents a woman who believed she was infested with
"termites" that would periodically emerge from the joints of
her arms and legs.  Her physician sent her home to "collect
some specimens" and a week later she returned with a plastic
bag full of "termites" which, upon microscopic examination,
proved to be scabs and skin fragments she had picked or
scratched from her body.
Treatment of entomophobia and delusionary parasitosis is
difficult because the fear is irrational.  Some success has been
achieved with behavioral therapies that involve systematic
desensitization, starting first with imagery (pictures) and
gradually working up to more direct types of exposure.
 SCIENCE LABORATORY TECHNOLOGY

Pests of Medical Importance

It is impossible to measure the full impact of insects and

other arthropods on human health and welfare.  These
organisms have the capacity to inflict injury, disease,
discomfort, or distress.  They can be a direct cause of illness,
pain, and suffering through bites and stings, infested wounds,
or allergic reactions.  They feed on blood or body tissues and
they may transmit deadly pathogens or parasites.  Economic
losses associated with these pests are borne not only by the
affected individuals and their families, but also by human
society in general.  Losses include not only the direct costs of
medicine and health care, but also indirect costs resulting
from stress, absenteeism, and reduced productivity.  These
are costs that are not easy to measure in dollars and cents.

Biting Insects

Most mandibulate insects are not strong enough to pierce

human skin with their mouthparts.  Their bite is usually little
more than a pinch, serving primarily as a defensive behavior
(e.g. ground beetles and ants).  Even fire ants (Solenopsis
spp.), whose painful "bite" is well known, lack the ability to
penetrate skin.  In fact, their "bite" is actually caused by a
sting at the other end of the body!
All of the arthropods that can pierce human skin have
mouthparts that are especially adapted for piercing, cutting,
or burrowing.  These include:
 SCIENCE LABORATORY TECHNOLOGY

 Diptera (mosquitoes, black flies, horse flies, deer flies,

stable flies, sand flies, and various biting midges)
 Hemiptera, (bed bugs, assassin bugs, water bugs)
 Thysanoptera (thrips)
 Phthiraptera (sucking lice)
 Siphonaptera (fleas)
 the class Arachnida (spiders, mites, and ticks).

Most of these arthropods are hematophagous (they feed on

blood).  Their mouthparts are designed either to cut the skin
and induce bleeding (horse flies and stable flies) or to pierce
far enough under the skin to reach capillary blood
(mosquitoes, bugs, fleas, etc.).  Salivary enzymes and other
compounds, such as anti-coagulants, anesthetics, and
vasodilators also may be injected by the mouthparts during
feeding.  The localized reaction to an insect bite (pain,
swelling, redness, etc.) is usually a physiological
(inflammatory) response to these injected compounds.
Although the amount of blood taken by each insect may be
quite small, the cumulative effects of blood feeding by large
populations of hematophagous insects can be life-
threatening.  In the summertime, small herds of reindeer
living above the arctic circle have died from desanguination
(massive loss of blood) caused by hoards of mosquitoes and
black flies.
Some arachnids (e.g. spiders) and most water bugs
(Notonectidae, Belostomatidae, and Gerridae, for example)
bite only defensively.  Their strong, piercing mouthparts can
inject toxins or digestive enzymes that may cause pain and
localized swelling.  In North America, the black widow
 SCIENCE LABORATORY TECHNOLOGY

spider (Latrodectus mactans), the brown recluse spider

(Loxosceles reclusa), and two species of scorpions
(Centruroides sculpturatus and C. gertschi) are the only
arthropods with a venom that is toxic enough to cause serious
illness or death.
Itch mites, mange mites, and harvest mites (chiggers or
redbugs), burrow into the skin of humans and other animals
where they feed on body fluids and tissues.  These
infestations (variously known as acariasis, mange, or scabies)
cause redness, itching, and flaking of the skin.  The itch mite,
Sarcoptes scabiei hominis, causes a severe, itching rash in
humans; other subspecies of the same mite infest dogs, swine,
cattle, and sheep.  Infestations of various mites can become a
severe economic problem in commercial poultry and swine
production.  The northern fowl mite, Ornithonyssus
sylviarum, is a fast breeding pest that lives around the neck,
vent, and tail of chickens.  Under ideal conditions, this
species completes its life cycle in less than seven days. 
Population growth is explosive, and often difficult to control.
Thrips have rasping/sucking mouthparts capable of abrading
the skin and causing irritation.  These insects are primarily
herbivores, but they may be quite annoying to people who
work near infested plants.  Occasional reports of predatory
thrips that suck blood are unsubstantiated.
 SCIENCE LABORATORY TECHNOLOGY

Stinging Insects

Ants, wasps, bees, and scorpions are the only arthropods that
have a true stinger.  Some predatory and parasitic insects
sting to kill or immobilize their prey, whereas most other
species sting only as a defensive behavior to kill or drive
away potential predators.  In all of the stinging (aculeate)
hymenoptera, the stinger is modified from structures of the
ovipositor.  The shaft of the stinger is hollow, occasionally
barbed, and connected internally to a venom gland that
produces a complex mixture of compounds that may destroy
cells (hemolytic and proteolytic enzymes), increase blood
flow (hemorrhagic enzymes), break down intercellular
connective tissue (hyaluronidase), and cause neurotoxic or
other pharmacologic effects on nerve cells.
Arthropod venom is rapid-acting and frequently associated
with considerable pain.  Most people experience an intense
local reaction that subsides after several hours and heals
within a few days.  But for other people (estimates range
from 2-5% of the U.S. population) a single sting may elicit
 SCIENCE LABORATORY TECHNOLOGY

anaphylactic shock, a life-threatening allergic reaction

caused by hypersensitivity to insect protein or any other
venom component.  Anaphylaxis is characterized by rapid
loss of blood pressure, fainting, and respiratory difficulty.  If
the patient does not receive prompt medical attention,
unconsciousness and death may occur within minutes.
Emergency treatment of anaphylactic shock usually consists
of deep subcutaneous or intramuscular injections of a cardiac
stimulant (such as norepinephrine) in an effort to counteract
the physiological reaction, increase heart rate, and stabilize
blood pressure.  Emergency bee sting kits, containing auto-
injectable norepinephrine, are available by prescription to
patients with known sensitivity to insect stings.  In the United
States, about 20 deaths per year are attributed to stinging
insects -- in nearly every case death results from anaphylactic
shock, not the sting itself.
The larvae of certain flannel moths (Megalopygidae) and slug
caterpillars (Limacodidae) do not have a stinger, but they also
inflict a sharp, stinging pain upon contact.  These insects have
specialized urticating hairs that inject a painful chemical
when touched.  The sensation, like that of a stinging nettle, is
an intense localized pain that gradually fades after several
hours.  Saddleback caterpillars (Sibine stimulea) are probably
the most conspicuous of the stinging caterpillars.  They feed
on a variety of trees and shrubs, including cherry, plum, elm,
and poplar.

Irritants and Allergens

Skin and eye irritation, respiratory inflammation, and various

types of chronic allergies may also be caused by insects and
 SCIENCE LABORATORY TECHNOLOGY

related arthropods.  An allergic reaction can be induced by

nearly any component of an insect's body, but hairs, scales,
exuviae, and fecal products are the most common culprits. 
Allergies to household dust, for example, can often be traced
to dander from fleas (Siphonaptera), cockroaches (Blattodea),
or house dust mites (Dermatophagoides spp.).  Larvae of the
browntail moth (Euproctis chrysorrhoea) have hairs that
cause an irritating rash on the skin of many people.
Human exposure to sensitizing antigens usually occurs in one
of four ways:

 inhalation of airborne particles

 ingestion with foods
 dermal contact, or
 unintentional injection (as by rubbing the eyes)

Immunological responses of the human body vary, but

sneezing, watery eyes, a runny nose, or skin rashes are
common manifestations.  Chronic asthma, allergic rhinitis,
and eczema are frequently aggravated by exposure to
arthropod antigens.
The prognosis for treatment of "arboallergies" is not very
promising.  One option, immunotherapy, involves successive
exposures to increasing concentrations of the offending
allergen in an effort to desensitize the immune system.  This
approach may provide some relief for some patients, but the
only "sure cure" is complete avoidance of exposure to the
allergens.

Myiasis
 SCIENCE LABORATORY TECHNOLOGY

Of all the arthropods, only the larvae of certain flies (Diptera)

are adapted to invade and consume the tissues of a vertebrate
host.  An infestation by any of these flies is known as
myiasis.  In North America, this type of parasitism is most
common among domestic animals (particularly sheep, cattle,
and horses), but it can also be a problem among the poor and
elderly of the human population where it is usually associated
with neglect and unsanitary conditions.
Some of these flies breed in carrion or manure; others live in
spoiled food.  Eggs or larvae may be ingested and survive in
the vertebrate's intestinal tract, or larvae may crawl into the
bowel through the anus.  The screwworm fly (Cochliomyia
hominivorax) lays its eggs in open, festering wounds.  Larvae
feed on these injured tissues and prevent healing. 
Screwworms are endemic to Mexico and the southwestern
United States where they are a major pest of sheep and
cattle.  A sterile-male release program (see Chapt. 19) has
eradicated screwworm populations from Texas and New
Mexico, but ranchers in southern Mexico still suffer heavy
losses from these flies.
The family Oestridae includes warble flies, cattle grubs, and
bot flies whose larvae also parasitize domestic animals. 
Cattle grubs (Hypoderma bovis and H. lineatum) lay their
eggs on the legs of cattle.  After hatching, larvae burrow
through the skin and migrate upward, first to the digestive
system and eventually to the back where they produce
swellings known as "warbles".  Full grown larvae cut their
way out of the warble leaving a hole that decreases the hide's
value when it is tanned for leather. The sheep bot fly
(Oestrus ovis) produces live young and deposits them in the
nostrils of sheep.  These larvae migrate into the sinus cavities
 SCIENCE LABORATORY TECHNOLOGY

where they complete development.  Large fly populations can

weaken or kill the sheep.

Larvae of flies in the family Gastrophilidae (the horse bot

flies) are internal parasites of horses and mules.  Most species
lay their eggs around the animal's nose or mouth.  After
hatching, the larvae burrow or crawl into the horse's mouth
and eventually move down the digestive tract, attaching
themselves to the walls of the stomach, duodenum, or
rectum.  The horse bot fly (Gasterophilus intestinalis) lays its
eggs on the legs, flanks, or shoulders of the horse.  These
eggs hatch immediately when they are licked and ingested by
the horse.  The presence of bots (fly larvae) in the animal's
digestive system often causes malnutrition, ulceration of the
digestive tract, and even complete blockage of the intestine. 
Mature larvae leave their vertebrate host by passing out as
excrement.  They pupate in the soil and emerge as adults
several weeks later.
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Transmission of Diseases

Infectious agents that cause illness or disease in other living

organisms are known as pathogens.  These agents may
include a wide variety of microorganisms (e.g. mycoplasmas,
bacteria, protozoa, spirochetes, and rickettsias) as well as
fungi, helminths (roundworms and flatworms), and viruses. 
A host is any living organism that is infected by a pathogen,
regardless of whether or not symptoms of the disease are
present.  Some hosts die quickly, others may kill or inactivate
the pathogen, and still others may retain the pathogen in a
condition of readiness to infect other hosts.  Whenever a host
serves as a source of new infections for other hosts (of the
same or different species) it is known as a reservoir.  Since
pathogens do not have legs or wings, they are often "carried"
from one host to another by vectors.  Insects and related
arthropods are among the most important vectors of
pathogens.  The term zoonosis (or zoonotic disease) refers to
any infectious disease that is transmitted (by a vector) from
an animal reservoir to a human being.
Although epidemics of arthropod-borne disease have been
well-documented throughout human history, it was not until
the late 1800's that insects and related arthropods were
convincingly linked to the spread of human disease.  In 1878,
Patrick Manson first demonstrated that mosquitoes can
transmit filariasis, a disease of the blood and lymph caused
by roundworms (nematodes).  His research was followed in
rapid succession by evidence that Anopheles spp. mosquitoes
carry malaria (Ronald Ross, 1897), fleas spread bubonic
plague (P. L. Simond, 1898), and mosquitoes, particularly
Aedes aegypti, transmit yellow fever (the U. S. Army Yellow
 SCIENCE LABORATORY TECHNOLOGY

Fever Commission headed by Walter Reed, James Carroll,

Jesse Lazear, and A. J. Agramonte, 1900).  
Today, we know of over 200 human diseases that can be spread
by insects and related arthropods.

Despite the efforts of modern medicine, spread of arthropod-

borne disease is still one of the most serious concerns facing
public health officials and the medical community in general. 
The World Health Organization (WHO) estimates that as
many as 4 million people die each year from the
consequences of arthropod-borne disease.  Obviously, the
problem is most severe in underdeveloped countries where
access to good medical care is limited.  But even here in the
United States, encephalitis (mosquito-borne), Lyme disease
(tick-borne), and Rocky Mountain Spotted Fever (tick-borne)
are still regarded as epidemic-scale problems.  The
Communicable Disease Control Center (CDC) in Atlanta,
Georgia estimates that eight out of every ten Americans will
be infected by an arthropod-borne disease sometime during
their lives.  Pathogens coexist in elaborate ecological
relationships with their hosts and vectors.  Without a vector,
the pathogen could not infect new hosts, and without new
hosts, the pathogen would eventually become extinct.  The
term biocenosis (pl. biocenoses) can be used to denote an
ecological group that includes a pathogen and all of its hosts
and vectors.
Some pathogens may have multiple hosts, reservoirs, or
vectors.  For example, Pasturella tularensis, the tularemia
pathogen, can be transmitted to humans by deer flies, ticks,
fleas, or body lice from reservoirs in rodents as well as other
humans.  Other pathogens, like the Plasmodium species that
 SCIENCE LABORATORY TECHNOLOGY

cause malaria, have a much narrower biocenosis -- they

survive only as long as they remain within their human hosts
and mosquito vectors.  Finally, there are pathogens (e.g. the
causal agents of cholera and amoebic dysentery) that can
survive for long periods outside the bodies of living hosts. 
These infectious agents are often spread to humans by flies or
other insects that visit sewage and garbage.
In some cases, pathogens may simply adhere to a vector's feet
or mouthparts, catching a quick ride to a new host.  This is
known as mechanical transmission.  Most of the pathogens
that are transmitted mechanically are able to survive short-
term exposure to the atmosphere and sunlight.  Many types of
arboviruses (short for arthropod-borne viruses) are spread
by mechanical transmission on the mouthparts of mosquitoes.

In contrast, biological transmission occurs when the

pathogen survives for a time inside the vector's body, and is
later spread to another host.  Some of these pathogens
relocate within the body of the vector, traveling from the gut
to the salivary glands, for example.  Others, such as the
rickettsia of rocky mountain spotted fever, remain dormant in
the vector's body and only become activated after feeding
commences.  Removing a tick within 1-2 hours of attachment
usually will ensure too little time for activation and
transmission of its pathogens.
 

Some pathogens can reproduce in the vector, and a few (the

plasmodium of malaria, for example) must complete a part of
their life cycle inside the vector's body.  
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Newly hatched insects are usually pathogen-free.  After

feeding on an infected host, the vector may be able to infect
new hosts immediately (in the case of mechanical
transmission) or there may be a waiting period (latency) of
days to weeks (in the case of biological transmission) while
the pathogen migrates or reproduces inside the vector's body. 
Once infected, a vector may retain the pathogen for the rest of
its life (persistent infection) or may eventually eliminate or
inactivate the pathogen (non-persistent infection).
Although vectors typically acquire a pathogen only by
feeding on an infected host, there are a few species that pass
infection through the egg stage from generation to
generation.  Such transovarial transmission occurs most
commonly in ticks.

Arthropod-borne diseases have shaped the course of human

history.  Ever since medieval times, epidemics of bubonic
plague have swept through the civilized world.  It is
estimated that nearly one-fourth of the population of Europe
(20-25 million people) died of plague (The Black Death)
during the 14th century.  Edgar Allan Poe described the
gruesome death of plague victims in his story The Masque of
the Red Death:
"No pestilence had ever been so fatal or so
hideous.  Blood was its Avatar and its seal -- the
redness and the horror of blood.  There were sharp
pains, and sudden dizziness, and then profuse
 SCIENCE LABORATORY TECHNOLOGY

bleeding at the pores, with dissolution.  The scarlet

stains upon the body and especially upon the face
of the victim, were the pest ban which shut him out
from the aid and from the sympathy of his fellow-
men."

Malaria and typhus, both arthropod-borne pathogens, played

major roles in the outcome of several wars.  In 1815, during
Napoleon's final campaign, his army suffered 105,000 war
casualties but lost 219,000 fighting men to typhus.  After
their rout at Waterloo, one disappointed Frenchman
complained that they had been defeated by "General Famine,
General Winter, and General Typhus".
Forty years later, in 1856, the Russian army, weakened by
tularemia after marching through fly-infested swamps in
Turkey, was soundly defeated by the allied forces of Europe
to end the Crimean War.  1.2 million cases of malaria were
treated during the American Civil War (1861-1865) and
8,000 soldiers died.  More than 100,000 troops were sidelined
with malaria during World War II, yet the Allied army was in
much better health than the Japanese largely due to
widespread use of a "new" insecticide (DDT) to control lice
and mosquitoes.  Many historians believe that, even without
the atomic bomb, Japan's army would not have survived
much longer against the onslaught of typhus and malaria.
Demographics and civilization have also been shaped by
arthropod-borne disease.  Deteriorating socio-economic
conditions during the Russian Revolution (1917) prompted
Lenin to declare, "Either socialism will defeat the louse, or
 SCIENCE LABORATORY TECHNOLOGY

the louse will defeat socialism."  Even today much of

equatorial Africa is uninhabitable because of endemic
sleeping sickness vectored by the tsetse fly.  The World
Health Organization estimates that more than 500 million
people throughout the world currently suffer from the
debilitating effects of arthropod-borne disease.

Plant Pests
Injury to Living Plants

Of all the insect species now living on earth, at least half of

them (400,000 - 500,000) feed directly on the tissues of living
plants.   It is probably no exaggeration to claim that every
plant species on earth serves as food for at least one species
of insect.   No parts of a plant are immune from attack:  
insect herbivores chew the leaves, suck the sap, mine the
cambium, gather the pollen, invade the buds, destroy the
flowers, and devour the fruit.   Even plants that manufacture
potent insecticides (such as the nicotine in tobacco,
pyrethrum in chrysanthemums, or rotenone in tropical
legumes) have insect pests that are especially adapted to feed
on their tissues and detoxify their chemical defenses.
Herbivores with chewing mouthparts consume a plant
directly.   They use mandibles to masticate (knead), triturate
(grind), or abrade (scrape) plant tissue into manageable bites.
Most species consume entire roots, stems, leaves, buds,
flowers, fruit, and/or seeds.   All members of the orders
Orthoptera and Phasmida feed in this manner, as do nearly all
larvae of Lepidoptera.   Chewing herbivores are also found in
 SCIENCE LABORATORY TECHNOLOGY

the order Diptera (e.g. larvae of Tephritid fruit flies), in the

Coleoptera (e.g. larvae and adults of weevils, scarab beetles,
leaf beetles, and many others), and in the Hymenoptera (ants,
some bees, and the larvae of all sawflies).   Immatures of
aquatic orders, such as Trichoptera, Plecoptera, and
Ephemeroptera, also feed by chewing plant tissues but they
are seldom regarded as major pests.
Not all chewing herbivores consume entire portions of their
host plant.   Leaf miners (represented by several families in
the Diptera and Lepidoptera) are specialized herbivores that
excavate galleries in mesophyll, the inner layer of cells
between a leaf's upper and lower epidermis.   Other species
concentrate on epidermal cells.   The pear slug (an immature
sawfly) skeletonizes the leaves of its host plant by abrading
only cells from the lower epidermal surface.   Many bark
beetles feed only in cambium, the layer of cells just beneath
the bark of a tree or shrub.

 
Plant tissue is also damaged by herbivores with piercing-
sucking mouthparts.   Some species (e.g., stink bugs) stab
their feeding stylets into individual plant cells, inject
digestive enzymes to liquify the substrate, and then suck out
the partially digested food.   Other species (e.g., aphids)
thread their stylets delicately through intercellular spaces, tap
into the plant's vascular system, and feed by withdrawing sap
from the phloem.   Most pest species with piercing-sucking
mouthparts belong to the orders Hemiptera (Heteroptera and
Homoptera) and Thysanoptera.   Damage from these insects
 SCIENCE LABORATORY TECHNOLOGY

is often less visible than that of chewing insects, but it may

still result in severe injury to the plant.
Gall-making insects are herbivores that live within the tissues
of a plant.   They secrete a hormone-like substance that
stimulates growth in surrounding plant tissue and induces the
host plant to produce a specialized shelter (gall) that
nourishes and protects the herbivore inside.   Gall-making
insects (sometimes called cecidozoa) include species in the
orders Hemiptera, Hymenoptera, Lepidoptera, and Diptera.  
Gall formation is also induced by certain species of mites,
fungi, and bacteria.   Gall makers are highly specialized
herbivores; each species is narrowly adapted to its host plant
and produces a distinctively shaped gall.
Herbivores may also injure their host plants during
oviposition; females often cut holes or slits in plant tissues as
they deposit their eggs.   Oviposition scars from cicadas,
crickets, and katydids may be severe enough to kill twigs or
stems.   This problem is most troublesome in nurseries and
greenhouses where ornamental plants with obvious injury are
less marketable.
Many insects that feed on plants also serve as vectors of plant
diseases. All major taxa of plant pathogens are spread by
insects, including viruses, mycoplasmas, bacteria, protozoa,
fungi, and nematodes.  
Plant pathogens may be carried externally on a vector's feet,
mouthparts, or ovipositors.   This mechanical transmission
has been well-documented in vectors representing at least
eight arthropod orders [Hemiptera (both suborders),
Thysanoptera, Orthoptera, Diptera, Coleoptera,
Hymenoptera, Lepidoptera, plus Acarina (mites)].
 SCIENCE LABORATORY TECHNOLOGY

Some pathogens even provide symbiotic advantages for their

insect vectors.   Bark beetles, for example, infect trees with a
pathogenic fungus whenever they establish new colonies.   As
the fungus becomes established in each new tree, it changes
the micro-environment of the wood and improves
reproductive success for the bark beetles.   A blue-stain
fungus carried by Ips spp. bark beetles and Dutch elm disease
spread by Scolytus spp. bark beetles are examples of this type
of symbiotic relationship between pathogen and vector.
Plant pathogens may also be carried internally in the salivary
glands, digestive tract, or reproductive system of insect
vectors (biological transmission).   These pathogens are
usually inoculated into new hosts during feeding or
oviposition, but they may also be spread through the insect's
feces.   Several families of vectors show strong affinity for
biological transmission of pathogens in specific taxonomic
groups.   Thrips, aphids, and whiteflies, for example, tend to
be vectors of virus pathogens, whereas leafhoppers are more
likely to transmit mycoplasma-like organisms (MLOs).

Damage to Stored Products and Structural

Materials
Virtually any product or commodity derived or manufactured
from plant or animal matter may be damaged by insects.  
Silverfish, ants, cockroaches, and psocids (e.g., booklice) will
feed on starch sizing, wallpaper paste, bookbindings, and old
photographs.   Ants, beetles, and moths will destroy animal
products such as furs, leather, shoes, stuffed museum
specimens, and even pinned insects!   Some beetles will chew
through inedible packaging just to reach the food inside.  
One species, known as the "short-circuit" beetle, became
 SCIENCE LABORATORY TECHNOLOGY

notorious for chewing through lead sheathing on telegraph

cables in order to eat the fiber insulator around the copper
wires.   Drugstore beetles, Stegobium paniceum, have been
reported feeding on at least 45 different types of drugs in
addition to nuts, ginger, chocolate, cork, and pepper.
Stored agricultural products such as seeds and grains, cheese,
cured meats, dried fruits, grain products (e.g. flour and corn
meal), hay and other forages, and even cured tobacco serve as
food for a variety of beetles, moths, cockroaches, ants, and
silverfish.   These insects inhabit grain bins, silos, elevators,
warehouses, grocery stores, and home pantries wherever food
is stored.   In addition to damage caused by feeding, these
pests also accelerate spoilage of agricultural commodities by
introducing bacteria, fungi, and fecal matter.
Textiles and natural fibers, including wool, cotton, silk,
hemp, kapok, raffia, jute, flax, and sisal serve as food for
carpet beetles and clothes moths.   These insects destroy
upholstery, carpeting, draperies, and clothing by consuming
the natural fibers.   Before the invention of nylon, clothes
moths were a major cause of damage to hemp and sisal rope
used in shipyards.
Lumber, wood products and wooden structures are subject to
attack by a variety of insects including termites, wood-boring
beetles, carpenter ants, and carpenter bees.  At least 10
species of termites are found in the United States, mostly in
southern or coastal regions.   The eastern subterranean
termite, Reticulitermes flavipes, is the most destructive
species.   It is distributed throughout the South and Southeast
from Texas to Pennsylvania where it causes more than $800
million in damage each year.   The National Pest Control
 SCIENCE LABORATORY TECHNOLOGY

Association estimates that nearly 700,000 infested structures

are treated for termites every year.
Wood-boring beetles (including members of the families
Anobiidae, Bostrichidae, Buprestidae, Cerambycidae,
Curculionidae, Lyctidae, and Scolytidae) are responsible for
extensive damage to lumber both before and after it is
harvested.   The lumber industry estimates that about 10
percent of its annual saw timber production sustains enough
insect injury to affect its grade.   Loss of value may be only
about 10 cents per board foot, but with annual timber
production at nearly 45 billion board feet, the total loss is
close to $450 million each year.

Pest Management

There are basically two venues for pest control -- agricultural

and urban/industrial.   In homes and businesses, tolerance for
insect pests is very low.  Pest control objectives are best
described as:  Locate and Kill.  Heavy emphasis is placed on
excluding or eradicating pests, or creating environments
where pest species cannot survive and reproduce.  In
agricultural settings, where exclusion or eradication is less
practical, the objective is often to reduce pest populations to
levels that are compatible with human interests and
economics.  The term pest management was coined in the
1960's to reflect this current emphasis on coexistence rather
than eradication.  Management strategies include not only the
 SCIENCE LABORATORY TECHNOLOGY

traditional physical and chemical weapons that kill insects,

but also a wide variety of non-lethal cultural and biological
tactics that will be discussed in the next chapter.

Effective pest management usually follows a logical

progression of steps to identify and solve a pest problem:

1. Identification -- Exactly which species is causing damage? 

The cause is not always obvious or easy to determine,
particularly if it involves transmission of disease.  Some
pest species are very similar in appearance to non-pest
species, and confusing the two may result in unnecessary
expense for pest control.
2. Quantification -- What is the density and/or distribution
of the population?  Use of survey and sampling techniques
allows an assessment of whether or not the problem is
severe enough to warrant the time and expense of control
efforts.
3. Specification -- What is the most reasonable and effective
course of action?   Find out what experts would
recommend.   Control recommendations are often
available from agricultural extension services
4. Application -- Implement the appropriate management
tactic.   Whenever chemical insecticides are used, it is
important that label directions be followed precisely.  
Adequate consideration must be given to safety of the
applicator as well as to protection of non-target organisms
and the general environment.
5. Evaluation -- How effective was the control operation?  
Assessment of pest population density must be conducted
 SCIENCE LABORATORY TECHNOLOGY

after the management tactic has had time to have its

impact on the pest.

Insect pests can be grouped into three general categories

based on how frequently humans must take control action
against them.   The numbers in parentheses at the end of each
group are estimates by the National Academy of Science
(1969) for the number of North American species that fall
into each group.

1. Severe or persistent pests.   Only a small number of insect

species are regular problems.   These pests are typically
small, mobile, and have a high reproductive potential.  
They reach injurious populations every year, and control
strategies must be implemented regularly and
consistently to prevent economic loss.   Pests that
transmit disease, injure high-value crops, or become a
special nuisance would also be grouped as severe pests.  
[150 to 200 species:   examples include the cotton boll
weevil (Anthonomis grandis), codling moths (Cydia
pomonella), Colorado potato beetles (Leptinotarsa
decemlineata), Mexican bean beetles (Epilachna
varivestis), and corn earworms (Helicoverpa zea).]
2. Occasional or sporadic pests.   These insects become
pests only at specific times, under special conditions, or in
certain places.   They do not always require control and
are often maintained at sub-economic levels by natural
predators and parasites.   Outbreaks typically occur
whenever changes in cultural, ecological, or
environmental conditions produce special opportunities
for reproductive success.   [400 to 500 species:   examples
include fall armyworms (Spodoptera frugiperda), green
stinkbugs (Acrosternum hilare), potato leafhoppers
 SCIENCE LABORATORY TECHNOLOGY

(Empoasca fabae), white-fringed beetles (Graphognathus

leucolema), hog lice (Haematopinus suis), and pharaoh
ants (Monomorium pharaonis).]
3. Uncommon or infrequent pests.   There are many insect
species which only become pests under the most unusual
circumstances.   These insects are not generally regarded
as pests because a.) they are rarely encountered by
humans, b.) they are not abundant enough to cause
serious injury, or c.) they do not injure commodities or
resources that are valued by humans.   [6,000 species:  
examples include nutgrass billbugs (Sphenophorus
cariosus), blueberry stem borers (Oberea myops), azalea
caterpillars (Datana major), smut beetles (Phalacrus
politus), and honeysuckle sawflies (Zaraea inflata).]

Pest Outbreaks

Since all insect populations rise and fall with changes in the
seasons, interspecific competition, food quality, and
numerous other variables, it is important to recognize which
ones are likely to become pests and to understand what
factors might precipitate their explosive growth.   Even the
worst pest species are not continuous problems. Their status
as pests is usually a function of abundance, distribution, or
density.   A few Japanese beetles on a grapevine would cause
little concern, but an infestation that skeletonizes the leaves
and attacks the fruit would not be tolerated.
Large, sporadic populations of insect pests are usually called
outbreaks.   They typically occur when the pest population
rises significantly above its general equilibrium level and
 SCIENCE LABORATORY TECHNOLOGY

becomes a threat to human interests or endeavors.   There are

five common scenarios that lead to these pest outbreaks:

1. environmental change
2. introduction from abroad
3. destruction of natural enemies
4. development of resistance
5. higher quality standards

Environmental Change
Most insect populations, with their large gene pool and high
reproductive rate, are well-adapted to exploit changing
conditions in the natural environment.   Pest species are no
exception.   Changes in climate, habitat, or community
structure (caused either by natural phenomena or human
intervention) may provide an insect population with a
reproductive opportunity that could change its status from
endemic to epidemic within just a few generations.
There are numerous examples of relatively minor insect
species that have become important pests as a result of
environmental change.  In the early 1800's, Leptinotarsa
decemlineata was an inconsequential beetle that lived in the
midwestern United States where it fed on buffalo burr, an
unremarkable weed in the potato family.  As European
settlers emigrated to the midwest, they introduced Irish
potatoes as an agricultural commodity.   Within a few years,
the prairie was dotted with potato fields and the little beetles
that lived on buffalo burr soon discovered this new and very
appetizing food source.   Settlers quickly came to dread this
native pest -- it destroyed their potato fields, spread
throughout other potato-growing regions, and is now
 SCIENCE LABORATORY TECHNOLOGY

recognized world-wide as a major pest:  the Colorado potato

beetle.
The alfalfa butterfly (Colias eurytheme) presents a similar
story.   As a native of the southeastern U. S., this innocuous
species fed on a variety of indigenous legumes until local
farmers began planting fields of alfalfa.   The butterfly
quickly adopted alfalfa as a preferred host plant and has been
an important pest of that crop ever since.
By its very nature, modern agriculture provides opportunities
for insect populations that they would never encounter under
"natural" conditions.   Cultivation of single plant species (e.g.
cotton, corn, or soybeans) over large acreages (crop
monoculture) promotes excellent survival and growth of pest
populations, often well beyond their reproductive potential in
natural communities or mixed-species plantings.   To insect
herbivores, these monocultures must seem like a Garden of
Eden!
Virtually any agricultural practice that modifies a natural
community is likely to create obstacles for some insect
species and opportunities for others.   Housing domestic
animals in modern rearing facilities, for example, may reduce
the potential for flea or lice infestations, but it may also
compound the problems associated with waste disposal and
fly control.   Likewise, greenhouse operators can successfully
exclude many insect pests, but species that do manage to
breach the glass walls find an environment where temperature
and humidity are nearly optimal throughout the year.  
Breeding crop plants for higher yields or better taste may
produce a more saleable product, but it may also reduce the
plant's natural (native) resistance to insects.   Even those piles
of old tires that farmers keep behind the barn have become
 SCIENCE LABORATORY TECHNOLOGY

insect habitat -- they hold just enough water to serve as

breeding sites for a newly imported pest, the Asian tiger
mosquito (Aedes albopictus).
Introduction from Abroad
We live in an increasingly mobile society.   Expansion of
international travel and trade continues to bring new
opportunities for spread of insect pests from one part of the
world to another.   Much like pilgrims who came to America
to escape religious oppression, these six-legged immigrants
find a New World of opportunity free from parasites and
predators.   Some introduced species stake claim to an open
niche in their new environment, others may outcompete or
displace their native counterparts, and still others may adapt
to new habitats or new host plants.

The spotted alfalfa aphid (Therioaphis maculata) is an

example of an opportunistic species that arrived in the United
States from Mexico in 1954.   Remarkable similarity in the
genetic makeup of all aphids in the U.S. population led
entomologists to speculate that the infestation may have been
started by a single parthenogenetic female that landed in
southeastern New Mexico, perhaps as a stowaway on an
airplane that landed at Roswell Air Force Base.   Enjoying a
favorable climate, abundant food, and no natural enemies, the
aphid population grew so rapidly that it spread into alfalfa
fields from coast to coast in only three years.
It is not hard to find examples of introduced species that have
become major pests in the United States -- at latest count,
more than 600 noxious species have invaded our shores.   But
 SCIENCE LABORATORY TECHNOLOGY

we have also exported our share of insects, and many of these

have become pests in foreign countries.  The Colorado potato
beetle and the apple maggot are two examples of North
American species that are now regarded as important pests
throughout much of Europe and Asia.
Destruction of Natural Enemies
Pest populations often reach outbreak proportions if their
native parasites and predators are suppressed or eradicated. 
Changes in management practices or alterations of the
microclimate can have a great impact on the survival of
natural enemies.  Removing the ground cover from beneath
apple trees, for example, can eliminate harborages and
overwintering sites for the predators that help control scale
insects and spider mites.
Beneficial insects are often unintended victims of insecticides
used to control pest outbreaks.  Destruction of these non-
target populations may result in outbreaks of new pest species
that were previously held in check by predation or
parasitism.  Following introduction of the cottony cushion
scale into California, citrus farmers began using insecticides
(primarily lime sulfur) to protect their crops from scale
infestations. But in addition to killing the scale insects,
insecticides also eliminated populations of natural predators
that had always controlled the citrus red mite (Panonychus
citri), a previously inconsequential herbivore.  Many farmers
had never seen red mite infestations until they began spraying
for cottony cushion scale.  Ultimately, the problem was
resolved when entomologists imported and released the
vedalia beetle (Rhodolia cardinalis) as a predator of the scale
insects.  Farmers were then able to eliminate most insecticide
 SCIENCE LABORATORY TECHNOLOGY

applications and mite predators eventually returned to the

orchards.
When the insecticides used to control a pest population also
kill or repel beneficial insects, it can be very difficult to
reestablish the original balance of nature.   In a sense, long-
term control agents (natural enemies) are traded for short-
term control agents (chemical insecticides).   Once the effect
of chemical control wears off, the pest population may
"bounce back", growing relatively unrestrained in an
environment free of natural enemies.   This phenomenon,
known as resurgence, often forces the continued use of
insecticides as a substitute for the missing natural enemies.  
In some cases, this vicious cycle can be hard to break.
Development of Resistance
Insect evolution did not stop at the end of the Mesozoic era.  
The forces of natural selection are just as active today in a
cotton field as they were millennia ago in the primeval forest.
When insect populations are exposed to selective pressures,
whether natural or artificial, they change and adapt.   Most of
the control tactics we use to suppress insects are not 100%
effective:   they leave small numbers of survivors who
manage to reproduce and pass on their genetic heritage to
subsequent generations.   When this genetic heritage includes
genes (or alleles) that confer resistance, our control tactics
will be less effective against future generations.   Resistant
populations may reach outbreak proportions quickly unless
we change to newer, more effective controls.   Resistance
may be biochemical (e.g. an enzyme that degrades or
detoxifies an insecticide), physiological (e.g. the ability to
withstand greater environmental stress), or behavioral (e.g.
the ability to avoid a poison or adapt to a new host plant).  
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The genetic basis for resistance may be conferred by a single

gene (dominant, co-dominant, or recessive), or it may be a
result of phenotypic interaction of several genes (polygenic).  
Dominant or co-dominant resistance can spread quickly
throughout a population.   Recessive or polygenic resistance
is often slower to develop, but it is also harder to eliminate or
overcome.
Insects that become resistant to one control strategy may also
be resistant to other, related controls.   This phenomenon,
known as class-resistance, is commonly found within classes
of insecticide.   Insects that become resistant to aldicarb
(Temik), for example, are usually also resistant to other
carbamate insecticides, such as carbaryl (Sevin), carbofuran
(Furadan), and propoxur (Baygon).
Just because an insect population is resistant to one group of
insecticides (or one type of control strategy) does not prevent
it from developing resistance to other compounds or tactics.  
Such multiple resistance is a growing problem among large,
highly adaptable species.   Potato growers in Long Island,
NY, for example, must face populations of Colorado potato
beetle that are resistant to four major groups of insecticides:  
carbamates, organophosphates, synthetic pyrethroids, and
chlorinated hydrocarbons.
Higher Quality Standards
Back in the "good old days" humans and insects competed for
food and resources on a fairly level playing field.   The
humans were bigger and stronger, but insects outnumbered
them.   The two groups generally managed to coexist.   But
the advent of synthetic organic pesticides and their
widespread use after World War II prompted a revolution in
 SCIENCE LABORATORY TECHNOLOGY

pest control technology.   Humans now had a powerful new

weapon and they used it indiscriminately to eliminate the
competition.   As pest populations were suppressed,
Americans gradually became accustomed to higher living
standards, better food quality, and improved public health.  
They were no longer willing to tolerate worms in the apples,
flies in the soup, and roaches in the cupboard.
Higher standards of purity and sanitation are transformed into
dollars and cents in the marketplace.   Blemished fruit that
could have been sold in the 1800's or early 1900's will now
rot on the grocer's shelf.   For many types of insect pests there
is a zero tolerance.   As a result, producers are forced to apply
more stringent pest control just so they will have a
marketable commodity.   More intensive pest control drives
up the costs of production, and those costs are passed on to
consumers in the form of higher retail prices.   As long as
consumers are willing to pay the price for blemish-free
produce, the agricultural community will continue to give
them what they want.

The Economics of Pest Control

The scientific study of pests and pest control strategies is
often called economic entomology in recognition of the
financial impact insects have on industry, agriculture, and
human society in general.   To be sure, economically
important insects are not always pests; we have already
stressed their value as pollinators, natural enemies, producers
of silk, honey, etc.   But wherever pest populations develop,
their impact always results in monetary loss, either directly or
indirectly.   In most cases, losses from insect pests are
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directly proportional to the density of the pest population --

high density increases the extent or severity of damage and
makes the need for control more critical.

Injury vs. Damage

Many people use the terms "damage" and "injury"
interchangeably, but entomologists usually make an
important distinction between them.

 Injury is defined as the physical harm or destruction to a

valued commodity caused by the presence or activities of
a pest (e.g., consuming leaves, tunnelling in wood, feeding
on blood, etc.).
 Damage is the monetary value lost to the commodity as a
result of injury by the pest (e.g., spoilage, reduction in
yield, loss of quality, etc.).

Any level of pest infestation causes injury, but not all levels
of injury cause damage.   Plants often tolerate small injuries
with no apparent damage and sometimes even
overcompensate by channelling more energy or resources
into growth terminals or fruiting structures.   A low level of
injury may not cause enough damage to justify the time or
expense of pest control operations.   These sub-economic
losses are simply part of the cost of doing business.
But at some point in the growth phase of a pest population it
reaches a point where it begins to cause enough damage to
justify the time and expense of control measures.   But how
does one know when this point is reached?   (How many boll
weevils, for example, does it take to make a cotton farmer
 SCIENCE LABORATORY TECHNOLOGY

hook up his sprayer?)   To a great extent, the answer depends

on two fundamental pieces of economic information:

 A. How much financial loss is the pest causing?   and

 B. How much will it cost to control the pest?

A pest outbreak, by definition, occurs whenever the value of

"A" is greater than the value of "B".   Actual losses are
relatively easy to measure in agricultural or industrial settings
because commodity values are well established by commerce
and trade.   But losses from household insects, vectors of
human disease, and nuisance pests can be much harder to
quantify.   In these cases, estimates of damage are often based
on potential loss (from disease, contamination, etc.) rather
than on actual or expected loss.

Economic Injury Level

The break-even point, where "A"="B", is known as the
economic injury level.   This is the population density at
which the cost to control the pest equals the amount of
damage it inflicts (actual or potential).   Below the economic
injury level, it is not cost-effective to control the pest
population because the cost of treatment (labor plus
materials) would exceed the amount of damage.   Above the
economic injury level, however, the cost of control is
compensated by an equal or greater reduction in damage by
the pest.
The economic injury level (EIL) is often expressed
mathematically by the formula:
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where:

 "C"   is the unit cost of controlling the pest    (e.g.,

$20/acre)
 "N"   is the number of pests injuring the commodity unit   
(e.g., 800/acre)
 "V"   is the unit value of the commodity    (e.g., $500/acre)
  "I"   is the percentage of the commodity unit injured   
(e.g., 10% loss)

For the example given above, the economic injury level

would equal 320 insects per acre:

The economic injury level is usually expressed as a number

of insects per unit area or per sampling unit.   Occasionally,
when the insects themselves are difficult to count or detect,
the economic threshold may be based on a measurement of
injury (e.g., leaf area consumed or number of dead plants).
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It is important to recognize that the economic injury level is a

function of both the cost of pest control and the value of a
commodity or product.   Some commodities may be worth so
little that it is never worth saving them from insect injury (my
wife feels this way about the books I store in my attic).  
The value of other commodities may be so great that any
level of infestation is worthy of control (my wife feels this
way about the food she stores in her cupboard).  Because of
its dependence on both cost and value, the economic injury
level can be calculated only after establishing a value for the
damaged commodity or product.  In practice, this is a difficult
task because different people have different values.
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Economic Thresholds

The economic injury level is a useful concept because it

quantifies the cost/benefit ratio that underlies all pest control
decisions.   In practice, however, it is not always necessary or
desirable to wait until a population reaches the economic
injury level before initiating control operations. Once it is
determined that a population will reach outbreak status,
prompt action can maximize the return on a control
investment.   Since there is usually a lag time between the
implementation of a control strategy and its effect on the pest
population, it is always desirable to begin control operations
before the pest actually reaches the economic injury level.
Consequently, entomologists define a point below the
economic injury level at which a decision is made to treat or
not treat.   This decision point is called the economic
threshold, or sometimes the action threshold.   It is the
decision point for action -- the pest density at which steps are
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first taken to ensure that a potential pest population never

exceeds its economic injury level.   The economic threshold,
like the economic injury level, is usually expressed in units of
insect density or in terms of an injury measurement.   The
economic threshold is always lower than the economic injury
level in order to allow for sufficient time to enact control
measures.

Surveillance of Pest Populations

Effective use of economic thresholds in the management of
insect populations depends on accurate measurements of
population density as well as reliable predictions of
population growth trends.   Since it is not practical to count
all the flies in the barnyard or all the boll weevils in the
cotton field, entomologists depend on sampling strategies to
estimate density and distribution.   Hundreds of sampling
methods have been devised and entomologists continue to
develop and refine their techniques.
An "ideal" sampling strategy requires minimal effort and
gives an accurate and reproducible measure of the density
and/or distribution of an insect population.   In practice, such
"ideal" methods do not exist.   Every technique is inherently
biased in some way.   One method may be better than another
for a particular pest or situation, but no sampling process is
totally random, objective, and repeatable.   The most widely
used techniques, such as sweep nets or bait traps, do not
measure absolute density of pest populations, they are only
relative measures (yardsticks, in a sense) that may be used as
estimates of population density once they are properly
"calibrated" through exerimentation and comparison with
other sampling techniques.
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Sex pheromone traps, for example, may attract male

peachtree borers from several miles downwind.   Without
compensating for such immigration, trap catch data would
greatly exaggerate the size of a local moth population.  
Similarly, sweepnet samples of alfalfa weevils tend to
underestimate the numbers of small larvae in a field (relative
to adults and large larvae) because early instars hide within
the plant's terminal growth and are not easily knocked out
during sweeping.

Pest Control Tactics

The history of pest control probably began with the first
human who ever swatted a mosquito or picked off a louse.  
From the fossil record, we know that all major taxa of biting
flies and external parasites already existed by the time Homo
sapiens first appeared on earth.   Phthirus and Pediculus, the
two genera of lice that feed on humans, have a host range that
is limited to primates (apes and monkeys).   And we suspect
that human fleas (Pulex irritans) and bed bugs (Cimex
lectularius) adopted cave-dwellers as hosts because these
insects are most closely related to other species that live on
bats.   But since our primitive ancestors were hunters and
gatherers, they probably found that insects were more useful
as food than they were troublesome as pests.   (Even today,
people in some primitive cultures eat the lice they pick from
one another's hair).   It was probably not until the dawn of
organized agriculture, when insects attacked the plants we
 SCIENCE LABORATORY TECHNOLOGY

grew for food, that we first recognized them as a potential

threat to our own survival.
Pest control tactics were mentioned occasionally in writings
of the ancient Chinese, Sumerian, and Egyptian scholars.  
Many of these tactics were embedded in religion or
superstition, but a few had real scientific merit.   Predatory
ants, for example, were used in China as early as 1200 B.C.
to protect citrus groves from caterpillars and wood boring
beetles.   Ropes or bamboo sticks tied between adjacent
branches helped the ants move easily from place to place. A
passage in Homer's Iliad (8th century B.C.) describes the use
of fire to drive locusts into the sea, and the ancient Egyptians
organized long lines of human drovers to repel swarms of
invading locusts.   Pythagorus, a Greek philosopher and
mathematician, was credited with clearing malaria from a
Sicilian town during the 6th century B.C. by instructing its
residents to drain the marshes.   Chemical substances that
purportedly killed or repelled insects were in common usage.
Many of these were of questionable value, but some worked,
and a few are still in use today.   Some of the inorganic
compounds, such as sulfur and arsenic, have well-established
insecticidal activity.   And modern science has only recently
come to recognize that many plant extracts used by ancient
apothecaries (e.g, lemon oil, wormwood, hellebore, fleabane,
etc.) do indeed contain compounds with useful activity
against insects.
There was very little progress in pest control during the dark
ages.   Ignorance and superstition abounded.   For what it was
worth, St. Bernhard excommunicated the flies of his parish in
1121.   In a book entitled "Natural History", Ferrante
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Imperato (1599) gave a prescription for eliminating flies from

a dwelling:
"... draw the image of a fly ... on a copper plate during the
second half of the constellation of Pisces ... then bury it in the
center of your house (during) the first half of the constellation
of Taurus."
With the Renaissance, people began to view insects less as a
punishment from God and more as members of a natural
world that could be studied and controlled.   A renewed
interest developed in insects both as organisms and as pests.  
More accurate observations of natural history and behavior
led to more inventive control practices.   Hand labor was used
extensively in early pest control, but cultural, physical, and
chemical practices also evolved.   Franz Ernst Brückmann, a
German physician who lived in the early 1700's, designed the
first mechanical traps for various insects.   His fly traps
consisted of a wooden box baited with a sweet attractant and
equipped with a spring-loaded lid.   When several flies had
settled inside the trap, the lid was snapped shut to trap the
flies inside.   The volume of the trap was then decreased by
sliding the ends of the box together and squashing the flies
against their bait.  Brückmann also designed flea traps.  They
were hollow, perforated cylinders, baited with blood or
honey, and worn around the neck as a pendant.   For a time,
these flea traps, crafted from ivory or silver and often ornate
in design, were popular fashion accessories worn by the
aristocracy of western Europe.

Since the late 1800's, entomologists and chemists have made

outstanding progress in the technology of pest control. 
 SCIENCE LABORATORY TECHNOLOGY

Today's arsenal of weapons is large and diverse,

encompassing legal, cultural, physical, genetic, and biological
tactics, in addition to the well-known chemical pesticides.  In
general, all of these tactics work in at least one of the
following ways:

 They kill the pest directly -- usually by exposing it to lethal

substances or unsuitable environmental conditions.
 They reduce the reproductive potential of a pest
population -- often by modifying its environment (biotic or
abiotic) or by restricting its movement.
 They modify the pest's behavior to make it less
troublesome (attract it, repel it, confuse it, exclude it,
mislead it).

Most of the control tactics that are commonly used today can
be grouped into two broad categories:   natural controls and
artificial controls.   By definition, a natural control may be
any environmental factor that keeps a pest population below
its economic injury level.   Examples might include
geographic barriers, cold temperatures, or natural enemies
that keep population growth in check.   Artificial controls, on
the other hand, employ products or processes of human origin
to modify a pest's distribution, behavior or physiology.   Fly
swatters and insecticides are two obvious examples.   But as
is often true in biology, there are some control strategies that
seem to straddle the line, exhibiting characteristics of both
groups or not fitting neatly into either category.   In fact,
some of our most effective tactics are natural controls that we
can improve, enhance, accelerate, or augment by appropriate
human intervention.
 SCIENCE LABORATORY TECHNOLOGY

Cultural Control
Surprisingly simple modifications of a pest's environment or
habitat often prove to be effective methods of pest control.  
As a group, these tactics are usually known as cultural control
practices because they frequently involve variations of
standard horticultural, silvicultural, or animal husbandry
practices.   Since these control tactics usually modify the
relationships between a pest population and its natural
environment, they are also known, less commonly, as
ecological control methods.   Simplicity and low cost are the
primary advantages of cultural control tactics, and
disadvantages are few as long as these tactics are compatible
with a farmer's other management objectives (high yields,
mechanization, etc.).   Unfortunately, there are still a wide
variety of insect pests that cannot be suppressed by cultural
methods alone.

Crop rotation
Crop rotation is one of the oldest and most effective cultural
control strategies.   Growing a single crop year after year in
the same field gives pest populations sufficient time to
become established and build up to damaging levels.  
Rotating the field to a different type of crop can break this
cycle by starving pests that cannot adapt to a different host
plant.   Farmers in the Midwest, for example, can reduce
populations of wireworms (Elateridae) and rootworms
(Diabrotica spp.) in corn fields by switching to oats, wheat,
or legumes.   Similarly, the clover root curculio (Sitona
hispidula) that feeds exclusively on legumes can be
 SCIENCE LABORATORY TECHNOLOGY

eliminated by switching from clover or alfalfa to corn or

small grains.   Rotation schemes have also proven successful
for controlling pests in pasture lands.   Texas cattle fever, a
protozoan disease transmitted by cattle ticks, Boophilus
annulatus, has been eliminated from the southern United
States due, in part, to a diligent campaign of pasture rotation
designed to protect beef and dairy herds from tick infestation.

Crop rotation schemes work because they increase the

diversity of a pest's environment and create discontinuity in
its food supply.   As a rule, rotations are most likely to be
practical and effective when they are used against pests that:

 attack annual or biennial crops

 have a relatively narrow host range
 cannot move easily from one field to another, and
 are present before the crop is planted

Intercropping
Intercropping (also known as mixed cropping) is another
way to reduce pest populations by increasing environmental
diversity.   In some cases, intercropping lowers the overall
attractiveness of the environment, as when host and non-host
plants are mixed together in a single planting.   But in other
cases, intercropping may concentrate the pest in a smaller,
more manageable area so it can be controlled by some other
tactic.   Strips of alfalfa, for example, are sometimes
interplanted with cotton as a trap crop for lygus bugs
(Miridae).   The alfalfa, which attracts lygus bugs more
 SCIENCE LABORATORY TECHNOLOGY

strongly than cotton, is usually treated with an insecticide to

kill the bugs before they move into adjacent fields of cotton.

In some crops, it is possible to create discontinuity in the

pest's food supply simply by altering the time of year for
planting or harvesting.   This strategy, often known as
phenological asynchrony, allows farmers to manage their
crop so it remains "out of phase" with pest populations.  
Sweet corn, for example, can escape most injury from corn
earworms (Helicoverpa zea) if it is planted in early spring
and harvested before larvae mature.   In the Midwest, farmers
delay planting winter wheat until after the "fly-free date" to
protect their crop from injury by the Hessian fly (Mayetiola
destructor).   These planting dates are calculated to ensure
that wheat is not mature enough to attract egg-laying flies, yet
still has enough warm weather to grow sufficiently before
winter.   Careful timing of harvest dates in alfalfa can cause
high mortality in populations of alfalfa weevil (Hypera
postica) and alfalfa caterpillar (Colias eurytheme) by
removing edible foliage before the larvae have completed
development.   Harvest timing is often the most practical
method available to foresters for controlling bark beetle
(Scolytidae) infestations in pine plantations.   Since trees
become more susceptible to beetle outbreaks as they grow
older, good management dictates that timber stands be
harvested for lumber before the trees reach full maturity.

Managed application
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Managed application of water or fertilizer can have a big

impact on the survival of pest populations in some crops.  
Annual flooding, for example, is a cultural practice that
eliminates many potential pests in cranberry bogs.   Regular
rainfall (or overhead irrigation) can significantly reduce
infestations of twospotted spider mites (Tetranychus urticae)
in tree fruits.   Good irrigation and appropriate fertilization
keeps plants healthy, vigorous, and more resistant to insect
injury.   It is not unusual for small amounts of injury to
actually stimulate compensatory growth in healthy plants.

Sanitation
Sanitation is another cultural control strategy that may be
highly effective for some pests.   Removing crop debris from
cotton fields after harvest eliminates overwintering
populations of pink bollworms (Pectinophora gossypiella),
European corn borers (Ostrinia nubilalis), and sugarcane
borers (Diatraea saccharalis).   Collecting dropped fruit from
beneath an apple tree reduces the next season's population of
apple maggots (Rhagoletis pomonella), codling moths (Cydia
pomonella), and plum curculio (Conotrachelus nenuphar).  
Shredding or burning the pruning wood from a peach orchard
kills shothole borers (Scolytus rugulosus) and lesser
peachtree borers (Synanthedon pictipes) that would otherwise
emerge and reinfest the orchard.   Clean cultivation is often
recommended as a way to eliminate shelter and/or
overwintering sites for pest populations.   Simply tilling or
plowing a corn field before winter may disrupt a pest's life
cycle by causing mechanical injury, by increasing exposure
to lethal cold temperatures, by intensifying predation by birds
or small mammals, or by burying the pests deep beneath the
 SCIENCE LABORATORY TECHNOLOGY

soil surface.   Populations of corn earworms and European

corn borers have been greatly reduced in recent years by
community-wide efforts to plow under corn stubble after
harvest.

Although it works well in corn and cotton, clean cultivation is

not always the best solution for eliminating pest populations.
In some cases, ground cover or crop debris shelters natural
enemies that are important members of the agroecosystem.  
Without adequate shelter these beneficial populations would
die or move away.

Biological Control
Natural control strategies that employ biological agents for
pest suppression are generally classified as biological control
tactics.   In conventional usage, this term usually refers to the
practice of rearing and releasing natural enemies:   parasites,
predators, or pathogens.   A slightly broader definition of
"biocontrol" includes any related management activity that is
designed to protect or conserve natural enemies.
Biocontrol agents include a wide variety of life forms,
including vertebrates, invertebrates, fungi, and
microorganisms.   These beneficial species are common in
most natural communities and, although their presence is
often unnoticed, they help maintain the "balance of nature"
by regulating the density of their host or prey population.  
Insect species often become "pests" when this ecological
balance is disrupted by natural events or human intervention.
Biological pest control strives to reestablish this balance in
one of three ways:
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1. Importation.   Foreign exploration is conducted to identify

and collect natural enemies in the country from which an
exotic pest has been introduced.   Following the discovery
of a potential biocontrol agent, it undergoes extensive
evaluation to insure that its ecology and host range are
compatible with the community to which it will be
introduced and that it will not become a pest once it is
released.   Suitable candidates are reared and released in
the new habitat in hopes that they will become
established and suppress the pest population.
2. Conservation.   A variety of management activities can be
used to optimize the survival and/or effectiveness of
natural enemies.   Conservation activities might include
reducing or eliminating insecticide applications to avoid
killing natural enemies, staggering harvest dates in
adjacent fields or rows to insure a constant supply of
hosts (prey), or providing shelter, over-wintering sites, or
alternative food sources to improve survival of beneficial
species.
3. Augmentation.   Natural enemies that are unable to
survive and/or persist in a new environment can
sometimes be reared in large numbers and periodically
released to suppress a pest population.   In some cases,
small numbers of a beneficial species are released in
several critical locations to suppress local pest outbreaks
(an inoculative release).   In other cases, larger numbers
are released in a single location to flood the pest
population with natural enemies (an inundative release).

  Just because an insect is a predator or a parasite does not

necessarily make it a good biological control agent. Since the early
1900's over 600 species of beneficial insects have been introduced
 SCIENCE LABORATORY TECHNOLOGY

into the United States. Of these, only about 20% have been
outright successes as biological control agents; another 35% have
been partially successful, and the other 45% never became
established or failed to have any significant effect on pest
populations. From these successes and failures, entomologists
have concluded that introduced biocontrol agents are most
effective when they exhibit the following characteristics:

1. Narrow host range. Generalized predators may be good

natural enemies but they don't kill enough pests when
other types of prey are also available.
2. Climatic adaptability. Natural enemies must be able to
survive the extremes of temperature and humidity that
they will encounter in the new habitat.
3. Synchrony with host (prey) life cycle. The predator or
parasite should be present when the pest first emerges or
appears.
4. High reproductive potential. Good biocontrol agents
produce large numbers of offspring. Ideally, a parasite
completes more than one generation during each
generation of the pest.
5. Efficient search ability. In order to survive, effective
natural enemies must be able to locate their host or prey
even when it is scarce. In general, better search ability
results in lower pest population densities.
6. Short handling time.   Natural enemies that consume prey
rapidly or lay eggs quickly have more time to locate and
attack other members of the pest population. Small
populations of efficient natural enemies may be more
effective biocontrol agents than larger populations of less
efficient species.
 SCIENCE LABORATORY TECHNOLOGY

7. Survival at low host (prey) density.   If a natural enemy is

too efficient, it may eliminate its own food supply and
then starve to death. The most effective biocontrol agents
reduce a pest population below its economic threshold
and then maintain it at this lower equilibrium level.

Just as predators and parasites may serve as effective

biocontrol agents for insect pest populations, so insect
herbivores have also proven effective as agents for
controlling weed populations.   Feeding injury by herbivores
may weaken a plant or increase its susceptibility to disease,
desiccation, freezing temperatures, or other herbivores.   In
any case, an injured plant is likely to be a poor competitor,
and in time, beneficial species will have an opportunity to
reclaim the vacated space.
    Biological control is a particularly appealing pest control
alternative because, unlike most other tactics, it does not always
have to be reapplied each time a pest outbreak occurs.   Once
natural enemies are released into a new environment, there is a
good chance they will become established and provide a self-
perpetuating form of control.   Biological control is also the only
control tactic that increases, rather than decreases, the species
diversity within an agroecosystem.   This increased diversity often
results in greater stability because wild fluctuations in population
density are less common in communities with a diverse food web.

    Biological control is not a "quick fix" for most pest problems.  

Natural enemies usually take longer to suppress a pest population
than other forms of pest control and farmers often regard this as a
 SCIENCE LABORATORY TECHNOLOGY

disadvantage.   It also may be difficult to "integrate" natural

enemies into a crop or commodity when pesticides are still in use.
Beneficial insects are often highly sensitive to pesticides and their
resurgence (recovery to pre-spray densities) is usually much
slower than that of pest populations.   Rapid pest resurgence
often leads to a vicious cycle of continued chemical usage that
prevents natural enemies from ever becoming reestablished.

Perhaps the greatest potential for future progress in biological

control lies in improving the success of microbial pathogens.
Many of these organisms are highly desirable as biocontrol
agents:   they attack a narrow range of insect hosts, they are
not hazardous to humans or domestic animals, and they do
not pose a threat to the environment.   As a group, though,
pathogens have never proven to be quite as reliable as other
forms of pest control.   They are vulnerable to desiccation,
ultraviolet radiation, and high temperatures.   They may not
survive long enough in the environment to encounter a host,
and even if they survive, their virulence may be too low to
overcome the host's defenses.
In the past few years, however, there has been encouraging
progress in the development of microbial insecticides.   These
are commercial suspensions of spores, toxins, or virus
particles that can be mixed with water and sprayed onto crops
just like conventional insecticides (see Table 2).   In many
cases, microbial insecticides are better than conventional
insecticides because they suppress pest populations without
eliminating natural populations of predators and parasites.
A great deal of research is still needed before we can begin to
benefit from the full potential of biocontrol.   Fortunately,
new developments in biotechnology may soon enable us to
 SCIENCE LABORATORY TECHNOLOGY

create new strains of microbial pathogens that are more

virulent, easier to mass produce, and less sensitive to
temperature and humidity.   Work is currently underway to
transform a common soil microbe, Pseudomonas fluorescens,
into a pathogen of soil-dwelling insects by implanting the
delta-endotoxin gene from Bacillus thuringiensis. Clearly,
there are major changes in biocontrol that lie just over the
horizon.

Breeding for Host Resistance

Breeding plants (or animals) for resistance to insects is really
just another form of biological pest control.   Rather than
finding insects to attack the pests, breeders look for genetic
traits (or combinations of traits) that reduce an organism's
susceptibility to attack or injury by its insect pests.
This idea was first tested in the 1870's by C. V. Riley, an
entomologist who successfully fought a French outbreak of
phylloxera (an aphid-like pest of grapes) with resistant North
American rootstocks.   Indeed, much of the pioneering work
in this field has been done with crop plants because they are
usually cheaper to breed and easier to manage than livestock.
Recent innovations in genetic engineering, however, are
likely to accelerate development of resistant genotypes in
poultry, swine, and other commercial livestock.
In general, there are three approaches that plant breeders use
to develop resistant cultivars:

1. Antibiosis.   Plants produce a wide variety of defensive

compounds (allelochemicals) that protect them from
herbivores.   These compounds may reduce growth,
SCIENCE LABORATORY TECHNOLOGY

inhibit reproduction, alter physiology, delay maturation,

or induce various physical or behavioral abnormalities in
herbivores.   By purposely selecting for plants with high
levels of allelochemicals, or by breeding such plants with
less resistant ones, it is often possible to develop new
cultivars that resist pest injury yet still retain desirable
horticultural characteristics.
2. Antixenosis.   A physical or chemical property of a plant
can make it so unpalatable that it is largely protected from
herbivore attack.   This type of resistance is often known
as nonpreference.   It may involve the presence of feeding
repellents (or the absence of feeding attractants), or it
may involve physical traits such as hairs, waxes, or a thick,
tough epidermis that do not provide the pest with a
desirable feeding substrate.   Alfalfa, for example, has
been bred with hairy leaves to deter feeding by the
spotted alfalfa aphid.
3. Tolerance.   Some plant genotypes are simply able to
"tolerate" injurious insects better than others.   Tolerant
cultivars may be exposed to the same pest populations as
susceptible ones, but they do not suffer as much injury.  
Many varieties of field corn, for example, have fairly
narrow, brittle stalks.   When attacked by European corn
borers, these stalks are further weakened and break easily
in a windstorm.   This wind damage, known as lodging,
makes the corn hard to pick with mechanical harvesters.  
Plant breeders have largely resolved this problem by
breeding for corn with thicker, stronger cornstalks.   These
tolerant cultivars are still attacked by corn borers, but
they "stand up" to the injury and insure a harvestable
crop.
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Developing resistant cultivars by traditional breeding

methods can be a slow and uncertain process.   But new
advances in biotechnology now make it possible to pluck
genes from one organism and insert them into the cells of a
completely different species.   For example, the delta-
endotoxin gene from Bacillus thuringiensis, an insect
pathogen, has already been isolated and inserted into crop
plants (notably cotton and tobacco).   Cells of the genetically
engineered plants can produce their own toxin, making them
"resistant" to certain insect herbivores (e.g., Lepidoptera).  
Although gene-splicing techniques are still in their infancy,
they offer exciting new possibilities for expanding the
diversity of resistant plant genotypes.

Legal & Regulatory Control

At first, it may seem ludicrous to even contemplate the use of
legal measures as pest control strategies -- it's easy enough to
pass laws, but how do you train the insects to obey them?  In
reality, legal sanctions are aimed, not at the insects
themselves, but at a range of human behaviors that are most
likely to affect the dynamics of pest populations.  Legal
control tactics, therefore, include all forms of legislation and
regulation that might prevent establishment or reduce spread
of an insect population.

The Plant Quarantine Act of 1912 was the first legal action
taken in the United States to prevent the introduction of pests
from foreign countries.  This law, and others that followed it,
established a network of inspection stations at major ports of
entry and gave the federal government authority to organize
 SCIENCE LABORATORY TECHNOLOGY

border quarantines, to inspect all agricultural products, and to

restrict entry of any infested goods.   Today, these inspections
stations operate under the jurisdiction of the Animal and
Plant Health Inspection Service (APHIS), a branch of the U.
S. Department of Agriculture.  APHIS operates about 85
inspection facilities, employs 400-500 inspectors, and
intercepts over 20,000 potential pest introductions in an
average year.   It also operates the Plant Quarantine Training
Center where inspectors from all over the world are trained to
spot problems before they cross international borders.  These
agents, now representing nearly 40 countries, cooperate in
efforts to limit the spread of indigenous pests under an
international agreement that requires inspection and
certification of nearly all agricultural commodities prior to
export.

Federal import/export laws have been enacted to establish

standards for a wide range of agricultural products.  Most of
the fruits and vegetables imported to the U.S., for example,
must receive some form of fumigation, heat treatment,
controlled atmosphere storage, or radiation exposure before
they are released from quarantine.  These post-harvest
treatments are designed to kill whatever pest species may be
present without affecting taste or quality of the produce.

Despite the best efforts of APHIS inspectors, some pests still

slip into the U. S. and become established (e.g., cereal leaf
beetle), or move naturally across international borders (e.g.,
imported fire ants and Africanized honey bees).  When this
happens, APHIS cooperates with state and local governments
to establish domestic quarantines for limiting the movement
of these pests within our borders.  Whenever appropriate,
 SCIENCE LABORATORY TECHNOLOGY

eradication programs may be launched together with

quarantine actions in an effort to destroy the entire pest
population while it is still small.

Inspections and quarantines are also operated by some state

and local government agencies.   The California Border
Patrol, for example, is notorious for its interceptions of
uncertified agricultural products at the state line.  The rigid
laws that prohibit private citizens from bringing a bag of
oranges into southern California are generally given credit for
excluding numerous pest species that might have caused
millions of dollars in losses.  The price tag for these
prevention efforts is only a fraction of the estimated $350
million Californians spent to clean up their most recent
infestation of the Mediterranean fruit fly (Ceratitis capitata).

Insects Currently Under Quarantine in the United

States

Since 1915, nearly 300 pest species have been under some
sort of domestic quarantine (see Table 1).   The success of
these programs has been erratic.   In some cases (e.g.,
screwworm flies, khapra beetles, and parletoria date scale),
eradication programs have managed to eliminate the pests.  
But for every noteworthy success, there has been a complete
failure (witness the cereal leaf beetle, the gypsy moth, and the
European corn borer).   In general, eradication efforts are
most successful when the pest population is relatively small,
does not disperse rapidly, and is highly susceptible to control
tactics.
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Even if a newly established pest population cannot be

eradicated, it can often be slowed down a bit by regulating
the movement of its host plants or animals, prohibiting sales
of infested products, and inspecting all shipments of affected
goods.   This type of containment strategy usually gives
growers and producers a short reprieve before invasion of the
pest and buys a little more time for researchers to find better
methods of control.   By using the containment approach in
Texas during a 1971 outbreak of Venezuelan equine
encephalitis, entomologists and veterinarians had enough
time to reduce populations of mosquito vectors and immunize
nearly 3 million horses before the disease could spread into
other states.

Licensing and certification are important regulatory tools

used in some commodities to ensure that infested or
contaminated material is not sold commercially or used as
breeding stock.   Maintaining disease-free mother plants in
strawberries, for example, can often prevent the spread of
insect-borne disease during the growing season.   Certified
seed or plant material is produced under strict guidelines
established by commodity organizations in their own
interests.   These products may be more expensive initially,
but eventual savings in pest control usually provide a good
return on the investment.

Chemical Controls:
Semiochemicals
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Most people understand that chemical pest control involves

the use of chemical substances to kill or disrupt the life cycle
of an insect pest.   But few people outside the circle of
entomology realize just how diverse these compounds are,
and in how many different ways they can be used.   Although
conventional insecticides, the poisons, are still a mainstay of
chemical control, they are gradually being superseded by less
toxic compounds that disrupt insect development or modify
behavior.   Some of these new chemical weapons are much
safer for the environment and more species specific than most
conventional insecticides.

Semiochemicals -- Chemical Control

of Behavior
Much of an insect's behavior is mediated by chemicals in its
environment.  By turning these chemicals to our own
advantage, it is often possible to attract pests to traps or baits,
or repel them from our homes, our crops, or our domestic
animals.  Behavioral messages are delivered by a wide array
of chemical compounds.   As a group, these compounds are
known as semiochemicals. In some cases, they may facilitate
communication between the members of a single species
(e.g., pheromones) or between members of different species
(e.g., allelochemicals).   Functionally, semiochemicals may
have a wide range of activity.   They may serve as attractants
or repellents, they may stimulate or inhibit feeding, they may
provoke flight or inhibit it, or they may simply elicit behavior
patterns at inappropriate times.
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Attractant pheromones and allelochemicals can be used as

lures or baits in a wide variety of insect traps, or they can be
mixed together with toxicants to produce an "elixir of death".
Protein hydrolysates, for example, serve as feeding attractants
for fruit flies (Rhagoletis spp.).  These chemicals can be
applied to sticky traps to improve catch, or combined with an
insecticide and sprayed on fruit crops to suppress active
infestations.   Phenethyl propanoate, eugenol, and geraniol
can be mixed in a 3.5:3.5:3 ratio and used as an attractant for
Japanese beetles (Popillia japonica).  These are the active
ingredients in the "floral attractant" found in popular bag
traps for Japanese beetles.   In some cases, chemists have
produced synthetic compounds that are even more attractive
than naturally occurring chemicals.   Trimedlure, a synthetic
substitute for alpha-copaene, is produced commercially as an
attractant for the Mediterranean fruit fly (Ceratitis capitata).  
Improved food lures and baits are among the most promising
new developments for controlling cockroaches (Blattoidea) in
homes and businesses.   These are the active ingredients in a
new generation of "roach motels" where the insects "check in
but don't check out."

Sex pheromones are among the most powerful of chemical

attractants.   Ever since they were first discovered by A. A.
Budenandt in 1959 (from silkworm moths, Bombyx mori),
these chemicals have aroused great interest because of their
potential as pest control agents.   During the past 30 years,
chemists have identified the sex pheromones for over 300
insect species.   Many of these compounds are now sold
commercially.   In some cases, pheromones are packaged (or
encapsulated) in slow-release dispensers (rubber septa,
hollow fibers, or rope wicks) that are used as lures in traps of
 SCIENCE LABORATORY TECHNOLOGY

various designs.   At low densities, these pheromone traps are

a valuable monitoring tool, providing information on the
density and distribution of pest populations.   At high
densities, they can be used for mass trapping sexually active
adults (usually males) in efforts to reduce population density
and lower a pest's reproductive potential.

Slow-release formulations of sex pheromones can also be

used for mating disruption.   By increasing the
concentration of pheromone in an insect's environment, it
may be possible to make everything smell like a prospective
mate.   Males wear themselves out courting inanimate objects
or become habituated to the odor and stop responding to it.  
This approach, variously known as air permeation or the
innundation technique, has shown promise for controlling a
number of fruit and vegetable pests, including the codling
moth (Cydia pomonella), the cabbage looper (Trichoplusia
ni), the oriental fruit moth (Grapholita molesta), and the
peachtree borer (Synanthedon exitiosa).

Chemical repellents and feeding deterrents are also useful

tools for managing insect behavior.   As their name suggests,
these compounds cause insects (or other arthropods) to
disperse or to discontinue normal feeding behavior.  
Repellents such as dimethyl phthalate, benzyl benzoate, and
N,N-diethyl-m-toluamide (DEET) have been developed to
protect humans from biting flies, ticks, and chiggers
(immature trombiculid mites).   Other compounds, like di-n-
butyl succinate or butoxypolypropylene glycol, are used as
fly repellents for cattle.   Moth balls and flakes
(paradicholorobenzene or alpha-naphthalene) are placed in
drawers and closets to prevent infestation by a variety of
 SCIENCE LABORATORY TECHNOLOGY

insects that feed on stored products and natural fibers.  

Woolen cloth can be manufactured with colorless dyes (e.g.,
Eulans and Mitin FF) that bond permanently to the fabric and
make it unpalatable to clothes moths and carpet beetles.  
Wood preservatives, such as pentachlorophenol, act as
feeding deterrents to termites and other wood-dwelling
insects.

The neem tree, Azadirachta indica (Meliaceae) is a promising

new source of feeding repellents that may be developed for
use on selected non-crop plants.   The leaves, twigs, and
seeds of this tree, which is grown commercially in India,
contain at least 25 biologically active compounds that act as
insect repellents, feeding deterrents, or growth regulators.  
Azadirachtin, the most abundant of these active ingredients,
is now commercially available in the United States.   Sold
under the trade name Margosan-O, this new feeding deterrent
has been approved for use on non-food greenhouse crops,
ornamentals, and turf.

Chemosterilants -- Chemical Control

of Reproduction
 SCIENCE LABORATORY TECHNOLOGY

There are over four hundred chemical substances that are

known to cause reproductive sterility in insects.  Some of
these compounds inhibit ovarian growth and development,
while others appear to induce fundamental changes in the
chemical structure of nucleic acids (DNA and RNA). These
changes (mutations) prevent cell division or obstruct normal
embryonic development.  Chemosterilants belong to several
major chemical groups (see Table 1).  These compounds are
applied directly to the insect or incorporated into food that
serves as a bait.

All chemosterilants are extremely hazardous compounds.  

Their effects are not restricted to insects; they also cause
cancer, birth defects, and other mutations in humans and
domestic animals  Clearly, these chemicals cannot be
dispersed in the environment like other pesticides.   Instead,
they must be applied under controlled laboratory conditions,
usually to insects that are mass reared and released as part of
a sterile release program.  Although there is much interest in
finding a chemosterilant whose effects are limited to insects,
no such compound has yet been found.

Insect Growth Regulators -- Chemical

Control of Development
The enzymes and hormones that regulate developmental
processes within an insect's body can sometimes be exploited
as chemical control weapons.   These compounds, often
known as insect growth regulators (IGRs), can be used to
stimulate development at inappropriate times or inhibit it at
other times.   The major groups of IGR compounds include:
 SCIENCE LABORATORY TECHNOLOGY

Chitin inhibitors.   These chemicals (e.g., diflubenzuron and

teflubenzuron) inhibit the molting process (apolysis) by
blocking the activity of chitin synthetase, an enzyme needed
by epidermal cells when constructing a new exoskeleton.  
Because of this mode of action, chitin inhibitors are highly
specific to arthropods.   They act rather slowly (2-5 days), but
eventually disrupt any process that involves construction of
new cuticle (e.g., molting, hatching, pupation).   They are
most effective when used against the immature stages of a
pest.   Diflubenzuron, currently registered under the trade
name Dimilin, is used for controlling gypsy moths, boll
weevils, and various other pests.

Molting Hormone Analogues.   Ecdysteroids stimulate the

molting process by mimicking the action of molting
hormone.   Applied to the surface of an insect's body or
incorporated into its food, these compounds work by
initiating premature ecdysis during the immature stages of
development.   Ecdysteroid-like compounds have been found
 SCIENCE LABORATORY TECHNOLOGY

in some plants where they evidently serve as a defense

against insect herbivores.   But despite their potential as
insect growth regulators, the ecdysteroids have never been
developed into commercial products.   Their chemical
structural is similar to that of human reproductive hormones
(estrogen, progesterone, and testosterone), and like many
other steroid compounds, they have the potential to cause
cancer and birth defects.

Juvenile Hormone Analogues.   Juvenile hormone (JH) and

related compounds act as insect growth regulators by
inhibiting the developmental changes associated with
embryogenesis, morphogenesis, and reproduction.   During
normal development, JH levels are elevated in larvae (or
nymphs) and decrease prior to pupation (or adult eclosion).  
Contact exposure to JH analogues during the egg stage or
after the last larval (or nymphal) molt can inhibit
development, delay maturation, and eventually result in
death.   Since the onset of mortality is usually quite slow
(days to weeks), JH analogues have limited utility in
agriculture.   But several compounds (e.g., hydroprene,
kinoprene, and methoprene) have been successfully
incorporated into household products for controlling ants,
fleas, and other household pests.
 SCIENCE LABORATORY TECHNOLOGY

Anti-juvenile Hormones.   These unique compounds (the

precocenes) were first isolated in 1976 from a common
houseplant (Aegeratum houstonianum).   Precocenes are
cytotoxic agents.   They become activated by enzymes in the
insect's corpora allata, selectively destroying these glands,
and preventing all subsequent production of juvenile
hormone.   In immature insects, exposure to anti-JH
compounds may result in premature (precocious)
development of adult structures or behaviors.   In adults,
precocenes can cause sterility because the presence of
juvenile hormone is necessary for normal production of eggs
and sperm.   Anti-JH compounds seem to be most effective
against Hemipterans.   Despite their unique mode of action,
these IGRs have never been developed into commercial
products because they break down too rapidly in the presence
of oxygen.

Conventional Insecticides -
The Killer Chemicals
Conventional insecticides are among the most popular
chemical control agents because they are readily available,
rapid acting, and highly reliable.   A single application may
control several different pest species and usually forms a
persistent residue that continues to kill insects for hours or
even days after application.   Because of their convenience
 SCIENCE LABORATORY TECHNOLOGY

and effectiveness, insecticides quickly became standard

practice for pest control during the 1960's and 1970's.  
Overuse, misuse, and abuse of these chemicals have led to
widespread criticism of chemical control and, in a few cases,
resulted in long-term environmental consequences.
The effectiveness of an insecticide usually depends on when
and where the pest encounters it.   Most insecticides are
absorbed directly through an insect's exoskeleton.   These
compounds are known as contact poisons because they are
effective "on contact".   Other insecticides act as fumigants.  
They are released in the vapor state (as gases) and enter the
insect's body through its tracheal system.   Fumigants are
most effective when they are used in an enclosed area such as
a greenhouse, a warehouse, or a grain bin.   Still other
compounds must be ingested before they have an effect.  
These are known as stomach poisons.   They often work more
slowly than fumigants or contact poisons, but they are still
useful for certain types of pest control in homes and
businesses.
Systemic insecticides are a special type of stomach poison.  
These compounds are absorbed by the tissues of a plant (or
animal) without ill effects.   Insect pests ingest the insecticide
when they feed on the treated organism.   Systemic
insecticides are sometimes included in the diets of domestic
animals to protect them from internal parasites (e.g., cattle
grubs and other bot flies).   Plant systemics can be
incorporated into the soil around ornamentals or bedding
plants.   The insecticides are absorbed by the roots and
translocated to leaves, stems, and flowers.   If the insect that
feeds on a treated plant doesn't acquire a lethal dose of
insecticide, it may at least be deterred from further feeding.
 SCIENCE LABORATORY TECHNOLOGY

Although systemic insecticides are commonly applied to

horticultural plantings, they are not as useful for many food
crops because the insecticide remains in the food after
harvest.
  Insecticides contain one or more active ingredients that serve as
toxicants (poisons).   In their purest form (technical grade), these
chemicals may be too toxic, too unstable, or too volatile to be
handled or applied safely.   Therefore, technical grade insecticide
is always mixed with other compounds, known as adjuvants, in
order to improve the performance, safety, or handling
characteristics of a commercial product.   These mixtures
(technical grade insecticide plus adjuvants) are known as
formulations.   Almost anything could be an adjuvant:   pumice,
ground walnut shells, buffalo gourd root powder, vegetable oil,
etc.   These compounds are usually listed on the label as "inert
ingredients", but they are certainly not inactive.   Many adjuvants
are proprietary products, protected by patents and closely
guarded as industrial secrets.   They may represent 90-95% of the
total volume of a commercial formulation (see Table 1).

An insecticide can be manufactured in a variety of

formulations, each tailored for specific applications. Some of
the more common formulations include:
Granules (G) or pellets (P) -- Coarse particles (e.g., clay,
ground corn cobs, or walnut shells) can serve as a carrier for
certain types of insecticides.   The toxicant slowly leaches out
of the carrier, minimizing its movement within the ecosystem
and maximizing its active life (persistence).   Granular
formulations are commonly used for controlling soil-dwelling
insects and in systemic insecticides (see above) that are
 SCIENCE LABORATORY TECHNOLOGY

applied around the base of plants.   Since granular

formulations do not blow or drift, they are considered
relatively safe from the standpoint of accidental human
exposure.
Dusts (D) -- These dry powders are usually formulated with
inert particles of ash, chalk, talc, or clay.   They are designed
to be sprinkled or blown onto target surfaces.   The powder
sticks to feet, legs, and other body parts of passing insects,
and the toxicant is eventually ingested when the insect cleans
itself.   Insecticides that act as stomach poisons are often
formulated as dusts.   These formulations are not always
suitable for outdoor applications because they have a
tendency to drift (blow away with the wind).

Soluble powders (SP) or wettable powders (WP) -- Dry

formulations that are mixed in water to form homogeneous
spray solutions.   A soluble powder dissolves completely in
water, whereas a wettable powder contains an emulsifier that
produces a uniform suspension (colloid).   Many foliar
insecticides are formulated as wettable (or soluble) powders
because they are easy to transport and generally have a long
shelf life.
Emulsifiable concentrates (EC) -- These liquid formulations
contain the toxicant(s) and an emulsifier dissolved in organic
solvent.   The concentrate is diluted with a large volume of
water to produce the final spray mixture.   Some foliar
insecticides are formulated as emulsifiable concentrates.  
Unlike most wettable powders, they do not leave a visible
residue on fruits and vegetables.   Sensitive plants, however,
may be injured by organic solvents in the mixture.  
 SCIENCE LABORATORY TECHNOLOGY

Emulsifiable concentrates are also commonly used in sprays

for urban and industrial pests.
Aerosols (A) -- Insecticides that are formulated together with
a solvent may be pre-packaged in pressurized spray cans or
sold unpressurized for use in special fogging machines.  
Spray cans are relatively expensive (per pound of active
ingredient) but they are convenient, easy to store, and have a
long shelf life.   Commercial foggers are typically used
indoors (e.g., greenhouses and warehouses) or for control of
biting flies in community-wide pest control operations.
Ultralow-Volume Concentrates (ULV) -- These highly
concentrated formulations (more than 8 pounds of active
ingredient per gallon) are designed to be used in specialized
spray equipment that atomizes the concentrate droplets.  
ULV spray equipment is used by most aerial applicators
(airplane sprayers) who treat forested lands or large
agricultural acreages.
Despite their many advantages, conventional insecticides are
not ideal pest control agents.   Indeed, one of their greatest
strengths, broad-spectrum activity, is also one of their
greatest weaknesses.   While it is certainly an advantage to
control multiple pest species with a single chemical
treatment, the non-specificity of most conventional
insecticides poses a serious threat to non-target organisms in
the environment.   High mortality among natural enemies can
have an enduring impact on the ecological balance of any
community.   In the absence of biocontrol agents, more
insecticide applications may be the only recourse available to
stop pest resurgence.   Once we step onto this "insecticide
treadmill", it can be very difficult to get off.
 SCIENCE LABORATORY TECHNOLOGY

The non-target effects of insecticides are not limited to

natural enemies.   Most of the organochlorine compounds
(e.g., DDT) have been banned from use in the United States
because of their indirect effects on reproduction in birds of
prey (e.g., eagles, ospreys, condors, etc.).   These
environmentally stable compounds are highly soluble in
lipids, making it possible for them to accumulate in the body
fat of non-target organisms.   Predators, particularly those at
the top of a food chain, amass pesticide concentrations many
times greater than anywhere else in the environment.   This
process, known as bioaccumulation (or biomagnification),
was responsible for high levels of DDT and related
 SCIENCE LABORATORY TECHNOLOGY

compounds in birds of prey during the 1960's and 1970's.  

These pesticides did not injure the birds themselves, but
caused thinning of their egg shells and high rates of breakage
during incubation.   Most organochlorine insecticides were
banned during the 1970's and 1980's and many of the
threatened bird species are now recovering from the brink of
extinction.
In some cases, sub-lethal concentrations of an insecticide can
stimulate rather than suppress the growth of a pest
population.   This phenomenon, known as hormoligosis, has
been observed in a number of pest species, including
twospotted spider mites (Tetranychus urticae), western corn
rootworms (Diabrotica virgifera), and brown planthoppers
(Nilaparvata lugens).   Low doses of pesticide seem to
improve the nutritional quality of host plants, thereby
increasing a pest's reproductive potential or decreasing its
time of development.
And finally, there is the problem of resistance to insecticides.
Insects are among the most adaptable organisms on the face
of the earth.   For the past 400 million years they have
managed to survive by adjusting to changes in their
environment, so it should come as no surprise that they can
also adapt to chemical pesticides.   Resistance has increased
exponentially since the the late 1940's, and today, there are
over 500 pest species that exhibit some level of resistance to
at least one type of insecticide.
Insects may become resistant to insecticides in several ways.  
Biochemical resistance usually involves changes in the
metabolic pathways that insects normally use to break down
plant defenses and other environmental toxins.   This
detoxication is facilitated by enzymes (esterases, hydrolases,
 SCIENCE LABORATORY TECHNOLOGY

transferases, and oxidases) that change the chemical structure

of toxicants before they cause physical harm.   Physiological
resistance involves functional changes in basic life processes
that alter the way toxicants interact with the body.   Some
German cockroaches (Blatella germanica), for example, have
become resistant to carbaryl (a carbamate insecticide) as a
result of genetic changes in the permeability of their cuticle.  
Behavioral resistance may occur as a result of any innate
change in behavior that reduces an insect's probability of
encountering a toxicant.   In some parts of Panama, the
mosquitoes that vector malaria (Anopheles albimanus) have
become hypersensitive to certain insecticides.   They manage
to escape lethal doses of insecticide by avoiding enclosed
areas and refusing to land on treated surfaces.
When an insect population becomes resistant to one
insecticide, it may also become less susceptible to other
toxicants in the same chemical family.   This phenomenon,
known as class resistance, is a common problem in all major
groups of insecticides (organochlorines, organophosphates,
carbamates, and synthetic pyrethroids).   When an insecticide
loses its effectiveness because of pest resistance, users
typically replace it with another compound from a different
chemical group.   But resistance to one group of compounds
does not prevent subsequent development of resistance to
compounds in other chemical groups.  
Over the years, some pests have developed multiple
resistance to nearly every insecticide that has ever been used
to fight them.   Colorado potato beetles, Leptinotarsa
decemlineata, on Long Island, N.Y. are probably the best
example of a population that has developed multiple
resistance.   It may never be possible to prevent insects from
 SCIENCE LABORATORY TECHNOLOGY

developing resistance, but there are ways to manage these

chemicals that will prolong their useful life.

Integrated Control
Just as ground, air, and naval forces are integrated to achieve
military objectives, the tactical weapons of pest control can
also be integrated to achieve more effective management of
pest populations.  Development of resistance, effects on non-
target organisms, and damage to the environment can all be
minimized with selective and judicious use of multi-faceted
control tactics.  This approach, commonly known as
integrated control, requires an understanding of ecological
principles as well as a thorough knowledge of the pest's life
history and population dynamics.

Integrated pest control is not a new concept.  It was

commonly practiced in the years before synthetic organic
insecticides became widely available.  But the old ways were
largely abandoned after World War II because chemical
weapons were so effective, convenient, and inexpensive. 
Once we recognized the dangers of over-dependence on a
single control strategy, the principles of integrated pest
control gained renewed acceptance.
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Today, integrated pest control forms the foundation of

Integrated Pest Management programs (IPM) that take a
comprehensive and multi-disciplinary approach to solving
pest problems.  Insects, weeds, plant diseases, and even some
vertebrate pests (e.g., birds and rodents) are included under
the IPM umbrella.  These programs emphasize management
rather than eradication.  They take a broad ecological
approach to pest problems, focusing on all members of a pest
complex in an effort to identify the optimum combination of
control tactics that will reduce pest populations below
economic thresholds and maintain these levels with the least
possible impact on the rest of the environment.  This
approach, often called biorational pest control, relies heavily
on cultural and biological tactics that are supplemented with
carefully timed applications of highly selective chemical
weapons.
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The complexity of modern IPM programs will continue to

increase as we add more knowledge about pests, develop new
management tactics, and learn how to optimize existing
control strategies.  Mathematical models of population
growth rates and plant-pest interactions are being developed
and incorporated into computer programs that help us
assimilate and interpret data from the many different
variables that affect pest population dynamics.  These
computer programs, usually known as expert systems, are
yet another tool in our expanding stockpile of pest control
technology.

Integrated pest management is also a major component of

Sustainable Agriculture, a holistic approach to modern
farming that encompasses not only pest control, but also
conservation of soil and water resources, and a wide range of
other management practices (e.g., composting, mulching,
green manure, waste disposal, etc.).  The goal of sustainable
agriculture is to satisfy human needs for food and fiber while
preserving nonrenewable resources, protecting environmental
quality for future generations, and safeguarding the
profitability and long-term viability of commercial
agriculture.

But regardless of what it is called or how it is packaged, the

basic steps of integrated pest control remain the same:

1. Identification.  Detecting the presence of a pest and

identifying it to species.  Closely related species may
be very similar in appearance, but have significantly
different pest potential.
 SCIENCE LABORATORY TECHNOLOGY

2. Quantification.  Sampling to measure population

density.  How is population density changing with
time?
3. Determination.  Finding out where the population
stands relative to economic injury levels.  How much
more growth potential is left in the population?
4. Specification.  What type of control is warranted? 
What tools or resources are needed to implement a
control operation?
5. Application.  Taking whatever steps are necessary to
suppress the pest population.
6. Evaluation.  Confirm efficacy of control tactics by
resampling.  Re-evaluate the situation and take
appropriate actions if needed.

Resistance to Pesticides
Although pesticides are designed to kill pest populations,
they are seldom 100% effective -- a few individuals usually
survive and reproduce.  These survivors may have a
behavioral trait that helps them avoid the pesticide, a
biochemical trait that allows them to detoxify the pesticide, or
some other genetic characteristic that reduces their
susceptiblity to the pesticide.  If these survivors mate and
pass on this "resistance" to their offspring, then subsequent
generations will contain fewer susceptible individuals. 
Eventually, the entire population may become "resistant".  
There are two major variables that determine the rate at
which a resistant trait is likely to spread throughout the
population:
 SCIENCE LABORATORY TECHNOLOGY

1. Its mechanism of inheritance (dominant, recessive, or

co-dominant; sex linked, pleiotropic, or polygenic),
and
2. The severity of selective pressure (what percentage of
susceptible individuals survive each generation and
whether mortality occurs before or after the
susceptible individuals reproduce).

In general, resistance will spread through a population most

rapidly when it is inherited as a single, dominant allele and
selective pressure is high (meaning very few susceptible
individuals escape and reproduce).

Class Resistance and Cross Resistance

When an insect population develops resistance to one
pesticide, it may also prove to be resistant to similar
compounds that have the same mode of action.  This
phenomenon, known as class resistance, occurs frequently in
pest populations that develop resistance to organophosphate,
carbamate, or pyrethroid insecticides.  In some cases, a
population may develop a form of resistance that protects it
from compounds in more than one chemical class.  This cross
resistance may produce a population that can no longer be
controlled with chemical insecticides.

Resistance Management
Frequent and/or indiscriminant use of chemical insecticides
typically leads to the development of resistance and to a need
for more powerful poisons.  But wise use of pesticides can
 SCIENCE LABORATORY TECHNOLOGY

delay (or maybe even prevent) the evolution of resistant

genotypes.  There are basically three strategies that can be
used to "manage" the resistance problem:

1.   Management by Saturation involves excessive, heavy,

or frequent use of a pesticide that is designed to leave
absolutely no survivors.  This strategy overwhelms a
population's resistance, suppresses any detoxification
mechanism, and precludes reproduction by survivors.  It is
most effective when the resistant gene is dominant and the
target population is small, isolated, or living in a limited
habitat (e.g. greenhouse).

2.   Management by Moderation uses only the minimum

control necessary to reduce a population below its economic
threshold.  Growers use low rates, short-residual compounds,
infrequent applications, and/or incomplete coverage to ensure
that significant numbers of susceptible individuals survive
and reproduce.  This strategy tries to ensure that susceptible
genes are never eliminated from the population.  It works best
when the susceptible trait is dominant over the resistant trait.

3.   Management by Multiple Attack involves the use of

several control tactics that work in different ways.  By
rotating insecticides with different modes-of-action or by
alternating chemical with non-chemical control tactics, a pest
population is exposed to selective pressures that change from
generation to generation.  Natural selection for a resistant
genotype is less likely to occur when selective pressures are
highly variable.

 
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CHARACTERISTICS OF RESISTANCE
MANAGEMENT STRATEGIES
Saturation Moderation Multiple Attack
Low rates
High rates
Infrequent
Frequent Rotate treatments
applications
applications
Use mixtures
Short residuals
Long residual
Alternate with
Apply after
Apply before non-chemical
reproductive
reproductive stage controls
stage
Eliminate refugia
Preserve refugia
SCIENCE LABORATORY TECHNOLOGY