ADAMA SCIENCE AND TECHNOLOGY UNIVERSITY

SCHOOL OF APPLIED NATURAL SCIENCE

PROGRAM OF APPLIED BIOLOGY

General Biology Practice (Biol1111)

Teaching Manual

Adama, Ethiopia
October 2017

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Preface
This Laboratory Manual was prepared for the course ‘General Biology Practice (Biol1111)’
which is given to all freshman prescience students joining the School of Applied Natural
Science, Adama Science and Technology University. The course has 10 laboratory studies with
different sessions. Authors for this manual recommend that the students should familiarize
themselves by reading the experiments to be performed in the manual. Therefore, the
experiments should be read before coming to the laboratory, but the practical exercise should be
done in the laboratory class itself. The students should individually prepare a report and submit
the report at the end of each experiment off course with the advice of an instructor. The
background given in each laboratory study will give sufficient theoretical information about the
lab.

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 Instruction to students

1. Come to the laboratory in time.

2. Always bring your lab manual, separate notebook for the course, pencil, eraser, and ruler.
3. If you miss a lab for reasons beyond your control, report the case to the lab research
assistant in time. Only reasonable excuses shall be accepted to arrange a make-up lab;
otherwise, you will receive zero for the lab you missed.
4. Most laboratory activities are group work. In such cases all the members of the group
should participate in the group activities.
5. You are responsible for all laboratory equipment provided for your use. If you observe
any damage on your equipment, report at the beginning of the period, otherwise you be
held responsible.
6. You individually write neat and readable lab report for each practical and submit it to the
instructor in time. The lab report writing format is attached at the end of this manual
(Appendix).

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 TABLE OF CONTENT
CONTENTS PAGE

LABORATORY STUDY NUMBER 1

GENERAL LABORATORY SAFETY RULES---------------------------------------------------------1

LABORATORY STUDY NUMBER 2

MICROSCOPES---------------------------------------------------------------------------------------------14

LABORATORY STUDY NUMBER 3

THE CELL AND CELL STRUCTURE------------------------------------------------------------------32

LABORATORY STUDY NUMBER 4

DIFFUSION AND OSMOSIS-----------------------------------------------------------------------------38

LABORATORY STUDY NUMBER 5

TESTING FOR BIOLOGICALLY IMPORTANT MOLECULES----------------------------------43

LABORATORY STUDY NUMBER 6

BIOCATALYSIS – ENZYMES---------------------------------------------------------------------------52

LABORATORY STUDY NUMBER 7

PHOTOSYNTHESIS---------------------------------------------------------------------------------------54

LABORATORY STUDY NUMBER 8

MICROBIOLOGICAL LABORATORY TECHNIQUES---------------------------------------------59

LABORATORY STUDY NUMBER 9

CELL DIVISION--------------------------------------------------------------------------------------------66

LABORATORY STUDY NUMBER 10

COLLECTION AND PRESERVATION OF PLANT AND INSECT SPECIMENS-------------69
REFERENCES------------------------------------------------------------------------------86
APPENDIX ---------------------------------------------------------------------------------87

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 LABORATORY STUDY NUMBER 1

GENERAL LABORATORY SAFETY RULES

1.1. Introduction
A laboratory is a facility that provides controlled conditions in which scientific research,
experiments, and measurement is performed. The scientific method is a process for
experimentation that is used to explore observations and answer questions scientifically.
Scientists use the scientific method to search for cause and effect relationships in nature. In other
words, they design an experiment so that changes to one item cause something else to vary in a
predictable way. Just as it does for a professional scientist, the scientific method will help you to
focus your science fair project question, construct a hypothesis, design, execute, and evaluate
experiments.

Safety rules are guidelines designed to help and keep you safe when experimenting. Some
equipment and chemicals in a laboratory can cause serious harms. It is always wise to follow all
laboratory safety rules.

The following laboratory safety rules are a sample of the most basic rules that should be
followed when in working in the laboratory. Most laboratories have the safety rules posted in the
laboratory and the instructor/trainer will most likely go over them, with you, before you begin
working.

1. Before you enter a laboratory, you should be prepared for knowledgeable about any
laboratory exercises that are to be performed. That means you should read your
laboratory manual to know exactly what you will be doing.
2.  Upon entering the laboratory, place all books, backpacks, and large personal items on the
designated shelves. Do not clutter your work area.
3. Locate fire extinguisher, eyewash, first aid kit, broken glass container, and clean up
materials for spills.
4. In case of fire, use the nearest exit to evacuate the building/room in a quick & calm
manner.  

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 5. If clothing should catch fire, smother it with a blanket or coat. NEVER RUN!!
6. When working in a laboratory, make sure you keep your area neat and organized. If you
happen to spill something, ask for assistance when cleaning it up. Also remember to clean
your work area and wash your hands when you have finished the activity.
7. Remove or tie back any article of clothing or jewelry that can hang down and touch
chemicals and flames. You will also want to wear proper shoes that can protect your feet
in case something gets broken. Do not wear sandals or any type of open-toed shoes in the
laboratory.
8. Wear eye protection when requested by your instructor.
9. Report all spills, accidents, and injuries to your instructor (wash with water or ask your
teacher for help)
10. Wash skin immediately with soap and water if contaminated with chemicals or
microorganisms.
11. You may be working with glass or sharp objects, so you don’t handle them carelessly.
12. Remove affected clothing to prevent reaction with your skin.
13. Biological waste must be disposed of properly in the Biohazard Waste Container.
14. Keep flammable liquids away from open flames and heat sources.
15. Upon completion of laboratory exercises, place all materials in designated areas.
16. Do not pour liquid wastes in the sink unless told to do so by your instructor
17. Wash your hands with soap and water prior to leaving laboratory.
18. Do not remove materials from the laboratory.

There are several things in a biology laboratory that you must avoid such as:
 Do not taste/smell any chemicals or substances you are working with
 Do not use your mouth for pipetting substances
 Do not handle broken glass with bare hands
 Do not pour chemicals down the drain without permission
 Do not operate laboratory equipment without permission
 Do not leave any heated materials unattended
 Do not perform your own experiments unless given permission
 Do not place flammable substances near heat

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  Do not eat, drink or chew gum in the laboratory

1.2 Some basic equipment used in biology experiments

The daily routine of a biologist involves the use of basic equipment in their biology experiments
- such as microscopes, test tubes, beakers, and Bunsen burners as well as high-tech scientific
equipment and computers. This equipment is the bare-bone basics that you’d find in any
laboratory. This equipment is necessary for the basic studies of biology: visualizing cells and
organelles, as well as preparing samples of cells or fluids for testing or visualization, dissecting
specimens, or mixing chemicals.

1. Magnifying glass (hand lens) - is a convex lens mounted in a frame. It is used to

produce a magnified image of an object
2. Petri Dish - it is used for holding a culture medium upon which cells, bacteria and
viruses can be grown and studied. Other uses of the Petri dish: to teach students about
seed germination, as the clear dish allows the observer to see every step of growth,
commonly used for dissection, because it is ideally sized to be placed under a microscope
and can also be used for basic experimental purposes like transporting liquids in sterile
containers or drying fluids for study.
3. Cover slip - it is a protector that goes over tissues with mounting media, this keeps the
tissue on the slide from degrading
4. Thermometer - it is used for measuring temperature or temperature gradient using a
variety of different principles
5. Test tube – is basically a narrow glass tube used for storing things and performing small
chemical reactions in a laboratory; it is used hold, mix or heat small quantities of solid or
liquid chemicals
6. Microrcentrifuge Holds Eppendorf, or microcentrifuge, tubes that can hold about 1.5
ml of liquid
7. Microscope - it is used for viewing objects that are too small to be seen by the naked
eyes.
8. Funnel - it is used to channel liquid or fine-grained substances into containers with a
small opening without spilling. Used for filtration or the delivery of liquids.

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9. Dissecting pan - it is a pan where you dissect organisms so the organs or fluid will not
spread away.
10. Glass slide - it is used to place a specimen on and put it under a microscope to view
11. Dropper – used for addition of liquids, drop by drop
12. Dissecting scissors - it is used for slicing a specimen during dissection
13. Forceps - it is used for grasping, manipulating or extracting, especially such an
instrument used by a surgeon; hold or pick up small objects that are too small for your
hands or fingers; hold tissue out of the way or to pick up a structure. Forceps is used to
hold or pick things that are too small for your hands or fingers.
14. Scalpel - it is a fixed blade used in surgery and dissection (is an extremely sharp bladed
instrument that can neatly split open skin and cut through muscle and organs)
15. Beaker - is a simple container for stirring, mixing and heating liquids commonly used in
many laboratories. It is also used for holding liquids in the laboratory, a liquid-measuring
container; used when the solution mixed in it is going to be poured into something else.
They have a lip on them for pouring.
16. Flasks- have a narrow neck and are used when the solution may splash out of a beaker or
when the container of solution needs to be plugged at some point in the experiment.
17. Test tube rack - it is used to hold the test tube so the solution/substance in it won’t
spread away
18. Dissecting pin - it is used to hold open the specimen that you are dissecting
19. Meter stick – it is a stick that is 100 centimeters long, roughly 39 inches. Generally used
to measure distance.
20. Bunsen burner – is heat source; a cylinder is attached to a gas line. it is commonly used
in scientific laboratories; consisting essentially of a hollow tube which is fitted vertically
around the flame & which has an opening at the base to admit air. When the gas line is
opened, a spark ignites a flame in the Bunsen burner, which is then used to heat solutions.
21. Triple-beam balance - it is used to find the mass of an object
22. Safety goggles - it is used as protection of eye from dust, flying debris, chemical
splashes, etc.
23. Pipette - A piece of laboratory glassware, shaped like a thin tube with a bulge in the
middle, that allows better accuracy when measuring certain volumes (hence the range of

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 sizes) e.g. when making solutions or samples for titration. They are used in a laboratory
to transport and/or measure a specific volume of liquid.
24. Safety mask – it is a mask used when doing experiment, dissection or surgery to protect
us from poisonous gasses, viruses and dusts.
25. Hot Plate -Used for heating liquids
26. Erlenmeyer Flasks- have a narrow neck and are used when the solution may splash out
of a beaker or when the container of solution needs to be plugged at some point in the
experiment; are used to measure, mix, and store liquids. May be used to hold liquids,
instead of beakers, when a smaller opening is preferred. The most common sizes of
erlenmeyer flasks probably are 250 ml and 500 ml. They can be found in 50, 125, 250,
500, 1000 ml.
27. Measuring cylinders –
28. Vortex mixer - rotates the bottom of a tube rapidly; setting up a vortex in the liquid that
rapidly mixes the content.
29. Tong- used to handle hot beakers
30. Test tube holder – spring metal used to hold test tubes or glasses
31. Dissecting pin - it is used to hold open the specimen that you're dissecting
32. Volumetric Flask - Often used when solutions of specific concentration are being made.
33. Graduated Cylinder- Used to measure the volume of liquids
34. Dropper Pipette - Used to transfer small amounts of liquid/ used to transfer measured
substances into another vessel
35. Glass Stirring Rod -Used to stir liquids
36. Autoclave –used for sterilization.
37. Centrifuge - is a machine that spins test tubes at a high speed. This centrifugal force
pulls particles to the bottom of the test tubes, separating parts of a colloid. It is used to
separate blood cells from the plasma. They are used in separating any substances.
38. Ultraviolet/visible (UV/VIS) spectrophotometer - measures absorbance of light in the
ultraviolet region of the spectrum (about 100-700nm)
39. Micropipettes – devices routinely used to measure and transfer liquids by drawing the
liquid on top

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 40. Electronic balances - have replaced most mechanical balances due to their greater
accuracy and ease of operation
41. Column chromatography – used for separation of different chemical compounds.
42. Incubator - is a device used to grow and maintain microbiological cultures or cell
cultures? The incubator maintains optimal temperature, humidity and other conditions
such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside.
43. Incubating shaker – incorporate O2 & evenly distribute nutrients throughout the culture
media
44. Water distillation unit - used for water purification and distillation
45. Biosafety cabinet - is an enclosed, ventilated laboratory workspace for safely working
with materials contaminated with (or potentially contaminated with) pathogens requiring
a defined biosafety level
46. pH meter - measures the amount of H+ ions in a solution and is a measure of how acidic
a solution is/ measures acidity of solutions
47. Refrigerator - used for the storage of the stock solutions, chemicals, kits and PCR
products that should be maintained at certain temperatures
48. Burette - a graduated glass tube, commonly having a stopcock (valve for controlling
flow) at the bottom, used for accurately measuring or measuring out small quantities of
liquid.
49. Water Bath - is a device that maintains water at a constant temperature (used for
regulating the temperature of substances subjected to heat). It is used in the
microbiological laboratory for incubations.
50. Aquarium - a container (as a glass tank) or an artificial pond in which living aquatic
animals or plants are kept
51. Thermal cycler (PCR machine). This device is used for the amplification of a specific
region of any DNA sample with polymerase chain reaction in a test tube. It is also used
for detection and constitution of genetically modified organisms, as well other genetic
analyses.
52. Laminar flow hood: provides an aseptic work area while allowing the containment of
infectious splashes or aerosols generated by many microbiological procedures.

Questions

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1. What are the proper practices for working safety in a laboratory?
2. Why might eating or drinking in the laboratory be dangerous?
3. Look around the room. What safety equipment do you recognize?
4. What kinds of measurements might you need to make in the laboratory? What kinds of
equipment would you need for these tasks?

Burette Centrifuge

Petri dish Magnifying glass (hand lens)

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 Cover slip Thermometer

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Test tubes Vortex mixer

Microcentrifuge Holds Eppendorf, or microcentrifuge Funnel

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Animal dissection on dissecting pan Dissecting scissors

Forceps (tweezers) Scalpel

Test tube rack Beakers

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Bunsen burner Pipette

Triple bean balance Hot plate

Erlenmeyer flasks Volumetric Flask

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Graduated cylinders Dropper pipette

Glass string rod Ultraviolet/visible (UV/VIS) spectrophotometer

Autoclave Electronic balance

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 Micropipettes Laminar flow hood

Incubator Incubating shaker

Water distillation unit Thermal cycler (PCR machine or DNA amplifier)

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 Water bath Tong

Test tube holder Dissecting pins

Figure 1.1: Some basic equipment used in biology lab

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 LABORATORY STUDY NUMBER 2

MICROSCOPES

2.1 Introduction
Microscopes are useful for viewing objects that are too small to see clearly without
magnification. Microscopes are the most important scientific instruments used mainly by
biologists. There are two different types of microscopes: light microscopes and electron
microscopes.

Light microscopes
Light microscopes have glass lenses which magnify objects and use light to illuminate the
objects being examined. The smallest size you can see with your naked eye is 0.2 mm, which is
equal to 200 micrometers. This size is equivalent to one ridge on your fingerprint. Light
microscopes magnify cells up to 1,000 times. Using the shortest ray of light, which allows the
highest resolution, light microscopes can view things as small as 0.2 micrometers in width, i.e.,
0.0002 mm.

You will be using two different kinds of light microscopes in this lab, the compound
microscope and the dissecting microscope.

Compound Microscopes
Compound microscopes are used to examine objects in two dimensions. They use the
magnifying powers of two convex lenses to produce a magnified image of a very small object.
Very small organisms or cross-sections of organisms are placed on clear glass slides; these
objects are viewed as light passes through them. The compound microscope is the most
commonly used microscope and it is powerful enough so that the user can view cells, even the
living ones. The magnification of this microscope is high but the resolution is quite low.

Dissecting Microscopes
Dissecting microscopes are used to observe material that is either too thick or too large to be
viewed with the compound light microscope. With these microscopes, you see the surface of
things that reflect the light. While the magnification and depth of field are smaller in the

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dissecting scope, the field of view is much larger. As its name implies, the dissecting scope is
often used to study structures (like antennae and legs of insects) look at parts of organisms (like
insects) as you dissect them, since it allows for manipulation of material. Most of the parts of the
dissecting microscope are the same as the compound microscope
Electron microscopes
For objects smaller than 0.2 micrometers, an electron microscope must be used. Electron
microscopes allow you to see objects that are as small as 0.2 nanometers (nm), which is equal to
0.000000002 mm. In comparison to a light microscope being able to magnify 1,000 times,
electron microscopes can magnify objects 200,000 times.

Electron microscopes use beams of electrons to examine incredibly small objects (like the
components of an individual cell) that have been specially prepared. In these microscopes a
beam of electrons (in place of light) and circular magnets (in place of glass lenses) permit the
resolution of structures in much finer detail than in an optical microscope.

There are two types of electron microscope. These are scanning electron microscope (SEM)
and transmission electron microscope (TEM).

Scanning Electron Microscope (SEM): a beam of electrons scans the surface of an opaque
object and produces an image of that surface. This is an expensive microscope which has high
magnification and resolution. The images are viewed on a cathode tube, or in pictures taken with
the microscope. Many of the photographs of cell structure used in your text were taken with an
electron microscope.

Transmission Electron Microscope (TEM): an electron beam passes through the specimen
and provides the user with a two dimensional view and high magnification and resolution.

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 A B

C D
Figure 2.1: Different types of microscopes. A. Compound light microscope; B. Dissecting light
microscope; C. Transmission electron microscope; D. Scanning electron microscope

Objectives:
At the end of this section the students will be able to:
 describe the parts and functions of the compound microscope

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  use the microscope by focusing with the different objectives

2.2 The parts and functions of a compound microscope

A compound microscope is so called because it uses a combination of lenses to magnify any sort
of images. Basically, there are two components - the structural components and the optical
components.

Structural components
As the name suggests, these are the components that make up the configuration of a compound
microscope. There are three basic structural components of a compound microscope.

1. Head or Body tube

Body tube or eyepiece tube is the part that connects the oculars to the objectives. In this, oculars
are placed at the upper portion above the objectives.

Base - Base is the bottom part, which supports the microscope. Illuminator is located in the base.
Arm - Arm supports the head component and connects it to the base.
It is always recommended to handle by the arm and support the base while carrying a compound
microscope.

2. Optical Components
Working of a microscope entirely depends on the optical components. The optical components
are fixed to the rigid arm of the microscope.
The microscope has two magnifying lenses: the eyepiece or ocular lens and the objective lenses
on a turret which revolves above the stage.
Eyepieces (Oculars)
Eyepiece or oculars are secondary lenses located at the top of the BODY TUBE, near the eye. It
is through this lens that we view an object in a microscope. The magnification power of eyepiece
lenses is usually 10X.

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Objective lenses
Objectives are the primary lenses of a compound microscope. The objective lenses (there are
four: 4X, 10X, 40X and 100X) are screwed into a revolving nosepiece. By changing the
objectives the effective power of magnification is changed. The total magnification observed is
the product of the power of magnification of the eyepiece and the objective. Only the 100X
objective is used immersed in a drop of special oil (between the lens and the slide; all others are
designed to be used with air between the object and lens surface. The power of magnification is
clearly indicated on each lens along with the numerical aperture of each lens. Depending upon
their design and quality, different objectives have different resolving distances. The latter is the
smallest distance between two points that allows both points to be viewed as separate. This
resolving distance is dependent upon the wavelength of light used as well as the construction of
the lens.

Examine the objectives from the side and note the magnification.

A. The low power objective - the shortest one with the largest lens opening), usually has a
magnification of four times and is labeled as 4X.
B. The middle power objective –is medium in length and often has a magnification of 10X.
C. The high power objective - is longer and has a magnification of 40X.
D. The oil immersion objective has a magnification of 100X.

3. Nosepiece - Nosepiece supports the objectives. The objectives are mounted on a rotating turret
so that any of the objectives can be conveniently used for viewing.
4. Adjustment Knobs - The microscope is provided with two focusing mechanisms, the course
adjustment and the fine adjustment. These are used for focusing, so as to get a clear effect. The
focusing knobs move the lens assembly up and down to bring the object in focus. The coarse
adjustment should only be used with the shortest, low power objective lens. The fine adjustment
(smaller knob) brings the object into critical focus. Notice that all objects are projected upside
down in the microscope field. It takes a little practice in using the mechanical stage to move the
slide where you want it.

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The adjustment knobs of your microscope are used as focusing devices because they can raise or
lower the body tube as necessary. In some microscopes (e.g., Reichert Neovar) it is the stage but
not the body tube that is lowered or raised in focusing.

Note that the positions of the adjustment knobs vary with the manufacturers. The Bausch and
Lomb microscope you possess has its course adjustment knob at the top of the ram and its fine
adjustment knob located at the pivot of inclination joint.

In some types of microscopes (e.g., Reichert Neovar) the coarse adjustment and fine adjustment
are in one piece. The inner larger part is the coarse and outer smaller part is the fine adjustment.
In still another type of microscope (e.g., Reichert Monopan) the coarse and the fine adjustments
are combined into one. In this case the direction of the movement (the rotatory or back and forth
movement) of the knob determines type of adjustment.

Stage
Stage is the platform, where the slide to be viewed is placed. There is an opening in the centre of
the stage (stage opening) through which light passes. On the stages there are two metal clips that
serve to hold the slide securely in place. Instead some microscopes are equipped with mechanical
device that holds the slide in place and also to move the slide sideways as well as back and forth.
For higher magnification studies, mechanical stage is used for fine movement of the slide.

Condenser
Microscopes contain elements designed to project parallel beams of light through the specimen
and into the objective. These include the projection lens which focuses light onto the condenser
lens. The condenser lens focuses light onto the object.

The Condenser is found immediately beneath the stage. This component of the microscope
causes the light rays to converge at the stage opening. At its lower end the condenser is equipped
with an iris diaphragm which can be opened and closed as necessary to control the amount of
light entering the body tube. The condenser may be fixed or it may be raised and lowered by a
sub-stage adjustment. Lowering the condenser decreases the amount of light entering the body
tube and ultimately reaching your eye.

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Aperture - The hole in the stage through which light reaches the object.
Illuminator - Illuminator is the source of light in a microscope. It is placed in the base
component.

Figure 2.2: Parts of a compound microscope

Microscope Handling
1. Always carry the microscope in a straight upright position with one hand around the arm and
the other hand under the base. The eyepieces are not attached and will fall out if the
microscope is carried at an angle or upside down.
2. Keep the microscope clean. When anything is spilled or otherwise gets on the microscope,
clean it up immediately.
3. When using the microscope start with the low power lens and work up to the desired
magnification. These microscopes are par focal, which means that all powers should be in
focus when the turret is rotated.

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4. Never move the stage upwards with the coarse adjustment while viewing through the
eyepieces. Get the lens close to the slide while viewing from the side to make sure that they
never touch. Then move the stage downward with the coarse adjustment while viewing
through the lens. This will prevent the possibility of ramming the lens into the slide, thereby
ruining a slide you have just made and, quite possibly, damaging the lens.
5. Do not touch the lenses
Clean the lenses with lens paper only. Do not clean the lenses with handkerchiefs, facial
tissues, paper towels, etc. - they will scratch the lenses. If your lenses are very dirty, obtain
some lens cleaning solvent from the instructor.
6. Moist, living or preserved materials must be observed through a covers lip. This protects the
lens as well as tends to make the object under view optically flat. Be sure to maintain a safe
distance between the coverslip and the objective lenses.
7. Remove the dust cover and store it properly. Plug in the scope. Do not turn it on until told
to do so.
8. Teachers and students should be sure to keep all Glass and Sharp instrument Safety Rules
that are given by the teacher and in all general laboratory oratory experiences when preparing
microscope slides.

9. Always clean slides and microscope when finished. Turn off light.
Cover the microscope with dust cover and return your microscope to the cabinet with
light cord wrapped around its base and with the lowest power objective lens in position, if
requested by teacher.

2.3 Preliminary use of the microscope

Setting-up microscope for use

In order to study your mounted dot or any other object under the microscope first you have to
set-up your microscope for use.
If you follow the following steps, they will enable you set up your microscope for use.

1. Place the microscope near the edge, not on the edge, of your laboratory bench with its
arm facing you.

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 2. Arrange your laboratory stool such that it allows you an easy look through the ocular of
your microscope without inclining it.

3. Make the diaphragm wide open

4. If the condenser is in a lowered position, raise it as near the stage as it will go

5. Turn on your microscope through and while looking into the microscope without the
ocular adjusts the concave surface of the mirror until the field of vision (the circular area
you see in the microscope) is uniformly illuminated. Note that once you have adjusted
the light you should not move the microscope or the lamp. Otherwise you may need to
readjust the light.

I. Practice in mounting and focusing

Materials and chemicals

Pen/pencil, cover slips, glass slides, microscope, Onion, methylene blue stain, water
1. Dry mount slide:
1. Place a sample in the center of the slide
2. Carefully lower the cover-slip onto the slide - do not drop the cover-slip
3. Secure your slide to the stage (with the spring clips)
4. Focus on the sample
2. Wet mount slide
Wet mounts are commonly used, for example, to view microscopic organisms that grow in pond
water or other liquid media, especially when studying their movement, size of cells and behavior
The specimen is placed in a drop of water or other liquid held between the slide and the cover
slip

1. Place a sample in the center of the slide

2. Add a drop of water on the sample
3. Add a small drop of methylene blue stain to the preparation and gently agitate
4. Hold the cover slip so that its edge touches one side of the fluid
5. Carefully lower the cover-slip onto the preparation - do not drop the cover-slip

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 6. Secure your slide to the stage (with the spring clips)
7. Focus on the sample through the eyepieces

Figure 2.3: Technique of applying cover slip on specimen on the glass slide

A. Focusing with low power

1. If the low power objective is not in position (i.e. if it is not right above the stage
opening) hold any two objectives between your thumb and the forefinger and turn the
piece until the objective clicks into positions.

2. Looking from the side, lower the body tube with the coarse adjustment to within 5mm of
the slide. The body tube of some types of the microscopes cannot be lowered to the extent
the low power objective touches the slide. Thus, if you cannot lower it further, do not
apply force.

 In some types of microscope it is the stage that is raised up towards the objective
instead.
3. Looking into the microscope about 2 cm away from the eye piece, slowly raise the body
tube with the coarse adjustment until the image comes into view. Do not plant your eye
right on the ocular for if you see anything you will only see a reflection of your eye and
eyelashes. Keep both eyes open while looking into the microscope.
 Spectacles used to correct near or far-sightedness need not be worn when using the
microscope. Only those who are very near-sighted may need spectacles to avoid
headaches.
4. If the image viewed is blurred, focus it with the fine adjustment until it becomes clear.
5. If the image is off the centre of the field of vision, move the slide very gently, while
looking into the microscope, until the image is in the centre of the field.

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 6. Adjust (increase or decrease) the diaphragm as necessary

B. Focusing with middle power

In order to focus the objectives (dot) under the medium power go to step 5 without changing the
focus under the low power.
7. If you have followed the above steps, your object is in focus under the low power. Now,
holding any two of the objectives. Between the thumb and the forefinger rotate the nose-
piece until the middle power objective clicks (i.e. you hear a clicking sound) into position.
8. Focus with the fine adjustment until you get a clear image.
9. Increase the diaphragm opening if more light is needed.

C. Focusing with the high-power

Now you can directly (i.e. without changing the focusing under the medium power), go to step 8
in order to focus the objective under the high power.

10. Your object is already in focus under the middle power. Taking hold of any two
objectives, rotate the nose piece until the high power objective clicks into position. Care
should be taken so that the objectives do not hit against the clips.
11. Focus with the fine adjustment until you get a clear image. Never use the coarse
adjustment when focusing with the high power objective.

12. If more illumination is needed the opening of the diaphragm may be increased. You can
practice focusing (steps 1-12 above) until you do it perfectly.

D. Some important points of caution in focusing

1. Never focus downwards without looking at the objectives from the side if your
microscope is the type that is focused by lowering or raising the body tube.
2. Never focus upwards without looking at the objectives from the side if your microscope
is the type that is focused by lowering or rising of the stage.

24
3. Never focus the high power objective or the oil immersion objective. If your microscope
has one, with coarse adjustment. Always use the fine adjustment. Otherwise, you may
break the slide and possibly damage the microscope itself.

4. Do not try to focus an objective directly with the high power objective or with the oil
immersion objectives. First locate or focus the object with the low power, then shift to
the medium power and then to the high power. Alternatively, you can focus it with low
power and then high power without focusing it with the middle power.

5. You should note which way to turn the adjustment knob to lower or raise the body tube
or to lower or raise the stage depending upon the type of your microscope.

25
2.4. Type of image formed, resolution and magnification

A. The resolving power of microscope

Resolution means the ability to separate fine details. For example, if the distance between two
dots is less than 0.1mm, the two dots would appear as a single dot to the unaided human eye. The
resolving power of the human eye is about 0.1mm.

The microscope magnifies and resolves. The resolving power of a microscope depends upon the
kind of illumination used because the resolving power is ½ x the wave length of the illumination
used. Therefore, even with the most perfectly ground lenses and with white light having an
average wave length of 5500 Angstroms (Å), the objective cannot resolve anything less than
2750 Å (i.e. ½*5500 or 0.275 microns). Since many parts of the cells have dimensions less than
0.275 microns (or 275 millimicrons) their presence remained undetected until a means of greater
resolution was found.

The electron microscope uses beam of electrons instead of light. When electrons are propelled by
a charge of about 50,000 volts they have a wave length of about 0.05Å. Therefore, theoretically
an electron microscope can resolve objects with a diameter of about 0.025Å (i.e. ½*0.05Å). Note
that hydrogen atom has a diameter of about 1.06 Å which is larger than the diameter of an object
that an electron microscope can theoretically resolve; but due to difficulties in the construction of
objectives the actual resolving power of the best modern electron microscope is about 10Å.
1mm = 1000 microns or 1 micron = 1000 millimicrons, 1 millimicron = 10 Å
B. The depth field

Depth of field (DOFi) is the area in front of and behind the specimen that will be in acceptable
focus. The Depth of Field (DOFi) of a lens is its ability to maintain a desired amount of image
quality (spatial frequency at a specified contrast), without refocusing, if the object is positioned
closer to and farther from best focus. DOFi also applies to objects with complex geometries or
features of different heights. As an object is placed closer or farther than the set focus distance of
a lens, the object blurs and both resolution and contrast suffer. Because of this, DOF only makes
sense if it is defined with an associated resolution and contrast.

26
Depth of focus is essential the same as depth of field but for one important difference, that being
magnification (M). With higher magnification depth of field becomes shorter, however higher
magnification increase the depth of focus for the image. This is because the magnification is
done with a projection lens.

Field of view (FOV) is how much of a specimen is visible at any given time in the lateral plane
(i.e., perpendicular to the optical axis). It can also be thought of as the diameter of the circle of
light visible when looking through a microscope. FOV is inversely proportional to the
magnification (as the magnification increases, the FOV decreases). Another way to understand
this is to consider that when a specimen is magnified, the microscope is zooming in on it and,
consequently, seeing less of it (but in greater detail). The size of the FOV or the diameter of the
visible circle of light (also known as the Field Size) is equal to the Field Number (FN) of the
eyepiece divided by the magnification of the objective lens (measured in millimeters).

C. Image formation
The compound microscope consists of two optical components (thus the term compound): the
objective lens system, which has a very short focal distance and is placed very close to the
object; and the ocular or eyepiece system, which has a longer focal length, lower magnification;
and which further magnifies and projects the image onto the retina of the eye. The objective
lens projects a real image (the intermediate image) of the object up in the body of the microscope
onto the objective conjugate image plane or primary image plane. The objective lens forms a
real image in the microscope body that acts as the object for the ocular lens. The real image
becomes the object for the eye itself and is projected onto the retina. The ocular in turn acts as a
simple magnifier to form a large virtual image at the distance of most distinct vision (25 cm)
from the eye.

27
 Figure 3.1. Image formation with the context of a real microscope

Objectives:
At the end of this section the trainees will be able to:
 between magnification and resolution.
 estimate the size of image seen under light microscope
Materials and chemicals: Microscope, microscope slide, cover slip, pencil, a piece of paper,
graph paper, water

D. Focusing

1. Study of a dot
You are provided with a piece of paper bearing a typed dot (full stop). If not, using a pencil make
a small dot on a small piece of paper.

1. Wet mount the dot

2. Study the dot under the low power and then under the middle power following the
focusing steps on the previous pages.
(i) Under which of these powers does the dot look more diffuse (loosely held together)

28
 (ii) Why does the dot look diffused under the microscope than when you look at it with
the unaided eye?

E. Type of image formed and the direction of image movement

1. Type of image formed

You are provided with a small piece of paper with the letter ‘e’ typed on it. Wet mount it and
observe it under the low power objective.
(i) Is the letter inverted from top to bottom?
(ii) Is it inverted from left to right?

(iii) If you were to mount the letter p, what the image would look like if seen under the
microscope

(iv) If the above object stands in front of a mirror show with a drawing what its mirror image
would look like.

(v) From your above observations how does the image of an object under a microscope
differ from its mirror image?

2. Direction of image movement

(i) Looking into the microscope move the slide towards the left. Which way does the image
move?
(ii) If you move the slide away from you which way does the image move?

F. How to calculate magnification and area of field of vision

Total magnification
In a compound microscope the image of an object that is magnified by the objective is again
magnified by the ocular. Therefore, the total magnification provided by a compound
microscope is the product of the magnifying power of the ocular and the particular objective
used.

29
 i. What is the magnification of the microscope when using the low power? The medium
power? The high power?
ii. If the ocular of your microscope is marked 7.5X, by how many diameters will an
object viewed under middle power be magnified?

iii. If an object looks 2 mm long under the middle power, what is its real length?

iv. If an object is 0.001 mm in diameter how big would it appear the high power of your
microscope?

Area of field of vision

The field of vision is the area visible when you look through the microscope. Knowing the size
of the field of view will enable you to determine the size of the object you are observing. Special
rulers are used to determine the field of view and measure objects under the microscope.

i. Wet mount a piece of graph paper on a glass slide and count the number of millimeter
squares visible under low power and middle power. Half or more than half squares are
counted as full squares and those less than half should not be counted.
ii. Determine the ratio of the areas of the two fields of vision (under low power and middle
power).

iii. How is field of vision related to magnification?

iv. Under which power (the low or the middle) can you see more parts (area) of an object at a
time?

v. Counting the squares across the middle of the field of vision, determine the diameters of
the low and middle power fields. Give your result in microns?

vi. What is the ratio of the diameter of the low power field of vision to that of the medium
power?

vii. From the ratio of the two diameters, determine the ratio of the magnifying powers of the
low and middle power objectives?

30
viii. Using the graph paper can you determine the diameter of the field of vision of the high
power? If no, why not? If yes, give the figures in microns. Remember that you should focus
the high power with fine adjustment only!

D. Microscopic examination of living organisms

A considerable amount of information can be gained by careful microscopic examination of
microorganisms. Living organisms can be studied to determine the natural size and shape of
cells, cellular arrangement, and motility, reactions to various chemicals or immune sera and
response to environmental factors.

Direct examination of living organisms including bacteria, protozoan and others can be studied
by two means: Wet mount and Hanging drop technique. Both are very useful in determining
size, shape and movement.
A wet mount is a faster way to observe bacteria. In Hanging drop technique, the application of
petroleum seal around the cavity reduces the evaporation of suspended fluid drop. It also makes
possible to observe larger microbes and motile organisms more easily because of greater depth
provided by the hanging drop.

Materials and chemicals: Microscopic slides, cover slips, hanging drop (cavity) slide,
inoculating loop, tooth pick, sprit lamp, broth culture, petroleum jelly (Vaseline), distilled water
in a dropper, oil immersion

1. Wet mount technique

Procedure:
1. Transfer a small drop of the bacterial culture given on the center of clean glass slides.
2. Handle the cover slip by its edges and place it on the drop
3. Press the cover slip gently with the end of a pencil

2. Hanging drop technique

Procedure:
1. With a toothpick, spread a small ring of Vaseline around the concavity of a depression
slide. Do not use too much Vaseline.

31
2. Use inoculating loop to aseptically place a small drop of the bacterial suspension in the
center of a cover slip.
3. Lower the depression slide, with the concavity facing down, onto the cover slip so that
the drop protrudes into the center of the concavity of the slide. Press gently to form a
seal
4. Turn the hanging drop slide over and place on the stage of the microscope so that the
drop is over the light hole.
5. Examine the drop by first locating its edge under low power and focusing on the drop.
Switch to the high‐power objective and then, use immersion oil objective. Be careful to
distinguish between motility and Brownian movement. Brownian movement results
from the random motion of water molecules bombarding the bacteria causing them to
move

Questions:
1. Why are microorganisms difficult to see in wet preparations?
2. What is the advantage of a hanging-drop preparation over that of wet mount
preparation?
3. Distinguish between ‘true motility’ and ‘Brownian movement’?

32
 LABORATORY STUDY NUMBER 3

THE CELL AND CELL STRUCTURE

3.1 Introduction
The cell is the structural and functional unit of life. On the basis of level of cellular organization
two types of cells are recognized. They are prokaryotic cells (Prokaryotic cells are found in
bacteria, including both Archaebacteria and Eubacteria, and including the blue-green algae) and
eukaryotic cells (are found in animals, plants, Fungi and protists). The latter are characterized
by the compartmentalization of its parts i.e. its parts are divided into separate compartments such
as nucleus, mitochondria, etc.

The protoplasm of a eukaryotic cell is divided into nucleus and cytoplasm. The nucleus is
enclosed by a nuclear membrane consisting of two concentric membranes. The nuclear
membrane is perforated by a number of nuclear pores (Annuli). The prominent body in the
nucleus is the nucleolus. There could be more than one nucleoli in a nucleus. Most of the space
in the nucleus is occupied by thin thread like bodies called chromatin. The chromatin is the
material of the chromosome and it consists of DNA and special proteins complexed with the
DNA. The ground material in the nucleus is called nucleoplasm.

In the cytoplasm there are numerous bodies of various sizes, shape and function; networks of
interconnecting tubules (channels) and various membranes bound vesicles and vacuoles.

33
Among the bodies in the cytoplasm are the mitochondria (the power house of the cell) and the
ribosomes. The ribosomes are sites of protein synthesis i.e. ‘benches’ up on which amino acids
are assembled into protein. The ribosomes are found either freely in the cytoplasm or attached to
the surfaces of membraneous channels.

The interconnecting tubules (channels) in the cytoplasm form what is called endoplasmic
reticulum. The endoplasmic reticulum and that which is free of the ribosomes is known as
smooth endoplasmic reticulum. In the cytoplasm there is also another vesicular structure
known as the (Golgi body or Golgi apparatus) named after Camillo Golgi, who first described
it. This body appears as a collection of closely attached stack of flattened sacs.

In the cytoplasm there are also enzyme-containing vesicles called Lysosomes and Peroxisomes.
The former contain various digestive enzymes and the latter contain enzymes responsible for the
production or degradation of proxides such as the enzyme catalase that degrades hydrogen
peroxide (H2O2) to water and oxygen.
Some vesicles are concerned with transportation of materials into and out of the cell. Endocytic
Vesicles are formed from the cell membrane and move materials into the cell. There are two
types- pinocytic vesicles (transport liquid) and phagocytic vesicles (transport solid material).
Exocytic vesicles bud of the Golgi apparatus and transport cell secretion to outside.

Animal cells capable of division have near the nucleus at least a pair of hallow cylindrical bodies
called centrioles. There are also fine tubules and filaments known as microtubules and
microfilaments respectively.

The ground substance in which the various organelles of the cell are suspended is called cytosol,
largely composed of water in which various inorganic and organic substances are dissolved or
suspended. The whole cell is enclosed by the cell membrane.

In addition to most of the structure shown in the animal cell, plant cells have certain structures
that are not found in animal cells. The important ones are plastids (e.g. chloroplasts), cell wall
and a large central vacuole which occupies as much as 80-90% of the total cell volume of a
mature cell.

34
Figure 3.1: The structure of a typical plant cell as revealed by electron microscope

35
Figure 3.2: The structure of a typical animal cell as revealed by electron microscope

A STUDY OF PLANT AND ANIMAL CELLS

Objectives:
After completing the activity trainees will be able to:
 mount plant cells and animal cells and observe the major visible structures
 distinguish between plant cells and animal cells.
Activity 1: Observing plant cells (onion epidermal cells)
An onion bulb is made up of several layers of modified leaves. Each leaf has delicate epidermal
layers which can easily be peeled off. The epidermal layer consists of cells that onion leaf can be
readily seen under the microscope.

36
Materials: Microscope, microscope slides (depression and flat), cover slips, paper toweling,
toothpicks (round end), typical animal cell slide, needle
Chemicals: Alcohol, iodine, Janus Green B stain, distilled water
Procedure:
1. Peel the whitish inner epidermis from one of the leaves.
2. Place the piece of the epidermis on a glass slide, add a drop of water and then a cover slip
(Note that when you cover the cover slip, try to avoid air bubbles that may mistake you
with cell organelles. Your instructor shows you how to avoid air bubbles).
3. Examine it under the middle power.
a. Is the nucleus clearly visible?
b. Is the cytoplasm thicker in the centre or in the periphery of the cell?
4. Remove the slide from the stage and place a drop of iodine solution at one end, hold a
piece of absorbent paper at the opposite end of the cover slip. Wait for about 5 minutes till
the cells get stained. (N.B. if your mount is not good you can mount fresh piece of epidermis
directly in a drop of iodine).

a. Examine under the middle power and draw two or more adjacent cells and label the parts.
b. Which part(s) of the cell became more deeply stained?
c. How do you account for the difference in staining?

Activity 2: Observing animal cells (cheek cells)

Materials: Microscope, microscope slides, cover slips, tooth pick, dropper,
Chemicals: Methylene blue

Procedure:

1. Gently scrape the inside of your cheek or the inside of your lower lip with the broad end of a
clean toothpick
2. Stir the scraping into a drop of water on a clean glass slide.
3. Place a cover slip on it and examine it under the middle power.
a. Are the parts of the clearly visible?
b. What is the color of the cell?

37
4. Place a drop of methylene blue at one end and hold a piece of absorbent paper at the opposite
ends of the cover slip. Wait for about 5 minutes until the cells get stained.
5. Examine your preparation under the middle power (or you can use the high power with good
care).
a. Can you now distinguish the nucleus and the cytoplasm?
b. Compare position of the nucleus in cheek cell (animal cell) with that of onion cell (plant
cell).
c. Draw two or more adjacent cells and label the part

38
 LABORATORY STUDY NUMBER 4

DIFFUSION AND OSMOSIS

4.1 Introduction
The random movement of molecules from a region of higher concentration to a region of lower
concentration is known as diffusion. It tends to distribute molecules uniformly. Brownian
motion (or Brownian movement) can be defined as "the random movement of microscopic
particles suspended in a fluid."  
Osmosis is the diffusion (net movement) of solvent particles (molecules) across a semi-
permeable membrane from a region of their higher concentration (i.e. from week solution) to a
region of their lower concentration (i.e. to strong solution). In biological systems since the
solvent is water and the membrane is the cell membrane, osmosis can be defined as the diffusion
of water molecules across the cell membrane from a region of higher concentration to a region of
their lower concentration.

The net movement of water into or out of the cell depends upon the relative concentration of the
protoplasm and the solution surrounding the cell. When osmosis results in the net movement of
the water out of a cell, the protoplasm of the cell shrinks. This shrinkage of the protoplasm as a
result of net loss of water is called plasmolysis. The shrinkage of animal cell is called crenation.
When osmosis results in, a net movement of water into a cell, the protoplasm swells. If it is a
plant cell, the swelling protoplasm exerts a pressure (turgor pressure) against the cell wall.
Eventually the net movement of the water into the cell is stopped when the pressure exerted
inwards by the wall and the pressure exerted outwards by the protoplasm is equalized. An animal
cell (e.g. blood cell) since it does not have a rigid cell wall; it may burst if it takes in excess
water.

Objectives:
After completing the activity students will be able to:
 describe the significance of diffusion and osmosis in living organisms
 explain why different substances with different molecular weights differ in their rate of
diffusion

39
I. Diffusion
Materials: Petri dish containing solidified agar, Cork borer, beakers
Chemicals: Potassium permanganate, methylene blue

Activity 1: Determination of the rate of diffusion

1. You are provided (in groups) with a Petri dish containing solidified agar.
2. Using a number 5 Cork borer, punch four holes in the agar
3. Fill one of the holes with one or two drops of potassium permanganate (molecular weight
= 158.05) and the other one with methylene blue (molecular weight = 319.86).
4. Periodically examine the Petri dish and record your final observation.
a. Which chemical diffused faster?
b. Which chemical travels the shortest distance?
c. How do you account for the difference in the diffusion rates of the chemicals?

Activity 2: Diffusion experiment by using plastic bag as representing cell membrane

Materials
• 400-mL beaker • twist tie
• 25-mL graduated cylinder • iodine solution
• 1% starch solution • forceps
• Plastic sandwich bag
Precaution: Iodine solution can irritate the eyes and skin and can stain clothing. Wear safety
goggles, gloves, and a laboratory apron while handling any solution that contains iodine. Rinse
off any solution that spills on your skin or clothing. Wash your hands thoroughly with soap and
warm water before leaving the lab.
Procedure
1. Put on your goggles, apron, and gloves.
2. Add about 200 mL of water to a 400-mL beaker.
3. Pour 25 mL of starch solution into the plastic sandwich bag.
4. Use a twist tie to tightly seal the bag.
5. Use tap water to thoroughly rinse the outside of the bag in case any starch solution spilled onto
the outside of the bag. Be sure to rinse the twist tie as well.
40
6. Place the plastic bag in the beaker so that the bag is completely covered with water.
7. Add 8 drops of iodine solution to the water in the beaker. Record your initial observations in
the data table. Wait 10 minutes, and then record your final observations.
8. Use a forceps to remove and dispose of the plastic bag as instructed by your teacher.
Questions:
1. After you placed the plastic bag in the beaker, what happened to the iodine? What happened to
the starch?
2. Use what you know about the structure of starch molecules to explain your results.
3. Did water move into the bag or out of the bag? Why?

II. Osmosis
Activity 1: Studying osmosis using onion epidermis
Materials: Microscope, microscope slide, cover slip, onion root, needle, dropper
Chemicals: 1N NaCl solution, water
Procedure:
1. Remove a small piece of onion epidermis and mount it in water. The outer, pinkish
epidermis may be preferred since the cytoplasm can easily be seen under the microscope
2. Observe your preparation under the middle power and draw 2 or 3 adjacent cells and
label the parts.
3. Using a clean slide mount a fresh epidermis in a drop of 1N NaCl (instead of water, use
NaCl for wet mounting). Observe it under the middle power.
4. After about 20 minutes observe it again and draw 2 or 3 adjacent cells and label the
parts.
Questions:
a. What major differences do you observe between the cells mounted in water and
those mounted in NaCl solution?
b. Explain how the difference could come about.

Activity 2: Investigating osmosis using potato cylinders/strips

Osmosis can be easily demonstrated in biological systems using potato strips, water and salt or
sugar solution.

41
Materials: Potato tuber, cork borer, beakers, ruler, electronic balance, tissue paper
Chemicals: Sucrose, distilled water

Procedure:
1. Using number 4 cork borer, take 7 potato cylinders from a fresh potato tuber.
2. Cut the potato cylinders each 4 cm long and weigh them separately, recording the initial mass
and length.
3. Observe each strip by feeling it with your fingers, noting whether it is turgid or flaccid.
Record this.
4. Take six beakers of 50 ml volume and label them as 1- 7.
5. Prepare 2, 4, 6, 8, 10, and 15 % sucrose solutions separately.
6. Add 30 ml of distilled water to beaker 1, and add 30 ml of each of the sucrose solutions
sequentially to beakers 2 - 7.
7. Place a strip in each beaker. Record the time.
8. After 3 hours remove each strip and dab it on tissue paper. Take care of not mixing them.
9. Weigh and measure each potato strip, recording the final mass and length.
10. Observe each strip by feeling it, noting whether it is turgid or flaccid. Record this.
11. Perform % difference calculations for the mass and length using the formula:
Final - Initial x 100%
Initial

Questions:
1. In which potato cylinders have you observed changes in length and mass?
Explain reasons for the change.
2. Which potato cylinders have become turgid? Which ones have become flaccid? Which ones
do you think have neither flaccid nor turgid? Explain your results briefly.
3. What is the semi permeable membrane in the cells of the potato?

42
43
 LABORATORY STUDY NUMBER 5

TESTING FOR BIOLOGICALLY IMPORTANT MOLECULES

5.1 Introduction

I. Carbohydrates

Carbohydrates are compounds of carbon, hydrogen and oxygen occurring in a verity of forms in
both plants and animals. They include such substance as sugar, starch, glycogen and cellulose.
Carbohydrates serve as structural substance. (E.g. Cellulose in plant cell wall), chitin in some
fungi, murin strengthen materials of bacterial cell wall. As well as immediate source of potential
energy reserve (E.g. Glucose).
On the basis of size (i.e., number of sugar units that they are composed of), carbohydrates are
known as monosaccharide, disaccharides, oligosaccharides and polysaccharides.
Glucose + Glucose Maltose (malt sugar)
Fructose + Glucose Sucrose (cane sugar)
Galactose + Glucose Lactose (milk sugar)

44
Oxidation/Reduction (Redox) Reactions: Oxidation is generally referred to as the loss of
electrons, while reduction is the gain of electrons.
Benedict’s reaction for Reducing Sugars is shown as:
Cu2+ + e– Cu+
Copper (II) ions (Cu2+) are reduced to copper (I) ions (Cu+) by gaining electrons from the
reducing sugars. The reducing sugar is oxidized as a result of giving up its electron.
It is often difficult to see where electrons are flowing when you can’t see an ion charge.
However, since the movement of hydro-gen often follows the electrons, we could describe
oxidation as the loss of hydrogen and reduction as the gain of hydrogen.

45
Numerous tests have been devised for the determination of the properties and for the
determination of carbohydrates. A brief description of the most common tests follows.
Objectives:
After completing the activities trainees will be able to:
 determine an unknown food sample (carbohydrate, protein, or lipid) by conducting food
test.
 differentiate between reducing sugars and non-reducing sugars.

I. Tests for carbohydrates

Activity 1: Molisch’s Test
Molisch’s test is a common test for all carbohydrates larger than tetroses. The test is on the basis
that pentoses and hexoses are dehydrated by concentrated sulphuric acid to form furfural or
hydroxymethylfurfural, respectively. These products condense with α-naphthol to form purple
condensation product.
Materials: Test tubes, test tube rack, test tube holder
Chemicals: Distilled water, Molisch’s reagent, concentrated sulphuric acid, Alpha-naphthol
solution
Procedure:
1. Add 2 drops of the α-naphthol solution (5% in ethanol prepare fresh) to 2 ml of test
solution in a test tube.
2. Carefully, pour about 1 ml of conc. H2SO4 down the side of the tube so as to form two
layers.
3. Carefully observe any colour change at the junction of the two liquids.
4. Repeat the test, using water instead of the carbohydrate solution.

46
 a. Did you observe any color?
b. What color did you observe?
c. If there is any color formation, where did it form?

Reducing and non-reducing sugars

Some sugars have the capacity to reduce certain compounds such as Cu 2+. Such sugars are called
reducing sugars. Reducing sugars include all monosaccharides, such as glucose, fructose, and
some disaccharides such as maltose. A non-reducing sugar is a carbohydrate that is not oxidized
by a weak oxidizing agent (an oxidizing agent that oxidizes aldehydes but not alcohols, such as
the Tollen’s reagent) in basic aqueous solution.  The characteristic property of non-reducing
sugars is that, in basic aqueous medium, they do not generate any compounds containing an
aldehyde group.
Example: sucrose, which contains neither a hemiacetal group nor a hemiketal group and,
therefore, is stable in water.

A. Test for reducing sugars

Benedict’s test
When a solution containing Benedict’s reagent and a reducing sugar are heated, the copper (II)
ions in are reduced to copper (I) ions and the solution changes in color. Benedict’s reagent
contains potassium thiocyanate and is used to determine how much reducing sugar is present.
This solution forms a copper thiocyanate precipitate which is white and can be used in a titration.
Materials: Test tubes, test tube holder, water bath
Chemicals: 5% of glucose or 5% of fructose or 5% of maltose, Benedict’s solution, distilled
water

Procedure:

1. Add 2ml of 0.1-1% of a solution of the reducing sugar to a test tube.

2. Add 2ml of distilled water to another test tube.
3. Add an equal (3ml) volume of Benedict’s solution to both test tubes.
4. Shake and boil the test tube in a water bath gently.
a. Did you note any color change?
b. If so, what color changes?

47
 c. In which test tube did you note the color change?
d. What is the source of the color change?

B. Test for non -reducing sugars

Materials: Test tubes, test tube rack, test tube holder, Bunsen burner
Chemicals: 5 % sucrose solution, pH paper/litmus paper, Benedict’s solution, Sodium hydrogen
carbonate, Hydrochloric acid, distilled water
Procedure
1. Add 2ml of sucrose solution to a test tube.
2. Add 1ml of dilute hydrochloric acid.
3. Boil for one minute.
4. Carefully neutralize with Sodium hydrogen carbonate (check with pH paper).
5. Carry out Benedicts test.
a.What color change did you observe?
b. Are the color changes different from the previous experiment?
c.What is the purpose of the hydrochloric acid?
d. What statement you can make from this experiment?

Test for starch (Iodine test)

Starch is only slightly soluble in water, in which it forms a colloidal suspension. It can be tested
in suspension or as a solid.
Materials: Test tubes, test tube rack, Test tube holder, Bunsen burner
Chemicals: 5 % starch solution, glucose solution, sucrose, iodine/potassium iodide solution, and
distilled water
Procedure:
1. Take four test tubes and label them as 1, 2,3 & 4
2. Add 2 ml of 1% starch solution to a test tube 1
3. Add 2 ml of glucose solution to test tube 2
4. Add 2 ml of sucrose to a third test tube
5. Add 2 ml of distilled water (as a control) to the fourth test tube
6. Add a few drops of iodine or potassium iodide solution to each test tube

48
 7. Mix the contents by shaking

Questions:
1. What color did you observe at the end of experiment?
2. Is there any difference between the two test tubes?

II. Tests for Proteins

Activity 1: Xanthoproteic test
The xanthoproteic test is a method that can be used to determine the amount of protein soluble
in a solution, using concentrated nitric acid. The test gives a positive result in those proteins with
aminoacids carrying aromatic groups, especially in the presence of tyrosine. If the test is positive
the proof is neutralized with an alkali, turning dark yellow. The yellow colour is due to
Xanthoproteic acid which is formed due to nitration of certain amino acids, most common
examples being tyrosine and tryptophan. This chemical reaction is a qualitative test, determining
the presence or absence of proteins. The amino acid tyrosine, phenylanine and to some extent
tryptophane are responsible for the xanthoproteic test.

Materials: Test tubes, test tube rack, Bunsen burner

Chemicals: Egg albumen/egg white, ammonia solution, nitric acid, distilled water
Procedure:
1. Add 2 ml of protein solution (egg albumen) in a test tube.
2. Add about 2 ml of concentrated nitric acid to the egg albumen. Mix carefully.
3. Heat the content carefully.
4. Cool the test tube under tap water and then carefully add 2-5 ml of ammonia solution
(15N)
a. What color did you note when you mix the egg albumen with concentrated nitric
acid?
b. Did you see any color change when you heat the solution?
c. What color did you finally get after you cooled the test tube and add ammonia
solution to it?
Activity 2: Biuret Test

49
The biuret test is a chemical test used for detecting the presence of peptide bonds. In the
presence of peptides, a copper (II) ion forms violet-colored coordination complexes in an
alkaline solution. Several variants on the test have been developed, such as the Modified Lowry
test. Although not very specific, it is a good general test for qualitative determination of protein.

Materials: Test tubes, test tube rack, Bunsen burner, test tube holder
Chemicals: Egg albumen/egg white, 5% of KOH/5% of NaOH, distilled water, 1% of CuSO4
solution or Benedict’s solution

Procedure:
1. Add 2ml of protein solution (egg albumen) to a test tube.
2. In another test tube, add 2ml of distilled water (as a control).
3. Add an equal volume of 5% NaOH solution and mix.
4. Add 2 drops of 1% CuSO4 solution or Benedict’s solution and mix. No heating is
required
5. Leave the content for 15-20 minutes.
a. Is there any color difference between the control and the protein solution?
b. What color did you observe in the protein solution? How did the color form?

Activity 3: Millon’s Test

Materials: Test tubes, test tube rack, Bunsen burner, vortex, test tube holder
Chemicals: Egg albumen/amino acid, tap water, Millon’s reagent
Procedure:
1. To 1ml of the egg albumen solution in a test tube, add few drops of Millon’s reagent and
use vortex shaker
2. Boil the contents over a Bunsen flame for 3 – 5 minutes.
3. Cool the contents under running tap water and add few drops of sodium nitrite solution
a. Is there color difference between the control and the protein solution?
b. What color did you observe in the protein solution? How did the color form?

III. Tests for Lipids

50
 Lipids are diverse group of organic compounds including fats, oils, hormones, and certain
components of membranes that are grouped together because they do not interact appreciably
with water. Molecules such as proteins, nucleic acids, and carbohydrates have an affinity for
water and are called hydrophilic (“water-loving”). Lipids, however, are not hydrophilic but
hydrophobic (“water-fearing”).
NB. Any heating in that has to be done in the following tests should be carried out in a water
bath at the boiling of water. Direct heating of test tubes should not take place since you may
break the test tube.

Activity 1: Sudan III test

Materials: Test tube, test tube rack, test tube holder
Chemicals: Sudan III reagent, oil, water

Procedure:
1. Add 2ml oil to 2ml water in a test tube.
2. Add a few drops of Sudan III and shake.
a. Which part (up or bottom) of the contents of the test tube is stained red?
b. Why is that so?
Activity 2: Emulsion test
Materials: Test tube, test tube rack, test tube holder
Chemicals: Ethanol, cold water, fat or oil
Procedure:
1. Add 2 ml of fat or oil to a test tube containing 2 ml of absolute ethanol.
2. Dissolve the lipid by shaking vigorously.
3. Add an equal volume of cold water.
a. What changes did you observe?
b. What effect does the alcohol have?

Activity 3: Grease spot test

Materials: A piece of paper, Bunsen burner

Chemicals: Ethanol, cold water, fat or oil
Procedure:

51
1. Rub drops of oil or fat on to a piece of paper.
2. Allow time for any water to evaporate. Gently warming will speed up the process.
a. What did you observe?
b. Why did that happen?

52
 LABORATORY STUDY NUMBER 6

BIOCATALYSIS – ENZYMES

6.1 Introduction

Catalysts are those substances that alter the speed of chemical reaction without themselves
undergoing permanent change. For instance, at room temperature hydrogen peroxide (H 2O2)
undergoes a spontaneous but very slow decomposition. Furthermore, this example shows that
neither platinum nor catalase initiates the reaction. In fact, what enzymes or catalysts do is to
accelerate the rate of another, otherwise immeasurably slow reaction. Hydrogen peroxide (H 2O2)
is produced in cell as a result of the oxidation of molecular oxygen in oxidation reaction. Since it
is toxic to cells it is immediately decomposed in to water and oxygen.
2H2O2 → 2H2O + O2

In cells the decomposition of hydrogen peroxide is catalyzed by an enzyme called catalase. This
enzyme is found in various tissues (E.g. Potato, liver, kidney, muscles)

Objectives:
After completing the activities trainees will be to:
 explain the effect of temperature on enzyme activity
 explain the effect of pH on enzyme activity
Activity 1: Action of catalase on hydrogen peroxide (H2O2)
Materials: Test tube, potato cubes (boiled and unboiled)
Chemicals: 3 % H2O2 solution, distilled water.
Procedure:
1. Place about 3ml of 3 % H 2O2 solution in one test tube and an equal amount of water
in another test tube.
a. To each of the test tube add a small cube of potato tissue
b. In which of the tube is a reaction (gas bubble forming) taking place?
c. What is the reaction and what is the product?
d. What is there in the potato cube that is responsible for the reaction?

53
Activity 2: Effect of high temperature on enzyme action
Materials: Test tube, unboiled potato tissue, boiled potato tissue.
Chemicals: H2O2
Procedure:
1. Take two clean test tubes and place about 3ml of 3 % H2O2 solution in each of them.
a. Place a cube of raw/unboiled potato tissue in one test tube and place a cube of
boiled potato tissue in the other tube.
b. In which of the tubes is a reaction taking place?
c. How do you account for the fact that a reaction is taking place in one but not in
the other test tube?

Activity 3: Effect of pH on Enzyme Action

Materials: Test tube, potato tissue (boiled and unboiled)
Chemicals: H2O2, 0.1N HCl, 0.1N NaOH, water
Procedure:
1. Take three clean test tube and place 3ml of 3% H2O2 solution in each test tube.
2. Label them as A, B and C.
3. Add two drops of 0.1N HCl in test tube A; 2 drops of water in B; and 2 drops of 0.1N NaOH
in C.
4. Prepare three potato cubes of equal size and add one cube to each of the test tube.
a. In which test tube is the rate of the reaction highest?
b. In which pH range (acidic, neutral, or alkaline) is the activity of catalase
highest?

54
 LABORATORY STUDY NUMBER 7

PHOTOSYNTHESIS

7.1 Introduction
Photosynthesis is the process by which plants, algae, some bacteria, and other organisms convert
light energy and carbon dioxide into oxygen and glucose. It is an essential part of the life cycle,
and the reason that plants are at the bottom of the food chain. Typically, the plants take energy
from the sun to convert the carbon dioxide molecules (carbon and two oxygen atoms) into
glucose molecules, which are made up of carbon, hydrogen and oxygen. Oxygen gas is given off
as waste during the process, and water is often used in the process as well.

Figure 7.1. Diagrammatic Process of Photosynthesis

Photosynthesis is divided into two sets of reactions. Some characteristics of these reactions are
compared as follows:

55
Photochemical (light) reactions Biochemical (dark) reactions
 Light-dependent Light-independent
 Fast (practically instantaneous) Slower, but still extremely fast
 Split water to release O2, Convert (fix) CO2 to sugar
Electrons and protons

Figure 7.2. Diagrammatic presentation of plant pigmentations

Objectives:
Upon completion of these activities, you are expected to:

 isolate and identify photosynthetic pigments in leaves

 calculate retention factor (Rf) values of photosynthetic pigments and graph the
absorption spectrum for each pigment

56
  investigate the importance of light and chlorophyll for starch production

Activity 1: Paper chromatography of leaf pigment

Materials:. Green leaves, chromatography chamber, mortar and pestle, scissors, pencil, capillary

tube, spatula, scale, filter paper strips, watch glasses, thread

Chemicals: Ethanol, petroleum ether/acetone

Procedure:

1. Prepare leaf extract

2. Cut out a strip of filter paper/ chromatography strip about 2 cm wide
3. Draw a horizontal line with a pencil about 2-3 cm away from the tip of the notch
4. Using a capillary tube, place a few drops of the extract onto the middle of the line and let
it dry.
Note: each drop should be allowed to dry before placing the next one.
5. Add 2 ml of chromatography solvent (9 parts petroleum ether: 1 part acetone)
6. Immerse the end of the strip in the chromatographic chamber just above the level of the
solvent and watch as the solvent moves up the paper.
The spot should not be under the surface of the solvent.
7. Remove chromatography strip before the solvent-front reaches the top of the strip (after 3
to 5 minutes). Mark with a pencil the distance that the solvent front travelled, and set the
strip aside dry. You will see four bands of colour on the strip: a yellow band of carotenes
near the top, followed by a yellow band of xanthophyllous, a blue-green band of
chlorophyll a, and a yellow-green band of chlorophyll b near the bottom.

Questions
1. Measure the distance of each pigment band from the loading spot and also the distance
travelled by the solvent. Calculate the Rf value using the equation and record the values
in the table.

57
Note: An Rf value that is close to 1 indicates that the pigment is very soluble in the solvent. An
Rf value that is very small indicates that the pigment is not very soluble in the solvent.

RF values Pigment identification

 β-carotene = 0.99 Chlorophyll a= blue-green
 Chlorophyll a = 0.30 Chlorophyll b = Olive green
 Chlorophyll b = 0.13 Xanthophyll = yellow
 Violaxanthin =0 .40 Carotene = orange yellow
 Lutein = 0.68

2. What does a small Rf value tell you about the characteristics of the moving molecules?
3. Why is chromatography useful?

Factors necessary for photosynthesis

Chlorophyll, light, Carbon dioxide, water, and temperature are major factors important for
photosynthesis to take place.
Activity 1: Light – a factor necessary for photosynthesis
Materials: Partly masked leaf, Petri-dish
Chemicals: water, iodine solution, alcohol

Procedure:

1. You will be provided with a leaf which, while still attached to an intact plant is partly masked
for the last 24 – 48 hours partly clipped and covered.
2. Remove the cover and carry out the test for starch as described below.

3. Keep the leaf in boiling water for about 5 minutes. This kills the cells and renders them
highly permeable.

4. Transfer the leaf in warm alcohol; and keep it there until it is decolorized. The alcohol is kept
warm in a water bath, or it may be directly warmed over a ‘controlled’ oven or hot-plate.

58
5. Soak the decolorized leaf in water. Then put it a Petri dish and cover it with 0.05 iodine
solution.

6. Allow the leaf to remain in the iodine solution for 5 minutes and then rinse (wash) it with
water.

Questions:

1. Which part of the leaf, the masked or the unmasked, is positive for starch?
2. What does this result show about the importance of light for starch production?

Activity 2: Chlorophyll - a factor necessary for photosynthesis

Materials: Variegated leaf, Petri dish

Chemicals: water, iodine solution, alcohol

Procedure:

1. You will be provided with a variegated leaf. Make a drawing of the distribution of colors in
the leaf.
2. Test the leaf for starch.
3. Make the drawing of the distribution of starch in the leaf and compare it with first drawing.
Questions:
1. How do the distribution of chlorophyll and of starch in the leaf are compared?

2. Is chlorophyll important for photosynthesis? Explain this in line with your result.

59
 LABORATORY STUDY NUMBER 8

MICROBIOLOGICAL LABORATORY TECHNIQUES

8.1 Introduction
In the laboratory, the nutrient preparations that are used for culturing microorganisms are called
media (singular, medium). Three physical forms are used: Liquid (broth) media, semi‐solid
media and solid media. The major difference among these media is that solid and semi‐solid
contain a solidifying agent called agar, whereas a liquid medium does not. Agar is
polysaccharide extracted from marine seaweeds such as red algae. Agar has some unique
properties that make it useful in culture media. Only few microbes can degrade agar so it remains
solid during microbial growth. It liquefies at 100 oC and remains in a liquid state until cooled to
40oC. 1.5 % agar produces a firm gel at 32‐39oC but, once solidified, it will not melt until 85oC is
reached. This means you can incubate plates at any temperature up to 85 oC. You can add
organisms to molten agar cooled to a temperature (40‐45oC) which will not damage the majority
of organisms. This allows the pour plate technique to be carried out.

While in the liquefied state, solid media can be poured into either a test tube or Petri plate. If the
medium is allowed to harden in a slanted position, the tube is designated as Agar slant. If the
tube is allowed to harden in an upright position, the tube is designated as Agar deep tube and if
the agar is poured into a Petri‐plate, then the pate is designated an Agar plate.

Most chemo-heterotrophic bacteria are routinely grown on complex media. Organic carbon,
energy, nitrogen sources are usually supplied by protein in the form of meat extracts and partially
digested proteins called peptones.

The preparation of media from commercial dehydrated products is simple and straightforward.
Each bottle of dehydrated medium has instructions for preparation on its label. Media must be

60
sterilized after preparation. The most common method of sterilizing culture media that are heat
stable is steam sterilization, or autoclaving, using steam under pressure. During this process,
material to be sterilized is placed in the autoclave and heated to 121oC at 15 pounds of pressure
for 15 minutes.

Objectives:
After completing the activity students will be able to:
 Prepare different culture media
 describe the importance of sterilization in microbial techniques
 isolate bacteria using different techniques to get pure culture
 prepare a bacterial smear properly
 stain bacteria and observe them under the microscope

Activity 1: Preparation of culture medium

Materials: Petri plate, autoclave, erlenmeyer flask, triple beam balance, incubator, aluminum
foil, test tubes, hot plate, magnetic stirrer, heat proof gloves, safety hood/safety
cabinet, cotton, sprit lamp, metal inoculating loop, permanent marker, l-shaped glass
rod, volumetric pipettes, micropipette, pipette filler, spreading wheel, centrifuge,
bottles (reagent bottles)
Chemicals: Nutrient Agar, Nutrient Broth, Potato Dextrose Agar, Alcohol

Procedure
1. Weigh 7 grams of the powder of the Nutrient agar given (as mentioned on the label of the
bottle) and add it to 500 ml flask.
2. Add 250 ml of distilled water
3. Use Aluminum foil or cotton to cover the mouth of the flask.
4. Heat the solution until it begins to boil (Make sure to use magnetic stirrer or to shake the
bottle periodically until it boils)
5. Load the autoclave with the prepared culture media and autoclave for 15 minutes or more
at 121oC
6. When the period of sterilization is completed and the pressure in the chamber reads 0,
carefully open the door and remove the containers.

61
 Before opening the autoclave, you should wear heat‐proof gloves, stand at arm’s length,
and slowly open the door.
7. Wait until the temperature drops approximately to 48‐50oC
Pour 15‐20 ml of the medium to sterile, label (indicating the name of the medium) Petri‐ plates in
a Laminar flow following aseptic procedures. Wait until the agar solidifies and invert the plates
upside down.

Questions:

1. Why are culture media sterilized before use?

2. Why is agar Petri‐plates inverted after they have cooled?
3. Why is culture medium cooled to about 48oC to 50oC before pouring?
4. What are the three main types (in terms of their physical forms) of microbiological culture
media?

Activity 2: Isolation techniques of pure bacterial culture

A. Streak plate technique

In nature most microbes are found growing in environments that contain many different
organisms. However, mixed cultures are of little use in studying microorganisms because of the
difficulty they present in determining which organism is responsible for any observed activity. A
pure culture is a culture that contains only one kind of organism. A pure culture is required in
order to study concepts such as growth characteristics, pathogenicity, metabolism, and antibiotic
susceptibility and any biochemical tests. There are three dilution methods commonly used for the
isolation of bacteria: the streak plate, the spread plate, and the pour plate.
In the streak plate technique, a loop is used to streak the mixed sample many times over the
surface of a solid culture medium in a Petri‐plate. At some point on the streaks, individual cells
will be removed from the loop as it glides along the agar surface and will give rise to separate
colonies. One assumes that one colony comes from one cell. The principle is that by streaking, a
dilution gradient is established on the surface of the plate as cells are deposited on the agar
surface. Streak plate is the most common isolation technique in use today.
Materials: Sprit lamp, inoculating loops, permanent marker
Chemicals: 24‐48 hours mixed nutrient broth cultures, nutrient agar plates
Procedure:

62
1. Label all the plates, on the bottom, with the name of the organism(s) or sample code to be
inoculated, the date and your name with marker.
2. Flame the inoculating loop to redness, allow it to cool, and aseptically obtain a loopful of
one broth culture.
3. To streak a plate, lift one edge of the Petri plate cover, and streak the first sector by
making as many streaks as possible without overlapping previous streaks. Do not gouge
the agar while streaking the plate.
4. Flame your loop and let it cool. Turn the plate so the next sector is on top. Streak through
one area of the first sector, and streak a few times away from the first sector.
5. Flame your loop, turn the plate again, and streak through one are of the second sector.
Then streak the third sector.
6. Flame your loop, streak through one area of the third sector, and then streak the
remaining area of the agar surface, being careful not to make additional contact with any
streaks in the previous sections. Flame your loop before setting it down. (Why?)
7. Incubate the plates in an inverted position in the 35oC incubator (or at room temperature,
depending on the inoculum) for 24‐48 hours.

63
Figure 8.1: The streaking technique for pure isolation of bacterial culture

Questions:

1. What is the need to label the plates on the bottom?

2. Why is it necessary to incubate inoculated agar plate cultures in an inverted position?

Activity 3: Smear preparation and simple staining

A. Smear preparation
Materials: Microscope, microscopic slides, cover slips, inoculating loop and needle, sprit lamp,
permanent marker, forceps, blotting paper
Chemicals: Agar plate culture, sterile distilled water in a dropper, oil immersion, crystal violet
and methylene blue
Procedure:

1. Mark the name (Code) of the bacterial culture in the far corner on each of the slides.
2. For a broth culture, shake the culture tube and, with an inoculating loop, aseptically
transfer 1 to 2 loopfuls of bacteria to the center of the slide. Spread this out to about half
inch (1.27 cm) area. When preparing a smear from a slant or plate, place a loopful of
water in the center of the slide. With the inoculating needle, aseptically pick up a very
small amount of culture and mix into the drop of water. Spread this out as above.
3. Allow the slides to air dry.
4. Pass the slides through a Bunsen burner or sprit lamp flame three times to heat fix and
kill the bacteria.

B. Simple staining
Procedure:
1. Place the fixed smears on a staining loop or rack over a sink or other suitable receptacle.
2. Stain the slide with any of the three stains given (i.e., crystal violet (20 to 30 seconds
staining time), Carbol fuchsin (5 to 10 seconds staining time), or alkaline Methylene blue
(1 minute staining time)).
3. Wash stain off slide with water for a few seconds.
4. Blot slide dry with blotting paper. Be careful not to rub the smear when drying the slide
because this will remove the stained bacteria.

64
 5. Examine under the oil immersion lens.

Questions:
1. What would happen if you apply to much heat while heat fixing the smear?
2. Mention three importance of heat fixing?
3. What is the advantage of simple staining over wet mount?
4. What is the biochemical basis for simple staining?
C. Gram’s staining
Bacteria differ from one another chemically and physically and may react differently to a given
staining procedure. This is the basis for differential staining. Gram stain is a differential stain as
it differentiates bacteria in two groups ‐ gram negative and gram positive. The Gram stain
(named after Hans Christian Gram, Danish Scientist and physician) is the most useful and widely
employed differential stain in bacteriology. Gram staining is the single most useful test in the
clinical microbiology laboratory.

The first step in the procedure involves staining with the basic dye crystal violet. This is the
primary stain. It is followed by treatment with an iodine solution, which functions as a mordant;
that is, it increases the interaction between the bacterial cell and the dye so that the dye is more
tightly bound or the cell is more strongly stained. The smear is then decolorized by washing with
95% ethanol. Gram positive bacteria retain the crystal violet‐ iodine complex when washed with
the decolorizer, whereas gram–negative bacteria lose their crystal violet–iodine complex and
become colorless. Finally, the smear is counterstained with a basic dye, different in color from
crystal violet. This counter stain is usually safranin. The safranin will stain the colorless, gram
negative bacteria pink but does not alter the dark purple color of the gram positive bacteria.

Gram stain is more consistent with young cultures (8‐16 hours old). Old cultures may show
variation in Gram staining. Besides, some bacterial species are gram variable. That is, some cells
in the same culture will be gram positive and some, gram negative. Hence, indistinct gram‐ stain
results can be confirmed by a simple test using KOH. Place a drop of 10% KOH on a clean glass
slide and mix with a loopful of bacterial paste. Wait 30 seconds then pull the loop slowly through
the suspension up and away from the slide. A gram negative organism will produce a mucoid
string whereas gram‐ positive organism remains fluid.

65
Materials: Microscope, glass slides, inoculating loop, sprit lamp, blotting paper, staining rack
Chemicals: Solutions of Crystal violet, Gram’s iodine ( 2g KI in 300 ml Distilled water plus 1g,
iodine crystal), 95% ethanol, Safranin, KOH (3%), 18‐24 hour nutrient broth cultures of
Gram positive and Gram negative bacteria, immersion oil
Procedure

1. Prepare heat‐fixed smears of the given culture and mark circle around each smear on the
reverse side of slide with marker.
2. Place the slides on the staining rack.
3. Flood the smears with crystal violet and let stand for 30 seconds.
4. Rinse with water for 5 seconds.
5. Cover with Gram’s iodine mordant and let it stand for 1 minute.
6. Rinse with water for 5 seconds.
7. Decolorize with 95% ethanol for 15‐ 30 seconds. Do not decolorize too long. Add the
decolorizer drop by drop until the crystal violet fails to wash from the slide.
8. Rinse with water for 5 seconds.
9. Counter stain with safranin for about 60‐80 seconds.
10. Rinse with water for 5 seconds
11. Blot dry and examine under oil immersion. Gram positive organisms stain blue to purple;
gram negative organisms stain pink to red. There is no need to place a cover slip on the
stained smear.

Questions:
1. What is the basis for the Gram stain technique?
2. Why is the Gram stain technique known as a differential technique?
3. What do you conclude if you find red and purple cocci in pure culture?

66
 LABORATORY STUDY NUMBER 9

CELL DIVISION

9.1 Introduction
Cell division is a process where cells reproduce and multiply their number. Mitosis is a nuclear
division, which produces two complete sets of chromosomes. Each set is later separated from the
other by cytokinesis. The newly formed daughter cells are identical to one another. This division
also called equational division, as the chromosomes are equally distributed among the two
daughter cells. This division ensures that an identical genetic constitution is maintained for all
the cells of the body.

Most of the diploid organisms undergo reduction division during gametogenesis i.e., they
produce gametes that are haploid. The cell division that brings about this reduction from two sets
of chromosomes (diploid) to one set of chromosomes (haploid) is called meiosis. Meiosis
comprises of two divisions- Meiosis I & Meiosis II. At the end of meiosis four daughter cells are
produced, each daughter cell having half the chromosome complement –of the parent.

Objectives:
 After completing the activities trainees will be able to
 describe the process of mitotic cell division
 describe the process of meiotic cell division
 discuss the differences between mitotic cell division and meiotic cell division

Activity 1: Experiment on Mitosis

Materials: Microscope, slides and cover slips, germinating onion or garlic, test tubes, test tube
holders, watch glass, Bunsen burner
Chemicals: Distilled water, acetocarmine/haematoxylin, 0.1N HCl, 20 % acetic acid, Cornoy’s
fluid (30ml of 100% of Ethyl alcohol, 5ml of glacial acetic acid,15 ml of chloroform)

Procedure:

67
 1. Separate the garlic cloves (or use onions) and place them in test tubes containing water,
such that their bases are dipping in water.
2. Within 2-3 days root tips start appearing.
3. Cut the root tips and fix them in carnoy’s fluid (100% ethyl alcohol-30ml; glacial acetic
acid-5ml; Chloroform-15ml), for 18-24hrs at room temperature or 4 oC. Then transfer
them to preservative such as sprit or formalin
4. Remove the root tips and keep them in water in a watch glass for a few minutes.
5. Remove the water and add 0.1N HCl and heat it in water bath (about 60 0C) and wait for
about 10 minutes until the root tips become soft. (Do not allow the material to boil).
6. Remove the HCl and wash the material with distilled water, 2-3 times till all a trace of
the acid is removed.
7. Add the stain-acetocarmine/haematoxylin and allow the material to remain in the stain for
about 10-15 minutes.
8. Cut the swollen (stained) root tips and place them on a clean slide. Add 2-3 drops of 20%
acetic acid. Cover it with a cover slip and tap gently to make a squash.
9. Add a drop of fresh stain on the root tip and cover it with cover slip
10. Tap on the cover slip gently with the rubber end of your pencil. This will break the tissue
apart.
11. Place a folded piece of soft paper on the cover slip and squash the root tip by applying a
uniform pressure on the cover slip with the ball of your thumb.
 If you find your preparation unstained, restain it using the following steps:
12. Place a drop of the stain at one edge of the cover slip
13. By holding a piece of soft paper or any absorbent paper at the opposite end, draw the
stain under the cover slip
14. Observe the slide under the microscope and identify the different stages of mitosis.
NB. When you observe the dividing cells under the microscope, take the following into
consideration:
a. The treatment has killed the cells. Therefore, one cell shows only one phases i.e. the
phase in which it had been before the treatment.
b. You may not find all the phase in any single flied of vision

68
 c. Do not expect to find the phases in the order Interphase, Prophase, Metaphase and
Telophase
d. In any one field of vision there are a large number of cells, using the low power or the
middle power, scan through the field with your eyes. When you find a cell in the phase
you are looking for, bring it to the center of the field. Then shift the high power objective.

Questions:
1. Why you used the swollen root tip to observe the stages of cell division?
2. What is/are the stage(s) of mitosis observed?

Activity 2: Experiment on: Meiosis

Materials: Maize or testis of grasshopper, (fresh anthers from unopened buds can also be used),
Microscope, slides and cover slips, Bunsen burner
Chemicals: Carnoy’s fluid or formalin-acetic/prop ionic-alcohol (90 ml of 50% of ethyl
alcohol), 5ml of glacial acetic acid or 5ml of Prop ionic acid,5ml of formalin,
acetocarmine/haematoxylin

Procedure:
1. Fresh or fixed anthers of maize or the testis of grasshopper is taken on a clean slide.
2. The material is crushed with a scalpel or another slide such that the cells are spread or
smeared in a single layer.
3. If a fresh material is used, invert the slide over a Petri dish containing the fixing and
killing agent (Carnoy,s fluid or FAA), such that the material is immersed in it. Allow it to
remain for 10-15 minutes.
4. Add a few drops of the stain on the smeared material. Remove the large pieces and
debris. After a few minutes, fresh drops of the stain can be added.
5. Heat the slide gently over the flame and add a few drops of 20 % acetic acid or use the
stain itself. Cover the material with a cover slip.
6. Observe the slide under the microscope and identify the different stages of meiosis.

69
 LABORATORY STUDY NUMBER 10

COLLECTION AND PRESERVATION OF PLANT AND INSECT

SPECIMENS

10.1 Introduction
Collections of plants and animals serve as the scientific researchers, artists, and educators.
Collections may include a variety of preparation types emphasizing preservation of leaf, stem,
soft tissues, teeth, skin or some combination of these.

Objectives:

After completing the activities students will be:

 able to describe appropriate techniques for collecting and preparing herbarium and insect
specimens
 begin their own plant and animal collection
 acquainted to basic equipment used for insect collection
 acquainted to methods employed to kill and preserve insects
 acquainted to correct pinning, mounting, labeling and storing techniques

I. Plant collecting
Plant collecting is the acquisition of plant specimens for the purposes of research, cultivation, or
as a hobby. Plant specimens may be kept alive, but are more commonly dried and pressed to
preserve the quality of the specimen.

The purposes of collecting:

There are two main reasons for collecting plants. The first is to obtain records and specimens of
plants, either for a personal collection or to be stored in a herbarium. A herbarium specimen is a
pressed and/or dried sample of a plant or fungus that can be stored for future reference.

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What to look for in a specimen:
Specimens for collection should be as complete as possible. Ideally flowers and fruit should be
included, as well as vegetative parts
What to collect/plant specimens:
Each specimen should consist of a stem with attached leaves and, if at all possible, flowers
and/or fruits; these are usually critical for identification. With smaller plants, such as grasses,
rushes, sedges, irises and lilies, collect whole plants (or a number of entire plants) including
underground parts (i.e. bulbs, corms, rhizomes) still attached to aerial parts of plant. In the case
of very large trees, shrubs, or vines, pieces should be selected to illustrate to the greatest extent
possible the overall characteristics of the plant and the range of variation in flowers, leaves, and
other structures. Make specimens large enough to present a fair sample of the plant, its manner of
growth, branching and so on. Mosses and lichens should also be taken whole.
How to collect:
Ideally, collections of vascular plants should be put immediately into a field press (if you have
taken a plant press along), because this produces the best looking specimens. Collecting into
plastic bags is another option and the specimens will be pressed later on after the field.

Notes to take:
Every specimen should be accompanied by comprehensive notes retained in a collecting note
book. The notes should contain the following information.
1. Collection number: This is a serial number specific to a collector and a specimen. The
number may start at 1 and continue through the collector's life time.
2. The name of the plant: This is important as it helps the collector remember the individual
specimen even if the labels are accidently lost or mixed. Even if the collector has no idea
what the specimen is, it is sometimes useful to give a completely arbitrary name.
3. Locality: This should be as detailed as possible, including the name of towns, roads lakes
and so on in the vicinity, as well as Township, County or District, the latitude and
longitude.
4. Description: This should include everything about the plant. Essential items are the
height, type of bark, whether the stem is upright, sprawling or drooping, obvious smells,
whether the plant is clumped, single or growing in patches, and the presence of creeping

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 or underground stems. Flower and fruit colour should also be noted as these often fade on
dried specimens.
5. Habitat: This should include the general habitat as well as more specific details of micro-
habitat. Important points are type of soil or other substrate (sand, clay, granite, dead
wood, other vegetation), associated species, moisture and aspect (fully exposed on a
south facing bank; in a damp hollow under dense scrub, etc).
6. Date of collection
To avoid confusion, write the date in full (August 7, 2018). Note that ‘‘8/07/2018’’ could
be interpreted as August 7, 2018 or July 8, 2018.
7. Name(s) of collector(s)
Record all significant members of the collecting party. In the future, if one collector is
unavailable to answer questions, the others may be contacted.

Preparing plant specimens

After collection plants are pressed and dried, mounted and stored.
1. Pressing and drying plant specimens
Specimens should be pressed when fresh (i.e. in the field). This results in better herbarium
specimens, making them easier to identify. When pressing a specimen, carefully spread out
structures (i.e. leaves, flowers) so that diagnostic features are clearly evident. Make sure that
both the upper and the lower leaf surface are visible by turning over some leaves. Specimens are
pressed in a plant press, which consists of a wooden frame (for rigidity), corrugated cardboard
ventilators (to allow air to flow through the press), blotter paper (to absorb moisture), and folded
paper, typically a newspaper (to contain the plant material). The plant press is tightened using
straps with buckles or bolts with wing nuts. The objective of pressing plants is to extract
moisture in the shortest period of time, while preserving the morphological integrity of the plant,
and to yield material that can be readily mounted on herbarium paper (an acid-free cardstock) for
long-term storage.

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Figure 10.1: Plant pressers

How to press and preserve plants

 To press the specimen, clean up the plant. Brush off loose soil and blot off moisture.
 Arrange the plant on a sheet of newspaper. Next to it, place the identification tag with its
name, a number you have assigned to it, the location where it was collected, when it was
collected, and by whom. Make sure the same information is in your journal. Place another
piece of newspaper on top of the plant.
 Make layers. Place the pieces of newspaper with your specimen inside between two pieces of
blotting paper, then between two pieces of corrugated cardboard, to allow air to circulate.
 Place the resulting package in the plant press and gently screw it down. As an alternative,
you can hold it securely together with straps, or place some heavy objects (books, bricks) on
top.
 You can dry several plants in the press at one time. Each should be arranged in the same
layers as described above.
 Check the plants every two or three days, and replace the damp papers with dry ones. It will
take from two to four weeks before the specimens are completely dry.

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Figure 10.2: Pressing of plants

Processing dried specimens involves 3 major steps:

 identification
 label preparation
 mounting
 storage
Identification: Specimens need to be correctly identified. If you cannot identify it yourself or
have any doubt, send a duplicate specimen to a taxonomic expert for confirmation.

Label preparation: Labels (notes taken during collection) for the dry specimens can be prepared
before the plants are identified, recording information known at that time. The genus/species and
identifier can be typed in later. Note that any information on characters and field observations
that cannot be observed from the pressed specimen:
 Habitat: include a brief description of where the plant is growing
(e.g.Themeda triandra grassland; grazed paddock; weedy roadside, etc.) and a list of
other plants growing in association, if known.
 Habit: record the growth form (e.g. tree; shrub; vine; herb) and height (e.g. dense shrub to
2 meters high; sprawling herb). For trees, record the bark type and extent (e.g. rough bark
up to 2 meters on main trunk, smooth above). Bark type is especially important

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 in Eucalyptus. Also record the colour of fresh stems, leaves, flowers (for plants) or pileus,
stipe and lamellae (for fungi).
 Abundance: number of plants at site. Frequency in the area (rare, occasional,
frequent/common or abundant).
 Substrate: for bryophytes, fungi and lichens, include a description of the substrate on
which the specimen is growing.
 For algae, collecting labels should include information on water type (saline, brackish or
fresh) and quality (such as muddy or polluted), as well as whether the alga is free or
attached to a substrate.

Mounting specimens: Once material is pressed and thoroughly dried, it is mounted on

herbarium sheets. The standard size for these sheets is 420 x 297 mm. They should be made from
stiff, acid-free, paper or cardboard of good quality so that they will not turn yellow or crack with
age.

Storage of specimens: In order for plant specimens to be kept in good condition for future
reference, adequate storage conditions are needed. The storage space should be kept in a low
light, low humidity environment. The temperature of the storage space should be kept cool,
between 50 – 65 0F.

It is important to keep the storage space free of harmful pest. It is recommended to protect the
specimens by sheathing the sheets in sealed plastic bags. Various pesticides may also be used to
protect the storage space from pest infestation. If pest infestation has already occurred, the
samples should be frozen for three to four days. Freezing new additions of plant samples is a
suggested preventative measure against the introduction of pest to the storage space.

Activity 1: Plant collection

Each student will collect 5 plants which include grasses, shrubs, herbs, and trees. The plants will
be pressed and dried and submitted to the instructor with all necessary information.

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II. Insect collection
Introduction: The best way for a student to know and appreciate insects is by collecting all
sages of insects from different habitats at different seasons of the year. Short visits to known
habitats may not yield representative species of insects. Therefore prolonged search in likely
habitats would definitely result in success.

A keen student, in addition to what he/she learns in class, would come to know more about
insects as a result of regular collection from different habitats. A student may learn a lot about
the life and daily activity of insects by simply observing them in their natural habitats, or he/she
may systematically collect and prepare them for permanent display and identification. As a result
of such activity a student would develop much more interest in insects as well as appreciate the
role they play in nature.

Some butterflies and beetles are simply collected for their beauty. Other insects may be collected
to determine the species that may be of medical, veterinary or economic importance. Still many
other insects may be collected to know the insect fauna of a given area.

A student should therefore know some of the methods and techniques that are employed for
collecting different types of insects from different habitats. In addition, the student has to be able
to carefully process, mount, preserve, identify (where possible) and label his/her collection.
It will be difficult in this introductory class or laboratory session on entomological technique to
list all types of method for collecting and mountings insects. Entomological techniques actually
involve several other matters. The student is advised to consult various books and manuals to
learn more about the various methods used in the collection and preparation of insects. Standard
identification keys or even simple keys that are found in books could be used to identify insects
to the family. Identification of specimens to the genus or the species is usually very difficult and
may require the help of a specialist

Where to look for insects

Insects are found almost everywhere. Millions of insects could be collected from a small plot of
land. They are found under stones, logs, fallen leaves and rotting vegetation. A large number of

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insects are found on the flowers, leaves, branches and stems of trees or on different parts of small
plants or cultivated crops. Many are found in soil, and among grass. Numerous insects fully or
partially live in temporary or permanent bodies of water. Adult and immature stages of insects
tend to live in different habitats. For instance, the larvae and pupae of Culex quinquefasciatus
live in drainage systems, septic tanks, etc., while the winged adult mosquito flies into nearby
houses, caves and nests to feed on blood of man, various rodents or birds.

Some insects are active during the day (diurnal) while others tend to be active at night
(nocturnal). Therefore, the habitats as well as the time of the day are important to collect
different stages and species of insects.

Basic equipment for collecting insects

1. Insect nets
Aerial nets: These are used to collect insects in flight. A net bag made of translucent netting so
one can see what’s inside; it can be used as a beating net if needed.
Beating (or sweeping) net- A heavy cloth bag, perhaps with small netted area at the bottom; it is
used to sweep “like a broom” through vegetation many times. It is used for taking insects that
live among vegetation.
Aquatic net or water net: It is used to collect insects from aquatic habitat.

2. Aspirators
A tube plugged with a rubber cork in which are inserted two tubes: one bent and used to point at
tiny insects; the other connected to a rubber tube for inhaling quickly to suck the insect into the
tube. The latter one has a tiny screen attached to the inside end to prevent insects from getting
into one’s mouth. Aspirators are used for catching small insects such as mosquitoes, sand flies,
black flies midges, etc.

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Figure 10.3: Aspirator
3. Berlese funnel
A commercial funnel of any size is needed, equipped with a screen inserted just above the
narrow spout to prevent material from falling out. Leaf litter, birds’ nests, and other organic
matter are put into the funnel, which is mounted on a rack or ring stand. A light bulb is placed
over the top to dry out the organic material, driving arthropods downward as they seek moisture.
This is a simple method of collecting insects (e.g. spring tails) out of soil, leaf litter, etc. As the
material dries with electric light bulb the insect moves down the funnel and gets collected in a jar
containing 70% alcohol. The Berlese funnel is left in place until the organic matter is completely
dried out.

4. Light traps
Various types of light traps (e.g. CDC light trap, New Jersey light trap, Monk’s Wood trap, etc.)
are used to collect small insects such as culicoides, sand flies, mosquitoes and black flies. Light
traps are normally used at night and are effective in catching insects that are active at night.

5. Floatation technique
Some insects and related arthropods can be collected from soil, debris or leaf litter using this
technique. Solution of sugar or magnesium sulphate could be placed in a bucket. Stir or shake a
sample of soil in this solution or in water. Various mites, larvae, pupae and adults of insects
could float to the surface from where they could be easily removed using camel-hair brush or
forceps.

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 6. Sticky plates
These are formica plates or simple non-absorbent paper plates of known size that could be
smeared with sticky material such as castor oil. The plates will be left overnight in likely habitat
to catch or entrap small insects such as sand flies and ants.
7. Human bait
Human beings could serve as bait to collect anthropophilic species and haematophagous insects
(e.g. mosquitoes, sand flies, black flies, etc.).

Insect killing jars/bottles

A glass or plastic jar of desired size (to collect small and large insects) can be made into a killing
jar by putting about a 1-cm layer of plaster of Paris in the bottom, or use just a pad of absorbent
material such as cotton or soft tissue. A fluid killing agent such as ethyl acetate is added to be
absorbed by the plaster or other material. Be sure not to have any fluid on the walls of the jar, or
specimens will be spoiled. If you use cotton or other absorbent material, cut a cardboard disk to
separate the insects from the pad of killing agent.

1. Cyanide bottles (Sodium or Potassium cyanide)

Toxic cyanide vapors are used commonly by entomologists to kill adult insects. Cyanide is an
insecticide which is a deadly poison. Sodium or potassium cyanide covered with sawdust and
plaster of Paris in killing bottles would serve in the killing of different species of insects. This
arrangement should be prepared in a tightly corked bottle. Calcium cyanide bottle is prepared in
a different way. Here instead of plaster of Paris, cotton and cardboard are used to cover the
cyanide in the killing bottle.

2. Ethyl acetate bottle

A bottle partly filled with plaster of Paris could be used for this purpose. Dry the plaster and then
saturate it with ethyl acetate. This killing bottle must be kept tightly corked. The ethyl acetate
will remain effective for months.

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 Figure 10.4: A killing jar

3. Other killing agents

Ether, chloroform, carbon tetrachloride and benzene are also used by collectors to kill insects.

Mounting and preserving insects

Once the specimens are dead, you can mount them. Remember to take them out as soon as they
are dead. Leaving them in the jar too long will damage their color. Insects are usually mounted
on the right side (when you look at it from behind).

1. Pinning

A Guide to mounting insects on pins

The groups normally mounted on insect pins are:  Odonata, Orthoptera, Dermaptera, Hemiptera
(Heteroptera), Homoptera (Auchenorrhyncha), Neuroptera, Coleoptera, Mecoptera, Lepidoptera,
Diptera, and Hymenoptera. Most small insects that can be pinned are at least 5 mm in length,
with a thorax big enough to hold the pin. Most medium and large insects are pinned
entomological pins of size 1 to 3. Sizes 4 to 7 are sometimes available for large specimens.

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 Figure 10.5: A variety size of entomological pins
 Beetles - pin on the upper quadrant of the right wing (elytra)
 Wasps, bees, and flies - pin to the right of the thorax, near the base of the wings.
 Moths and butterflies - pin in the middle of the thorax; this is to balance out the insects
for spreading their wings.
 Grasshoppers - pin to the right of the thorax.
 Dragonflies and Damselflies - are not usually pinned. They are kept in envelopes.
Pointing: Insects that are too small to be pinned are mounted on points made with paper. Make
triangular points out of a non-acid paper (such as artist canvas). The points should be only 7-
10 mm long and the width of the thickest end 2-3 mm wide.

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Diagram of pinning locations

A. Flies and bees

B. Butterflies and moths
C. Grasshoppers and crickets
D. True bugs
E. Dragonflies and damselflies
F. Beetles and weevils

Figure 10.5: Correct pinning location of some insect groups

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 Large Insects

Pin the insect, dorsal side up, so that precisely 10 mm of the shaft is free above the
specimen.  Generally, the pin should pass through the insect's mesothorax, slightly to the
right of center (see Diagram of Pinning Locations for different body types).  The insect's
ventral surface should be perpendicular to the pin.  Consider visibility, breakage, and
space when positioning parts.  Lepidoptera and Odonata are best positioned on a spreading
board. With Lepidoptera spread so that the trailing edge of the fore wing just overlaps the
leading edge of the hind wing, the line of juncture being perpendicular to the longitudinal
axis of the body.

Figure 10.6: Correct pinning of large insects like Orthoptera

Small Insects

Insects too small to receive the shaft of a #3 pin may be glued to pinned paper points. Using clear nail
polish as glue, firmly attach the point to the insect's right side just above the middle right leg (mesothorax),
dorsal side up. Align labels 5 mm apart as shown to protect the specimen and conserve space.

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Figure 10.7: Correct pinning of small insects like small ants

Data labels

Use index card stock or equivalent.  Labels should be lettered in black, waterproof ink or printed
with a laser printer.  A crow quill or Rapidograph pen (01 point) is adequate for most hand
lettering.
Size: no larger than 8 x12 mm, 1-4 lines of writing per individual label.
Format: Write data (where, when, and by whom collected)

1. Top level: Date/locality label (general to specific)

 Lines 1 & 2:  Country, State, County, City
 Line 3:  Date collected (day, month in Roman, year in full as 26-VII-2011)
 Line 4:  Collector's name (initials and last name)

2. Second level: Identification label

 Give family name for pinned insects; order and family for insects in alcohol

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 3. Third level (optional): Ecological label

Medium and large-sized insects such as butterflies, beetles, and grasshoppers can be pinned. The
pins should be the right size and should not be pinned on the part of the body that would not
make identification difficult. The labels are attached to the pins or slides with mounted
specimens. With preserved specimens the labels are written in pencil and kept in bottles with the
specimens and the preservative (e.g. 70% or 80% alcohol).

2. Other methods of mounting

Very small insects are mounted on a card point or on microscopic slides.
3. Spreading insects
Insects such as butterflies are prepared on spreading board before they are ready for display.

Preserving insect specimens in alcohol

Insects that are too small (e.g. small beetles, ants, many bugs, etc.), or the bodies of which are
too brittle or soft, should not be pinned. They are killed and stored in glass vials in 70% or 80%
ethanol.

Relaxing
Fresh specimen of insects can easily be mounted. Dried specimen are difficult to mount can
easily break. such specimen are relaxed by putting them in relaxing chamber containing wet sand
or cloth for a day or two. Carbolic acid is added to prevent mould.

Insect boxes
Prepared specimens are kept in boxes (large or small) to protect them from damage or insect
pests. Labeled specimens kept in insect box are either stored in a safe place for future use or are
put on display for use by students or the general public.

Activity 2: Insect collection

Collection instructions

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Each student should submit an insect collection from ten (10) orders. The collection must be
housed in Schmitt boxes or shirt boxes, and specimens must be mounted on insect pins (not
ordinary household straight pins) or preserved in vials of alcohol if soft-bodied.  Each pinned
insect and each vial of alcohol must contain a date/locality label which includes the following
labeling information:
1. name of specimens
2. place of collection (locality)
3. date of collection
4. name of collector of specimens

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 REFERENCES

Tayor, D. J., Green, N. P.O. and Stout, G. W. U. (1998). Biological Science 3rd edition. Pp
984 (Soper, R., ed).Cambridge University Press.

Http://www.sspca.org/general biology.html. Searched on September 5, 2017.

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 Appendix

Laboratory Report Writing Format

The purpose of writing a lab report is to determine how well you performed your experiment,
how much you understood about what happened during the experimentation process, and how
well you can convey that information in an organized fashion.

Lab Report Format

A good lab report format includes six main sections:
1. Title
2. Introduction
3. Materials
4. Methods
5. Results
6. Discussion
7. Conclusion
8. References

Keep in mind that individual instructors may have a specific format that they require you to
follow. Please be sure to consult your teacher about the specifics of what to include in your lab
report.

Title
The title states the focus of your experiment. The title should be to the point, descriptive,
accurate, and concise (ten words or less). In this course you use the given title for each activity in
the manual.
 Title page
Not all lab reports have title pages, but if your instructor wants one, it would be a single page
that states:
 The title of the experiment
 Your name and the names of any lab partners

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  Your instructor's name
 The date the lab was performed or the date the report was submitted

Introduction
It is usually several paragraphs long (but should not be too lengthy). The introduction of a lab
report states the purpose of your experiment, why the study was undertaken (objectives of the
study); give a brief summary of relevant background information; and end with a statement of
the specific problem you examined.

Materials
List everything needed to complete your experiment (Materials and chemicals).

Methods
Describe the steps you completed during your investigation. This is your procedure. Be
sufficiently detailed that anyone could read this section and duplicate your experiment. Write it
as if you were giving direction for someone else to do the lab.

Results
The results section should include all tabulated data from observations during your experiment.
This includes charts, tables, graphs, and any other illustrations of data you have collected. You
should also include a written summary of the information in your charts, tables, and/or other
illustrations. Any patterns or trends observed in your experiment or indicated in your illustrations
should be noted as well. Sometimes the Results section is combined with the Discussion (Results
& Discussion).

Discussion
This is where you interpret your data/results. It is also where you would discuss any mistakes
you might have made while conducting the investigation. In discussion you convince your reader
that you are interpreting the data fully and intelligently. What did you learn? Were there any
errors? Longer Discussion over 1-2 pages is not always better!

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Conclusion
Most of the time the conclusion is a single paragraph that sums up what happened in the
experiment, whether your hypothesis was accepted or rejected, and what this means. Sometimes
Conclusion can be merged with Discussion as ‘Discussion and Conclusion’.

Citation/References
All references used should be included at the end of your lab report. References include any
books, articles, lab manuals, etc. that you used when writing your report.

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