MICROBIOLOGY AND

PARASITOLOGY
DIPLOMA NOTES
PREPARED BY
MADAM CYNTHIA CHEPKOECH
 CHAPTER ONE
INTRODUCTION TO FOOD MICROBIOLOGY AND
PARASITOLOGY.

 Microbiology. Is the study of all living organisms that are too small to be visible
with the naked eye. This includes bacteria, archaea, viruses, fungi, prions, protozoa and
algae, collectively known as 'microbes'.
 Microorganism or microbe is an organism that is so small that it is microscopic
(invisible to the naked eye).

HISTORY OF MICROBIOLOGY
With the development of microbiology came 4 important concepts:

1) Discovery of microorganisms -

2) Pure culture concept

3) germ theory of disease
4) role in chemical transformations

I. Discovery
- 1665 Robert Hooke - observed cells in sections of cork
~ (1680s) Leeuwenhoek - “animalcules” observed in rain water, other sources
Spontaneous Generation = life from non-life, disproving this belief:
~ 1670 Redi - demonstrated larva present on meat was from eggs
~ 1765 Spallanzani and others used sealed flasks and treated air to prevent microbial growth
however, others such as Needham claimed a vitalistic force in air was necessary for
spontaneous generation to occur.
- 1860’s Louis Pasteur - “Father of Microbiology” used swan necked flasks (Fig 1-2, p2)
to disprove spontaneous generation. Also, observed that heat could kill these microbes. This led
to the development of Pasteurization. (also see Tyndall)

II. Pure Culture Concept

pure culture = 1 type of organism grown in culture, once this was accomplished,
microbes could be studied individually.

III. Germ Theory of Disease - proved role of microorganisms in causing disease.

A) - 1870’s Robert Koch - demonstrated 6 different infections in mice caused by 6 different
bacteria; demonstrated anthrax was caused by bacteria
1884 - 4 postulat
1) organism present in diseases cases, absent in healthy animals
2) can be isolated from diseased animal and grown in pure culture
3) disease can be reproduced by inoculation of healthy animal with cultured organism
4) organism can be re-isolated from experimentally infected animal and returned to culture
(pure)

B) Koch’s work and others led to the “Golden Age of Microbiology”

1) causes of many diseases were determined
2) improved culture methods
3) improved staining techniques

C) Lister in 1860s, developed aseptic surgical techniques, used carbolic acid

(phenol) to treat dressings.

IV. Cell Theory

V. Role of Microorganisms in Chemical Transformations

A) 1837, demonstrated that Yeast generated alcohol

Glucose + H2O ---> ethanol + CO2

B) 1857 - 1876, Pasteur demonstrated such fermentations were microbial processes

C) 1897 Buchner discovered enzymes

VI. Areas developed from Microbiology

Development of microbiology led to the following areas:
1) Immunology
2) Chemotherapy
3) Virology
4) Molecular Biology

A) Immunology
1) 1796 Jenner - immunity to smallpox conferred by inoculation with cowpox
2) 1881 Pasteur - used “attenuated” virus for immunization against rabies
3) 1884 Metchnikoff - observed phagocytosis = cellular theory of immunity
4) 1890 Emil von Behring - immunity in cell free portions of blood = humoral theory

B) Chemotherapy
1) 1908 Ehrlich - treatment for syphilis (salvarsan = arsenic derivative)
2) 1929 Fleming - penicillin
3) 1944 Waksman - streptomycin
4) 1950’s - many antibiotics discovered

C) Virology
1) 1892 Iwanowski - showed an agent passing through a filter caused a disease in plants
2) 1931 - cultivation of viruses in chick embryos
3) 1949 - cultivation in cell cultures

D) Molecular Biology
1) Monoclonal antibodies - 1975 Kohler and Milstein fused myeloma and antibody producing
cells = hybridoma which generates monoclonal antibodies
2) Genetic engineering - 1973 Chakrabarty transferred genes from one organism to another

Importance of Microbiology in Food Industry

Microbiology also helps to keep our food safe. Microbiology helps us to identify the
microorganisms that exist in food. With a better understanding of these microorganisms, help the
biologists to find out the ways for preventing the food from spoilage and make food safe.
Scientists use good bacteria against pathogenic bacteria to prevent food contamination.
Following are the merits of microbiology in the food and agricultural industry.

 Microbiologists specializing in food-based bacteria can work with the Food and Drug
Administration to help identify food products that can pose a risk to human health. They
also investigate the food poisoning outbreaks, aiming to identify their causes in order to
prevent recurrence.
 Microbiology also provides us with clean water. These living bacteria help to keep water
clean through sewage treatment. Bacteria break down the organic matter in sewage,
helping to clean the water before this water released back into the environment.
 Microbiologists investigate the vital role of microbes in the soil. Some concentrate on
plant pests and diseases, developing ways to control them or even use microbes to control
insect pests and weeds. Others research the microbes that cause diseases in farm animals.
Moreover, microbiology helps farmers to optimize nitrate levels and maximize the
output.
 It helps to find out the natural pesticides. Few microbes like bacteria and virus are
exploited against pest attacking farm crops. Hence they are called natural pesticides.
They are so specific to the pests or insects and do not cause any harm to the plant or
animals and humans.
 Help out in the natural manures. Few microbes like algae and bacteria are grown up to
enhance soil fertility by fixing nitrogen and also water retaining the capacity of the soil.
Thus they also maintain soil microbiology suitable for plant growth. Crop rotation is a
technique adopted by farmers to enhance soil fertility by the use of microbes in the roots
of leguminous plants.
 Agricultural microbiology helps in decompose the waste. Microbes decompose the
synthetic pesticide residues and other toxic material in agriculture soil and thereby
protecting farms from toxin accumulation.
 An example of microbiology’s usage in the food industry is nisin; it is an antibacterial
agent used in cheese, meats and beverages to extend shelf life by suppressing the growth
of harmful bacteria.

CHAPTER TWO
 ROLE AND SIGNIFICANCE OF MICROORGANISMS.
 Significance of microorganisms
a). Food production and preservation through fermentation
b). source of food
c). production of antibiotics
d). production of single cell protein
e). decomposition of matter
f). food spoilage
g). Food poisoning and food borne diseases
h). pest control and purification of air.
 Roles of microorganisms to environment and human activities.
1. Environment
a). decomposition of organic matter
b). pest control and purification of air
c). They break down dead plant and animal tissues and make their nutrients,
including carbon and nitrogen, available to support plant growth.

2. Human activities

a). Food production through fermentation.

b). Food borne diseases and food spoilage.

c). Energy production

d). Food

e). Production of antibiotics.

CHAPTER THREE
 CELL AND STRUCTURE OF MICROORGANISM.

 Prokaryotic Cells

Prokaryotes, found in both Domain Archaea and Bacteria, are unicellular organisms that lack
membrane-bound organelles and a defined nucleus.

A prokaryotic cell is a simple, single-celled (unicellular) organism that lacks a nucleus, or any
other membrane-bound organelle. Prokaryotic DNA is found in the central part of the cell: a
darkened region called the nucleoid.

All prokaryotes have plasma membranes, cytoplasm, ribosomes, a cell wall, DNA, and lack
membrane-bound organelles. Many also have polysaccharide capsules. Prokaryotic cells range in
diameter from 0.1–5.0 µm.

 Eukaryotic cells

Eukaryotic cells are defined as cells containing organized nucleus and organelles which are
enveloped by membrane-bound organelles. Examples of eukaryotic cells are plants, animals, protists,
fungi. Their genetic material is organized in chromosomes. Golgi apparatus, Mitochondria,
Ribosomes, Nucleus are parts of Eukaryotic Cells.

Eukaryotic cells contain a variety of structures called organelles, which perform various
functions within the cell. Examples of organelles are ribosomes, which make proteins,
the endoplasmic reticulum, which sorts and packages the proteins, andmitochondria, which
produce the energy molecule adenosine triphosphate (ATP). They also have a true nucleus,
which contains the genetic material DNA and is surrounded by a nuclear envelope. All of the
organelles are stabilized and given physical support through the cytoskeleton, which is also
involved in sending signals from one part of the cell to the other. In eukaryotic cells, the
cytoskeleton is composed mainly of three types of filaments: microtubules, microfilaments, and
intermediate filaments. The gel-like substance that surrounds all the organelles in the cell is
called cytosol.

 Bacteria
 Bacteria are single celled microbes. The cell structure is simpler than that of other
organisms as there is no nucleus or membrane bound organelles. Instead their control
centre containing the genetic information is contained in a single loop of DNA. Some
bacteria have an extra circle of genetic material called a plasmid. The plasmid often
contains genes that give the bacterium some advantage over other bacteria. For example
it may contain a gene that makes the bacterium resistant to a certain antibiotic.
  Bacteria are classified into 5 groups according to their basic shapes: spherical (cocci),
rod (bacilli), spiral (spirilla), comma (vibrios) or corkscrew (spirochaetes). They can
exist as single cells, in pairs, chains or clusters.
 Bacteria reproduce by binary fission. In this process the bacterium, which is a single cell,
divides into two identical daughter cells. Binary fission begins when the DNA of the
bacterium divides into two (replicates). The bacterial cell then elongates and splits into
two daughter cells each with identical DNA to the parent cell. Each daughter cell is a
clone of the parent cell.
 Fungi
 Fungi are eukaryotes and have a complex cellular organization.
 As eukaryotes, fungal cells contain a membrane-bound nucleus where the DNA is
wrapped around histone proteins.
 A few types of fungi have structures comparable to bacterial plasmids (loops of DNA).
Fungal cells also contain mitochondria and a complex system of internal membranes,
including the endoplasmic reticulum and Golgi apparatus.
 Unlike plant cells, fungal cells do not have chloroplasts or chlorophyll.
 Many fungi display bright colors arising from other cellular pigments, ranging from red
to green to black. The poisonous Amanita muscaria (fly agaric) is recognizable by its
bright red cap with white patches. Pigments in fungi are associated with the cell wall.
They play a protective role against ultraviolet radiation and can be toxic.
 The rigid layers of fungal cell walls contain complex polysaccharides called chitin and
glucans. Chitin, also found in the exoskeleton of insects, gives structural strength to the
cell walls of fungi. The wall protects the cell from desiccation and predators. Fungi have
plasma membranes similar to other eukaryotes, except that the structure is stabilized by
ergosterol: a steroid molecule that replaces the cholesterol found in animal cell
membranes. Most members of the kingdom Fungi are nonmotile.
.
 Virus

 All viruses contain the following two components: 1) a nucleic acid genome and 2)
a protein capsid that covers the genome. Together this is called thenucleocapsid. In
addition, many animal viruses contain a 3) lipid envelope. The entire intact virus is called
the virion. The structure and composition of these components can vary widely.

A: Viral Genomes: While the genomes of all known cells are comprised of double stranded
DNA, the genomes of viruses can be comprised of single or double stranded DNA or RNA. They
can vary greatly in size, from approximately 5-10 kb (Papovaviridae, Parvoviridae, etc.) to
greater than 100-200 kb (Herpesviridae, Poxviridae). The known structures of viral genomes are
summarized below.

DNA: Double Stranded - linear or circular

 Single Stranded - linear or circular

Other Structures - gapped circles

RNA: Double Stranded - linear

Single Stranded - linear : These single stranded genomes can be either + sense, - sense, or
ambisense The sense strand is the one that can serve directly as mRNA and code for
protein, so for these viruses, the viral RNA is infectious. The viral mRNA from - strand viruses
is not infectious, since it needs to be copied into the + strand before it can be translated. In an
ambisense virus, part of the genome is the sense strand, and part is the antisense.

The genome of some RNA viruses is segmented, meaning that a virus particle contains several
different molecules of RNA, like different chromosomes.

B: Protein Capsid
Viral genomes are surrounded by protein shells known as capsids. One interesting question is
how capsid proteins recognize viral, but not cellular RNA or DNA. The answer is that there is
often some type of "packaging" signal (sequence) on the viral genome that is recognized by the
capsid proteins. A capsid is almost always made up of repeating structural subunits that are
arranged in one of two symmetrical structures, a helix or an icosahedron. In the simplest case,
these "subunits" consist of a single polypeptide. In many cases, however, these structural
subunits (also called protomers) are made up of several polypeptides. Both helical and
icosahedral structures are described in more detail below.

1) Helical Capsids: The first and best studied example is the plant tobacco mosaic virus (TMV),
which contains a SS RNA genome and a protein coat made up of a single, 17.5 kd protein. This
protein is arranged in a helix around the viral RNA, with 3 nt of RNA fitting into a groove in
each subunit. Helical capsids can also be more complex, and involve more than one protein
subunit.

A helix can be defined by two parameters, its amplitude (diameter) and pitch, where pitch is
defined as the distance covered by each turn of the helix. P = m x p, where m is the number of
subunits per turn and p is the axial rise per subunit. For TMV, m = 16.3 and p= 0.14 nm, so
P=2.28 nm. This structure is very stable, and can be dissociated and re-associated readily by
changing ionic strength, pH, temperature, etc. The interactions that hold these molecules together
are non-covalent, and involve H-bonds, salt bridges, hydrophobic interactions, and vander Waals
forces.

Several families of animal virus contain helical nucleocapsids, including

the Orthomyxoviridae (influenza), the Paramyxoviridae (bovine respiratory syncytial virus), and
the Rhabdoviridae (rabies). All of these are enveloped viruses (see below).
2) Icosahedral Capsids: In these structures, the subunits are arranged in the form of a hollow,
quasi spherical structure, with the genome within. An icosahedron is defined as being made up
of 20 equilateral triangular faces arranged around the surface of a sphere. They display 2-3-5 fold
symmetry as follows:

- an axis of 2 fold rotational symmetry through the center of each edge.

- an axis of 3 fold rotational symmetry through the center of each face.

- an axis of 5 fold rotational symmetry through the center of each corner.

These corners are also called Vertices, and each icosahedron has 12.

Since proteins are not equilateral triangles, each face of an icosahedron contains more than one
protein subunit. The simplest icosahedron is made by using 3 identical subunits to form each
face, so the minimum # of subunits is 60 (20 x 3). Remember, that each of these subunits could
be a single protein or, more likely, a complex of several polypeptides.

Many viruses have too large a genome to be packaged inside an icosahedron made up of only 60
polypeptides (or even 60 subunits), so many are more complicated. In these cases, each of the 20
triangular faces is divided into smaller triangles; and each of these smaller triangles is defined by
3 subunits. However, the total number of subunits is always a multiple of 60. The total number of
subunits can be defined as 60 X N, where N is sometimes called the Triangulation Number, or T.
Values for T of 1,3,4,7,9, 12 and more are permitted.

When virus nucleocapsids are observed in the electron microscope, one often sees apparent
"lumps" or clusters on the surface of the particle. These are usually protein subunits clustered
around an axis of symmetry, and have been called "morphological units" or capsomers.

C: Viral Envelope

In some animal viruses, the nucleocapsid is surrounded by a membrane, also called

an envelope. This envelope is made up of a lipid bilayer, and is comprised ofhost-cell lipids. It
also contains virally encoded proteins, often glycoproteins which are trans-membrane proteins.
These viral proteins serve many purposes, such as binding to receptors on the host cell, playing a
role in membrane fusion and cell entry, etc. They can also form channels in the viral membrane.

Many enveloped viruses also contain matrix proteins, which are internal proteins that link the
nucleocapsid to the envelope. They are very abundant (ie, many copies per virion), and are
usually not glycosylated. Some virions also contain other, non-structural proteins that are used in
the viral life cycle. Examples of this are replicases, transcription factors, etc. These non-
structural proteins are present in low amounts in the virion.

Enveloped viruses are formed by budding through cellular membranes, usually the plasma
membrane but sometimes an internal membrane such as the ER, golgi, or nucleus. In these cases,
the assembly of viral components (genome, capsid, matrix) occurs on the inside face of the
membrane, the envelope glycoproteins cluster in that region of the membrane, and the virus buds
out. This ability to bud allows the virus to exit the host cell without lysing, or killing the host. In
contrast, non-enveloped viruses, and some enveloped viruses, kill the host cell in order to escape.

CHAPTER FOUR.
GROWTH OF MICROORGANISM.
Factors affecting growth of microorganisms

1. Solutes and Water Acidity:

Water is one of the most essential requirements for life. Thus, its availability becomes most
important factor for the growth of microorganisms. The availability of water depends on two
factors — the water content of the surrounding environment and the concentration of solutes
(salts, sugars, etc.) dissolved in the water.

Microorganisms show variability in their ability to adapt the habitats of low water activity.
Microorganisms like S. aureus can survive over a wide range of water activity and are called as
osmotolerant (as water activity is inversely related to osmotic pressure).

However, most microorganisms grow well only near pure water activity (i.e., around 0.98-1).
Thus, drying of food or addition of high concentration of salts and sugars is the most popular
way of preventing spoilage of food

2. Temperature:
All forms of life are greatly influenced by temperature. In fact, the microorganisms are very
sensitive to the temperature since their temperature varies with that of environment
(puikilothermic).

Temperature influences the rate of chemical reactions and protein structure integrity thus
affecting rates of enzymatic activity. At low temperature enzymes are not denatured, therefore,
every 10°C rise in temperature results in rise of metabolic activity and growth of
microorganisms.
Thermophiles are microorganisms that show growth optima at 55°C. They often have-growth
maxima of 65°C, while few can grow even at 100°C and higher temperatures. Their growth
minima are 45°C. The vast majority of thermophiles belong to prokaryotes although a few
microalgae (e.g., Cyanidium caldarium) and micro fungi (e.g. Mucor pusillus) are also
thermophiles.

A few microorganisms are hyperthermophiles as they possess growth optima between 80°C and
about 113°C. Hyper-thermophiles usually do not grow well below 55°C (e.g., Pyrococcus abyssi,
Pyrodictium occultum)

3. pH:
The pH is defined as a negative logarithm of hydrogen ion concentration:
The activity of microbial enzymes depends on the change present on the surface of
amino acids. Any change in the environmental pH may either enhance the enzyme
activity or inhibit the activity.

Thus, pH can dramatically affect the growth of microorganisms. Each species of microorganisms
shows specific pH growth range. Microorganisms can be classified as acidophilus, neutrophils
and basophiles (alkalophiles) on the basis of their requirement for particular pH in their
environment.

The acidophiles grow between the pH range of 0.0 to 5.5, neutrophils between 5.5 to 8 and
basophiles between 4.5 to 11.5. Most micro fungi are acidophiles as they grow in surroundings
having pH about 4 to 6. Most bacteria and protozoa are neutrophils

3. Oxygen Requirements:

Microorganisms capable of growing in the presence of atmospheric oxygen are called aerobes
whereas those that grow in the absence of atmospheric oxygen are called as anaerobes.
The micro-organisms that are completely dependent on atmospheric oxygen for growth are
called obligate aerobes whereas those that do not require oxygen for growth but grow well in its
presence are called as facultative anaerobes.

Aerotolerants (e.g. Enterococcus faecalis) ignore O2 and can grow in its presence or absence. In
contrast, obligate anaerobes (e.g., Bacteroids, Clostridium pasteurianum, Furobacterium) do not
tolerate the presence of oxygen at all and ultimately die. Few microorganisms (e.g.,
Campylobacter) require oxygen at very low level (2-10%) of concentration and are called as
microaerophiles (Fig. 19.16). The latter are damaged by the normal atmospheric level of oxygen
(20%).
5. Pressure:
Normal life of microorganisms on land or on the water surface is always subjected to a pressure
of 1 atmosphere. But, they are many microbes that survive in extremes of hydrostatic pressure in
deep sea. Others are there that not only survive rather grow more rapidly at high pressures (e.g.,
Protobacterium, Colwellia, Shewanella) and are called barophilic.

Some archaebacteria are thermobarophiles (e.g., Pyrococcus spp., Methanococcus jannaschii).

However, A barophile has been recovered from the depth about 10,500 m in sea near Philippines
and has been found unable to grow at 2°C temp, and below about 400-500 atmospheric pressure.

6. Radiation:

Some electromagnetic radiations, particularly the ionizing radiation (e.g., X-rays, gamma rays)
are very harmful to microbial growth. Low levels of these radiations may cause mutations and
may indirectly result in death whereas high levels may directly cause death of the microbes.

Ionizing radiation, however, destroys ring-structures, breaks hydrogen bonds, oxidizes double
bonds and polymerizes certain molecules. Ultraviolet (UV) radiation is lethal to all categories of
microbial life due to its short wavelength and high-energy; the most lethal UV radiation has a
wavelength of 260 nm.
Ultraviolet radiation primarily forms thymine dimers in DNA to cause damage. Two adjacent
thymines in a DNA strand join each other covalently and inhibit DNA replication and function.
Microbial photosynthetic pigments (chlorophyll, bacteriochlorophyll, cytochromes and flavins),
sometimes, absorb light energy, become excited or activated, and act as photosensitizers.

Microbial reproduction

Binary Fission
Binary fission is the simplest type of reproduction. Bacteria use this strategy to divide one cell
into two identical cells. Bacteria have a very basic structure, with a rigid cell wall surrounding a
thin plasma membrane to protect the cell. Inside, the DNA (genetic material) floats around in the
center.

1. In the first step of binary fission, the DNA duplicates.

2. Next, the cell starts to elongate and the DNA is moved to either side of the cell.
3. Once the cell is long enough, a new cell wall forms in the center and two new cells are
made.

Binary fission in bacteria

Bacteria divide incredibly quickly using this method, sometimes every twenty minutes. This
strategy allows pathogenic bacteria, likeStreptococcus pyogenes, the cause of strep throat, to
quickly colonize a human host.

2. Budding
Yeast use a more complicated method, as their cell structure is more complicated than bacteria.
Yeast is called eukaryotic cells, meaning they have a nucleus holding their DNA and lots of
little compartments inside the cell called organelles.
During budding a small outgrowth of the cell appears, like a bud from the main yeast cell. As
the bud grows bigger, DNA duplicates inside the nucleus and the nucleus buds as well, creating
new nuclei in the bud. When the process is complete, the bud detaches from the main cell and
forms a new cell.

Budding yeast

Yeast can be helpful to us, such asSaccharomyces cerevisiae, which is used to make beer and
bread. However, some yeast species like Candida albicans can cause illness, like the vaginal
yeast infection.

4. Sexual Reproduction
Although sexual reproduction might sound 'sexy', really it all comes down to combining
gametes, (sperm and eggs in humans). Even single celled microbes can do it! During sexual
reproduction each gamete has half of the original amount of DNA. When they combine, they
make a new organism with the correct total amount of DNA.
An example of this type of reproduction can be seen in the parasite Plasmodium falciparum,
which causes malaria. Malaria is transmitted through infected mosquitoes, which, after biting a
human, release the parasite into the blood stream.
An adult form of Plasmodium called the sporozoites are injected through the mosquito's salivary
glands and travel to the liver. There they create offspring called merozoites. The merozoites then
make male and female gametocytes, the sperm and eggs equivalent ofPlasmodium. When a
mosquito bites an infected person it picks up these gametocytes which then fuse during sexual
reproduction, creating new sporozoites and the life cycle begins again.

Life cycle of Plasmodium

For Plasmodium to reproduce it must have both mosquito and human hosts. The mosquitoes
provide a place for them to combine gametes and make new organisms. Humans continue the life
cycle, allowing thePlasmodium to duplicate itself and create new gametes.

Mitosis
Ciliates are single celled microorganisms with two different nuclei. They have smaller
micronuclei, which are involved in sexual reproduction, and larger macronuclei that are
responsible for daily activities such as producing energy and development. During asexual
reproduction, the micronuclei undergo mitosis, or cell division in eukaryotes.

Paramecium

Mitosis can be divided into four steps.

1. Prophase: the DNA condenses into chromosomes inside the nucleus. In most eukaryotes
the nucleus dissolves, releasing the chromosomes into the cell. But, in the ciliates, the
nucleus stays intact.
2. Metaphase: the chromosomes line up in the middle of the cell.
3. Anaphase: the chromosomes separate to either side of the nucleus.
4. Telophase: the micronucleus divides into two new nuclei.
 Mitosis in the micronuclei of ciliates

Bacteria growth curve

Bacteria require certain conditions for growth, and these conditions are not the bacterial growth
curve represents the number of live cells in a bacterial population over a period of time.

 Lag Phase: This initial phase is characterized by cellular activity but not growth. A small
group of cells are placed in a nutrient rich medium that allows them to
synthesize proteins and other molecules necessary for replication. These cells increase in
size, but no cell division occurs in the phase.
 Exponential (Log) Phase: After the lag phase, bacterial cells enter the exponential or log
phase. This is the time when the cells are dividing by binary fission and doubling in
numbers after each generation time. Metabolic activity is high as DNA, RNA, cell
wall components, and other substances necessary for growth are generated for division. It
is in this growth phase that antibiotics and disinfectants are most effective as these
substances typically target bacteria cell walls or the protein synthesis processes of DNA
transcription and RNA translation.
 Stationary Phase: Eventually, the population growth experienced in the log phase begins
to decline as the available nutrients become depleted and waste products start to
accumulate. Bacterial cell growth reaches a plateau, or stationary phase, where the
number of dividing cells equal the number of dying cells. This results in no overall
population growth. Under the less favorable conditions, competition for nutrients
increases and the cells become less metabolically active. Spore forming bacteria produce
endospores in this phase and pathogenic bacteria begin to generate substances (virulence
factors) that help them survive harsh conditions and consequently cause disease.

 Death Phase: As nutrients become less available and waste products increase, the
number of dying cells continues to rise. In the death phase, the number of living cells
decreases exponentially and population growth experiences a sharp decline. As dying
cells lyse or break open, they spill their contents into the environment making these
nutrients available to other bacteria. This helps spore producing bacteria to survive long
enough for spore production. Spores are able to survive the harsh conditions of the death
phase and become growing bacteria when placed in an environment that supports life.
 Cultivation of Microorganisms:
For cultivating microbes in laboratory, we require culture media. The various mixtures of
nutritive substances used for the laboratory cultivation of microorganisms are collectively
known as culture media. The culture media serve as soil in which bacteria are planted for the
purpose of study.

Culture Media:
Culture media must contain all the essential nutrients required by the organism for its growth
and reproduction. A suitable source of energy, building materials and growth factors must be
supplied in adequate amounts.

The different types of culture media employed fall into three groups:
1. Defined or synthetic media:
These are the media prepared from chemical compounds. They are highly purified and specific,
an investigator working in another laboratory can duplicate them.

2. Complex or non-synthetic media:

Media that are prepared from ingredients that have not been precisely defined. It contains
hydrolysed proteins and vitamin extracts. This type of medium cannot be duplicated by another
worker in another laboratory. Peptone is usually produced by boiling beef, by the hydrolysis of
its protein. Casein peptone and milk peptone are also used in complex media as the source of
amino acids and nitrogen.

3. Living cells:

These are used for the cultivation of viruses. For example, fertilized eggs incubated for 8 to 12
days are able to support the growth of many viruses.

In another classification culture media are grouped into following four types:
1. Natural media:
Includes substances occurring in nature, as 1) Milk 2) Eggs 3) Blood 4) Extract of plant and
animal tissues.

2. Derived media:
Includes known substances but the chemical composition of each is unknown. Examples are 1.
Nutrient broth (prepared by boiling beef to extract nutrients and adding an amino acid-nitrogen
source.) 2. Nutrient agar 3. Nutrient gelatin.

3. Chemically defined media:

Exact chemical composition known.

4. Special media:
Include combinations of the other three types of media.
There are four categories of media used in laboratory:
They are:
1. Enrichment, 2. Selective, 3. Differential and 4. Propagation.

1. Enrichment media:
They are prepared with ingredients that will enhance the growth of certain microbes. Enrichment
media encourage the growth of the suspected pathogen so that it will become the most pre-
dominant type of microbe in the culture. Two types of enrichment media are blood agar and
chocolate agar.

2. Selective media:
They are prepared with ingredients that inhibit the growth of unwanted microbes which might be
in the specimen. The inhibitor may be an antibiotic, salt or other chemical. Mixed culture of
microbes originally grown in enrichment media may be inoculated into selective media to isolate
the desired microbe.

3. Differential media:
They are designed to differentiate among microbes. Different bacterial species may produce
dissimilar colony colours when grown on differential agar. While in differential broth cultures,
the media change colour. Differential media are used to confirm the identity of a microbe that
has already been isolated by culturing in enrichment and selective media.

4. Propagation media:
They are used to propagate, or keep microbes growing for a long lime. Samples grown on these
media may be taken for analysis. The most common propagation media are nutrient broth and
agar.

Isolation methods are employed to isolate microbes from mixed cultures:

1. Streaking, 2. Plating,3. Dilution,4. Enriched procedure, and5. Single cell technique.

1. Streaking:
This is most widely used method of isolation. The technique consists of pouring a suitable sterile
medium into sterile petriplate and allowing the medium to solidify. By means of a sterile loope
or straight needle or a sterile bent glass-rod a small amount of growth preferably from a broth
culture or bacterial suspension is streaked back and forth across the surface of agar until about
one third of the diameter of the plate has been covered.

The needle is then flamed and streaking in done at right angles to and across the first streak. This
serves to drag bacteria out in a long line from the initial streak. When this streaking is completed
the needle is again flamed and streaking is done at right angles to the second streak and parallel
to the first.

2. Plating:

It includes diluting of a mixture of microorganisms until only a few hundred bacteria are left in
each millilitre of the suspension. A very small amount of the dilution is then placed in a sterile
petriplateby means of a sterile loop or pipette. The melted agar medium is cooled to about 45°C
and is poured into plate. The microorganism and agar are well mixed. When the agar is solidified
the individual bacterium will be held in place and will grow to a visible colony.

3. Dilution:

This method is used for the microorganisms which cannot be easily isolated by streaking or
plating method. Sometimes when several organisms are present in a mixture, with one organism
predominating, the predominating form may be isolated by this method. For example, when raw
milk is allowed to sour at room temperature it will, at the time of curding, have a mixture of
microorganisms with high percentage of Streptococcus lactis.

4. Enrichment Procedure:
This procedure involves the use of media and conditions of cultivation which favour the growth
of the desired species. For example, when a man suffers with typhoid, the intestinal discharge
posses small number of Salmonella typhosa when compared with E. coli and other forms.

5. Single Technique:
This is one of the most ideal and difficult method of securing pure culture. In this method a
suspension of the pure culture is placed on the under-side of a sterile cover-glass mounted over a
moist chamber on the stage of the microscope.

While looking through the microscope, a single cell is removed with the help of sterile
micropipette and transferred to a small drop of sterile medium on a sterile cover-glass and is
mounted on a sterile hanging drop slide, which is then incubated at suitable temperature. If the
single cell germinates in this drop, few cells are transferred into a tube containing sterile culture
medium which is placed in the incubator to obtain pure culture originated from single cell .

MICROSCOPIC COUNTING
 Hemocytometer: A device that counts microscopic particles. The hemocytometer works
by creating a volumetric grid divided into differently sized cubes for accurately counting
the number of particles in a cube and calculating the concentration of the entire sample.
 Streak plate: A petri dish with a growth medium.

Methods for Measurement of Cell Numbers

Measuring techniques involve direct counts, visually or instrumentally, and indirect viable cell
counts.

1. Direct microscopic counts are possible using special slides known as counting chambers.
Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted
(>107 cells per ml), but samples can be concentrated by centrifugation or filtration to increase
sensitivity.

Direct counting methods include microscopic counts using a hemocytometer or a counting

chamber. The hemocytometer works by creating a volumetric grid divided into differently sized
cubes for accurately counting the number of particles in a cube and calculating the concentration
of the entire sample. One can also quantify the number of cells in a culture by plating a known
volume of the cell culture on a petri dish with a growth medium, which is also known as a streak
plate. If the cells are distributed on the plate properly, it can generally be assumed that each cell
will give rise to a single colony. The colonies can then be counted and, based on the known
volume of the culture that was spread on the plate, the cell concentration can be calculated.
Bacterial colony counts made from plating dilutions of bacteria are useful to estimate the
strength of bacterial infections; for example, a urinary tract bacterial infection.

A variation of the direct microscopic count has been used to observe and measure growth of
bacteria in natural environments. In order to detect and prove that thermophilic bacteria were
growing in boiling hot springs, T.D. Brock immersed microscope slides in the springs and
withdrew them periodically for microscopic observation. The bacteria in the boiling water
attached to the glass slides naturally and grew as microcolonies on the surface.

2. Electronic counting chambers count numbers and measure size distribution of cells. For
cells the size of bacteria the suspending medium must be very clean. Such electronic devices are
more often used to count eukaryotic cells such as blood cells.

3. Indirect viable cell counts, also called plate counts, involve plating out (spreading) a sample
of a culture on a nutrient agar surface. The sample or cell suspension can be diluted in a nontoxic
diluent (e.g. water or saline) before plating. If plated on a suitable medium, each viable unit
grows and forms a colony. Each colony that can be counted is called a colony forming unit
(cfu)and the number of cfu's is related to the viable number of bacteria in the sample.

Advantages of the technique are its sensitivity (theoretically, a single cell can be detected), and it
allows for inspection and positive identification of the organism counted. Disadvantages are (1)
only living cells develop colonies that are counted; (2) clumps or chains of cells develop into a
single colony; (3) colonies develop only from those organisms for which the cultural conditions
are suitable for growth. The latter makes the technique virtually useless to characterize or count
the total number of bacteria in complex microbial ecosystems such as soil or the animal rumen
or gastrointestinal tract. Genetic probes can be used to demonstrate the diversity and relative
abundance of procaryotes in such an environment, but many species identified by genetic
techniques have so far proven unculturable.

Table 1. Some Methods used to measure bacterial growth

Method Application Comments

Enumeration of bacteria in Cannot distinguish living from
Direct microscopic count
milk or cellular vaccines nonliving cells
Enumeration of bacteria in
Viable cell count (colony Very sensitive if plating
milk, foods, soil, water,
counts) conditions are optimal
laboratory cultures, etc.
Estimations of large numbers Fast and nondestructive, but
Turbidity measurement of bacteria in clear liquid cannot detect cell densities less
media and broths than 107cells per ml
Measurement of total N or Measurement of total cell only practical application is in
protein yield from very dense cultures the research laboratory
Measurement of Biochemical Microbiological assays Requires a fixed standard to
activity e.g. O2 uptake CO2 relate chemical activity to cell
production, ATP production, etc. mass and/or cell numbers
Measurement of dry weight or probably more sensitive than
Measurement of total cell
wet weight of cells or volume of total N or total protein
yield in cultures
cells after centrifugation measurements

CHAPTER FIVE.
MICROSCOPY.

 Microscopy is the technical field of using microscopes to view objects and areas of
objects that cannot be seen with the naked eye (objects that are not within

the resolution range of the normal eye).

THE IMPORTANT MICROSCOPE PARTS.

1. Eyepiece: The lens the viewer looks through to see the specimen. The eyepiece usually
contains a 10X or 15X power lens.
2. Diopter Adjustment: Useful as a means to change focus on one eyepiece so as to correct
for any difference in vision between your two eyes.
3. Body tube (Head): The body tube connects the eyepiece to the objective lenses.
4. Arm: The arm connects the body tube to the base of the microscope.
5. Coarse adjustment: Brings the specimen into general focus.
6. Fine adjustment: Fine tunes the focus and increases the detail of the specimen.
7. Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the
nosepiece to select different objective lenses
8. Objective lenses: One of the most important parts of a compound microscope, as they
are the lenses closest to the specimen.
9. A standard microscope has three, four, or five objective lenses that range in power from
4X to 100X. When focusing the microscope, be careful that the objective lens doesn’t
touch the slide, as it could break the slide and destroy the specimen.
10. Specimen or slide: The specimen is the object being examined. Most specimens are
mounted on slides, flat rectangles of thin glass.
11. The specimen is placed on the glass and a cover slip is placed over the specimen. This
allows the slide to be easily inserted or removed from the microscope. It also allows the
specimen to be labeled, transported, and stored without damage.
12. Stage: The flat platform where the slide is placed.
13. Stage clips: Metal clips that hold the slide in place.
 14. Stage height adjustment (Stage Control): These knobs move the stage left and right or
up and down.
15. Aperture: The hole in the middle of the stage that allows light from the illuminator to
reach the specimen.
16. On/off switch: This switch on the base of the microscope turns the illuminator off and
on.
17. Illumination: The light source for a microscope. Older microscopes used mirrors to
reflect light from an external source up through the bottom of the stage; however, most
microscopes now use a low-voltage bulb.
18. Iris diaphragm: Adjusts the amount of light that reaches the specimen.

19. Condenser: Gathers and focuses light from the illuminator onto the specimen being
viewed.
20. Base: The base supports the microscope and it’s where illuminator is located.

Microscope Diagram

Types of Microscopes
Depending on the source of illumination, microscopes can be divided into two categories. They
are:

 Light Microscopes: These use light rays to illuminate objects. e.g. Dissection microscopes and
compound microscopes.
 Electron Microscopes: These illuminate objects with a beam of highly charged electrons. e.g.
Transmission electron microscope (TEM) and scanning electron microscope (SEM). These
microscopes provide better magnification than light microscopes.

CARE AND MAINTENANCE OF MICROSCOPE.

Routine care and proper maintenance of microscope will ensure good performance over the
years. In addition to this, a properly maintained and clean microscope will always be ready for
use at any time. Professional cleaning and maintenance should be considered when routine
techniques fail to produce optimal performance of the microscope.

Cleaning and maintenance supplies.

Dust cover: When not in use, a microscope should be covered to protect it from dust, hair, and
any other possible sources of dirt. It is important to note that a dust cover should never be placed
over a microscope while the illuminator is still on.

Lens tissue: Lint-free lens tissues are delicate wipes that would not scratch the surface of the
oculars or objective. Always ensure that you are using these types of tissues. Never substitute
facial tissue or paper towel, as they are too abrasive.

Lens cleaner: Lens cleaning solution assists in removing fingerprints and smudges on lenses and
objectives. Apply the lens cleaner to the lens tissue paper and clean/polish the surface.

Compressed air duster: Using compressed air to rid the microscope of dust particles is far
superior to using your own breath and blowing onto the microscope. Compressed air is clean,
and avoids possible contamination of saliva particles.

Maintenance tips

1. Whenever the microscope is not in use, turn off the illuminator. This will greatly extend
the life of the bulb, as well as keep the temperature down during extended periods of
laboratory work.
2. When cleaning the microscope, use distilled water or lens cleaner. Avoid using other
chemicals or solvents, as they may be corrosive to the rubber or lens mounts.
3. After using immersion oil, clean off any residue immediately. Avoid rotating the 40×
objective through immersion oil. If this should occur, immediately clean the 40×
objective with lens cleaner before the oil has a chance to dry.
 4. Do not be afraid to use many sheets of lens tissue when cleaning. Use a fresh piece (or a
clean area of the same piece) when moving to a different part of the microscope. This
avoids tracking dirt/oil/residue to other areas of the microscope.
5. Store the microscope safely with the stage lowered and the smallest objective in position
(4× or 10×). This placement allows for the greatest distance between the stage and the
objective. If the microscope is bumped, the likelihood of an objective becoming damaged
by the stage surface will be greatly minimized

CHAPTER SIX
LABORATORY EQUIPMENT AND
STERILIZATION.
BASIC LABORATORY EQUIPMENT USED IN MICROBIOLOGY

In the laboratory, eight main types of instruments are used:- 1. Balance 2. Centrifuge 3. Hot Air
Oven 4. Incubators 5. Water Bath 6. Microscope 7. Autoclave 8. Laminar Flow.
By advancement in technology to make the laboratory testings more accurate, fast, reliable and
cost effective, we require these instruments with its proper care and maintenance.

1. Balance:
A balance is used to find out the mass of a substance by comparing it with known masses. These
are used to weigh the chemicals accurately.
2. Centrifuge:
They are designed to accurate the sedimentation process by using centrifugal force.
Centrifuge is applying centrifugal force to separate the useful component in mixtures of liquids
and solids or liquids and liquids.
Centrifuge is mainly used to separate solids from liquids in suspension or separate two liquids
with different density and non-homogenous liquids, for example, separate cream form milk, and
also it can be used to remove liquids existed in solids, such as special speeding tubular
centrifuges can separate the mixed gas content with different density, depending different density
and particle size of solid particles in the liquid and different characteristics of the subsiding
speed centrifuge, the sedimentation centrifuge also can classified solids according to different
density and particle size.
3. Hot Air Oven:
This kind of dry heat sterilization is recommended when it is undesirable that steam make
contact with the material to be sterilized. This is true for certain glassware’s – Petri plates,
Pipettes as well as for substances like oil, powder, etc. The apparatus employed is an electric/gas
oven. Even the kitchen oven can be used. For laboratory glassware’s 1 hour exposure to a
temperature at 160°C is enough for sterilization.
4. Incubators:
Principles of Operation:
Light bulbs heat air in the bottom part of the incubator. The air passes over a container with
evaporating water, so that its humidity increases. The warm, humid air then flows upwards
(chimney effect) into the compartment. A thermostat in an exit hole compares the air temperature
with the desired temperature. If it is too high, the light bulbs will be switched off; if it is too low,
the bulbs will be switched on.
The incubator operates with a thermostat that keeps the temperature of the air at a constant,
adjustable value.
They are used for growing microorganisms on various culture media; it must be properly cleaned
before and after uses.
5. Water Bath:
It is used to carry out various chemical reactions at specific temperature. Depending upon the
requirement of an experiment. It is often essential to keep a liquid mixture at an exact
temperature without a gradient of heat (the bottom being hotter than the top) to make it “cook”
properly.
6. Microscope:
Microscope consists of two lens called eye lens and objective lens, objective lens is lens kept
behind object and eye lens is keep on the top of microscope i.e. on the place through which we
look, firstly the object is placed behind the objective of microscope which is turned into virtual,
erect and magnified image, later this image is thought to be the object for the eye lens and this
objects forms real, inverted and magnified image.
7. Autoclave:
Autoclaving is one of the techniques used in moist heat sterilization. The equipment used is
called as Autoclave.
The principle behind the working of an Autoclave is the higher the pressure created inside the
autoclave, the higher would be the attainable temperature inside it.
The boiling point of water at normal atmospheric pressure is 100°C.
When the free-flowing steam at a temperature of 100°C is subjected under pressure of 1
atmosphere above the sea-level pressure i.e. 15 pounds pressure per square inch, the temperature
inside the autoclave happens to rise up to 121°C, which is an usual and common parameters
employed in the moist heat sterilization.
Principle involved in Autoclave is moist heat sterilization. It causes coagulation of cell proteins
at a temperature lower than hot air oven. At the point of condensation steam liberates thermal
energy equal to its heat of vaporization. So autoclaving is mostly done at this temperature for a
period of 15-20 minutes under 15 lb pressure. As the temperature increases, steam gets energized
increasing its penetration capability.
8. Laminar Flow:
Laminar flow hoods are essential machines to medical research. They are an enclosed worktop
that scientists can keep entirely sterile. In the hoods, scientists can do various treatments on cells
or experimental animals, keeping both the test subjects safe from outside contaminants, and
scientists safe from possible threats inside the hood, like virus particles. However, they require
strict maintenance to ensure the region inside the hood remains sterile and safe.
9. Filters:
Laminar flow hoods are equipped with filters that trap particles that might flow into the hood and
contaminate the air in the sterile environment. These filters, ranging from basic air filters to the
more complex high efficiency particulate air, or HEPA, filters, must be changed on a regular
basis to ensure they are effective. Each type of filter has a different lifespan. For example, HEPA
filters can last from 3 to 5 years. Filters should be marked so that you know when to change
them.
Blower:
Most of the front of the flow hood is protected by a sliding glass door that lifts to allow a
scientist to reach in his or her hands. When the window is lifted up, a blower begins to push air
out, creating a barrier that stops environmental air from getting into the sterile hood.
The blower is an essential part of the flow hood’s effectiveness. Regular check-ups to ensure that
is blowing at a steady, strong rate are necessary. Most research institutions have a scheduled
cycle for flow hood blower maintenance in order to keep them in working order.
Surfaces:
Each time a researcher uses the hood, they need to make sure the surfaces within it are cleaned.
Strong acids and bases can eat away at the metal or acrylic that covers the worktop and walls,
and must be cleaned immediately. To keep the area free of bacteria or other foreign
contaminants, cleaning with a fast-evaporating alcohol, such as an ethanol mixture, is the best
way to keep the surfaces sterile.

STERILIZATION METHODS

 Sterilization can be achieved by a combination of heat, chemicals, irradiation, high

pressure and filtration like steam under pressure, dry heat, ultraviolet radiation, gas vapor
sterilants, chlorine dioxide gas etc. Effective sterilization techniques are essential for
 working in a lab and negligence of this could lead to severe consequences, it could even
cost a life.
1. Heat Method:
This is the most common method of sterilization. The heat is used to kill the microbes in the
substance. The extent of sterilization is affected by the temperature of the heat and duration of
heating. On the basis of type of heat used, heat methods are categorized into-
(i) Wet Heat/Steam Sterilization- In most labs, this is a widely used method which is done in
autoclaves.. Autoclaves use steam heated to 121–134 °C under pressure. This is a very effective
method that kills/deactivates all microbes, bacterial spores and viruses. Autoclaving kills
microbes by hydrolysis and coagulation of cellular proteins, which is efficiently achieved by
intense heat in the presence of water. The intense heat comes from the steam. Pressurized steam
has a high latent heat and at 100°C it holds 7 times more heat than water at the same temperature.

(ii) Dry heat sterilization- In this method, specimens containing bacteria are exposed to high
temperatures either by flaming, incineration or a hot air oven. Flaming is used for metallic
devices like needles, scalpels, scissors, etc. Incineration is used especially for inoculating loops
used in microbe cultures. The metallic end of the loop is heated to red hot on the flame. The hot
air oven is suitable for dry material like powders, some metal devices, glassware, etc.

2. Filtration
Is the quickest way to sterilize solutions without heating.This method involves filtering with a
pore size that is too small for microbes to pass through. Generally filters with a pore diameter of
0.2 um are used for the removal of bacteria. Membrane filters are more commonly used filters
over sintered or seitz or candle filters. It may be noted that viruses and phage are much smaller
than bacteria, so the filtration method is not applicable if these are the prime concern.
3. Radiation sterilization:
This method involves exposing the packed materials to radiation (UV, X-rays, gamma rays) for
sterilization. The main difference between different radiation types is their penetration and hence
their effectiveness. UV rays have low penetration and thus are less effective, but it is relatively
safe and can be used for small area sterilization. X-rays and gamma rays have far more
penetrating power and thus are more effective for sterilization on a large scale.
4. Chemical method of sterilization:
Heating provides a reliable way to get rid of all microbes, but it is not always appropriate as it
can damage the material to be sterilized. In that case, chemical methods for sterilization is used
which involves the use of harmful liquids and toxic gases without affecting the material?
Sterilization is effective using gases because they penetrate quickly into the material like steam.
There are a few risks, and the chances of explosion and cost factors are to be considered.
The commonly used gases for sterilization are a combination of ethylene oxide and carbon-
dioxide. Here Carbon dioxide is added to minimize the chances of an explosion. Ozone gas is
another option which oxidize most organic matter. Hydrogen peroxide, Nitrogen dioxide,
Glutaraldehyde and formaldehyde solutions, Phthalaldehyde, and Peracetic acid are other
examples of chemicals used for sterilization. Ethanol and IPA are good at killing microbial cells,
but they have no effect on spores.

CHAPTER SEVEN.
STAINING.
 Staining is a supplementary method that gives divergence to the microscopic image for
better vision under the microscope. It is a technique that is widely used for the
examination of cells, tissues and cellular components.
 STAINS/ DYES. They are colored organic compounds used for staining
microorganisms. Chemically,
Stains= Benzene ring+ Chromophore+ Auxochrome

IMPORTANCE OF STAINING

 Enables us to see the organism better: As microorganisms are a very minute creature as
well as transparent, so it makes the specimen easy to identify.
 Helps to differentiate organisms: Some microbes retain the colour of stain, and some don’t,
which helps to distinguish between two different groups of organisms.
 To identify a particular structure: For further study of microorganisms, it is also important
to study the various internal and external structure of organism like flagella, capsule, nucleus,
spores etc.
Types of dye
 1. Acidic Dyes: It is dye which has negative charge so they bind to positively charged cell
structures like some proteins. Acidic dyes are not very often used in Microbiology lab.except to
provide background staining like Capsule staining. Examples: Nigrosine, Picric acid, Eosin, Acid
fuschin, India ink etc.

2. Basic Dyes: This dye have positive charge & bind to negatively charged molecules(nucleic
acid, -COOH -OH). Since, surface of bacterial cells are negatively charged(due to Teichoic
acid), basic dyes are most commonly used in bacteriology. Examples: Crystal Violet, Methylene
Blue, Safranin , basic fuschin.

3. Neutral Dyes: They are usually formed from precipitation in which are produced when
aqueous acidic & basic stains are combined. Neutral dyes stains nucleic acids, & cytoplasm. Eg;
Eosinate of Methylene blue, Giesma stain.

STAINING TECHNIQUES

 Some staining techniques involve the application of only one dye to the sample; others
require more than one dye.
 In simple staining, a single dye is used to emphasize particular structures in the
specimen. A simple stain will generally make all of the organisms in a sample appear to
be the same color, even if the sample contains more than one type of organism.
 In contrast, differential staining distinguishes organisms based on their interactions with
multiple stains. In other words, two organisms in a differentially stained sample may
appear to be different colors. Differential staining techniques commonly used in clinical
settings include Gram staining, acid-fast staining, end spore staining, flagella staining,
and capsule staining. Table 3 provides more detail on these differential staining
techniques.
1. Gram Staining

The Gram stain procedure is a differential staining procedure that involves multiple steps. It
was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to
distinguish between bacteria with different types of cell walls, and even today it remains one of
the most frequently used staining techniques. The steps of the Gram stain procedure are listed
below and illustrated in Table 1.

1. First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the
cells a purple color.
2. Next, Gram’s iodine, a mordant, is added. A mordant is a substance used to set or
stabilize stains or dyes; in this case, Gram’s iodine acts like a trapping agent that
 complexes with the crystal violet, making the crystal violet–iodine complex clump and
stay contained in thick layers of peptidoglycan in the cell walls.
3. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Cells
that have thick peptidoglycan layers in their cell walls are much less affected by the
decolorizing agent; they generally retain the crystal violet dye and remain purple.
However, the decolorizing agent more easily washes the dye out of cells with thinner
peptidoglycan layers, making them again colorless.
4. Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized
cells pink and is less noticeable in the cells that still contain the crystal violet dye.

Gram-staining is a differential staining technique that uses a primary stain and a secondary
counterstained to distinguish between gram-positive and gram-negative bacteria.

The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red,
safranin-dyed cells are gram-negative. However, there are several important considerations in
interpreting the results of a Gram stain. First, older bacterial cells may have damage to their cell
walls that causes them to appear gram-negative even if the species is gram-positive.

2. Acid-Fast Stains

Acid-fast staining is another commonly used, differential staining technique that can be an
important diagnostic tool. An acid-fast stainis able to differentiate two types of gram-positive
cells: those that have waxy mycolic acids in their cell walls, and those that do not. Two different
methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique.

Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin
even after a decolorizing agent (an acid-alcohol solution) is applied. A secondary counterstain,
methylene blue, is then applied, which renders non–acid-fast cells blue.

The fundamental difference between the two carbolfuchsin-based methods is whether heat is
used during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the
carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. Both
techniques are important diagnostic tools because a number of specific diseases are caused
by acid-fast bacteria (AFB). If AFB are present in a tissue sample, their red or pink color can be
seen clearly against the blue background of the surrounding tissue cells.

3. Capsule Staining

Certain bacteria and yeasts have a protective outer structure called a capsule. Since the presence
of a capsule is directly related to a microbe’s virulence (its ability to cause disease), the ability to
determine whether cells in a sample have capsules is an important diagnostic tool. Capsules do
not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is
typically used for capsule staining. The dye stains the background but does not penetrate the
capsules, which appear like halos around the borders of the cell. The specimen does not need to
be heat-fixed prior to negative staining.

One common negative staining technique for identifying encapsulated yeast and bacteria is to
add a few drops of India ink or nigrosinto a specimen. Other capsular stains can also be used to
negatively stain encapsulated cells . Alternatively, positive and negative staining techniques can
be combined to visualize capsules: The positive stain colors the body of the cell, and the negative
stain colors the background but not the capsule, leaving halo around each cell.

4. Endospore Staining

Endospores are structures produced within certain bacterial cells that allow them to survive harsh
conditions. Gram staining alone cannot be used to visualize endospores, which appear clear
when Gram-stained cells are viewed. Endospore staining uses two stains to

differentiate endospores from the rest of the cell. The Schaeffer-Fulton method (the most
commonly used endospore-staining technique) uses heat to push the primary stain (malachite
green) into the endospore. Washing with water decolorizes the cell, but the endospore retains the
green stain. The cell is then counterstained pink with safranin. The resulting image reveals the
shape and location of endospores, if they are present. The green endospores will appear either
within the pink vegetative cells or as separate from the pink cells altogether. If no endospores are
present, then only the pink vegetative cells will be visible

CHAPTER EIGHT
CULTURING.
 Colony-forming unit (CFU, cfu, Cfu) is a unit used to estimate the number of
viable bacteria or fungalcells in a sample. Viable is defined as the ability to multiply
via binary fission under the controlled conditions.
 A culture media is a special medium used in microbiological laboratories to grow
different kinds of microorganisms. A growth or a culture medium is composed of
different nutrients that is essential for microbial growth.
TYPES OF CULTURE MEDIA

 A culture may be solid or liquid. The solid culture media is composed of a brown jelly
like substance known as agar. Different nutrients and chemicals are added to it to allow
the growth of different microorganisms.

Below given are some types of important culture or growth media used in microbiological
laboratories:
Agar Plate with bacterial colonies

1. The Preservation Culture Media

This is composed of all the basic nutrients required for a microbial growth and is used to
preserve a specific type of microorganism, preferably bacteria or a set of different microbial
entities for a long period of time.

The basic purpose of this culture is to let these microorganisms grow safely in an ensured
environment that has all the important nutrients and to protect them against any environmental
damage so these organisms can be used when needed.

2. The Enrichment Culture Media

This is a liquid medium which allows the microorganisms to multiply and has the essential
nutrients that are required for it.

It is usually composed of bacteria taken from a liquid source such as pond water. The basic
nutrient broth is the most commonly used.
Selective Media Plate

3. Selective Culture Media

This is a special type of media which allows the growth of certain microorganisms while inhibits
the growth of the others.

For example if we want to isolate a specific bacteria let’s say that can with stand an acidic
environment from a sample of pond water and get rid of others, a selective media with a low pH
will be taken which will allow the growth of only those organisms that can withstand acidity and
will kill the others that cannot. Examples of commonly used selective media includes: PALCAM
agar medium or Mac conkey agar medium.

4. Differential Culture Media

This is a media that is used for differentiating between bacteria by using an identification marker
for a specific type of microorganism.

The selective and differential culture media are opposites to each other in a way that one inhibits
the growth of other organisms while allowing the growth of some while the other does not kill
the others but only highlights one type.Blood agar is a common differential culture medium used
to identify bacteria that causes haemolysis in blood.

5. Resuscitation Culture Media

This is a special type of media which is used for growing microorganisms that are damaged and
have lost the ability to produce due to certain harmful environmental factors.

This culture allows the organisms to regain their metabolism by providing the nutrients that the
organisms had been deprived of. For example, a type of bacteria that requires histamine for its
growth is subjected to a medium lacking this essential component its growth will be inhibited.

If the same bacteria is then placed in a medium consisting of histamine it will start to grow again.
In this case the media containing histamine will act as resuscitation media. An example of a
commonly used resuscitation culture media is the tryptic soya agar.

Fermentation Media
 6. General Purpose Media

The general purpose media is a media that has a multiple effect, i.e. it can be used as a selective,
deferential or a resuscitation media.

7. Isolation Culture Media

An isolation culture medium is a simple agar containing solid medium that allows the growth of
microorganisms in the direction of the streaks.

For example the bacteria will only grow on the pattern made on the solidified agar during the
streak plate method. This is the most commonly used medium in microbiological labs.

8. Fermentation Media

The fermentation culture media is a liquid selective media which is used to obtain a culture of a
specific organism more likely yeast or a particular toxin.

The fermentation media can also be differential but mostly it is selective in nature that is
allowing the growth of one type while inhibiting the growth of others.

COMPOSITION OF CULTURE MEDIA

The formulation of all Oxoid culture media and the components can be divided into different
roles or functions:

1 Nutrients: proteins/peptides/amino-acids.

2 Energy: carbohydrates.

3 Essential metals and minerals: calcium, magnesium, iron, trace metals: phosphates, sulphates
etc.

4 Buffering agents: phosphates, acetates etc.

5 Indicators for pH change: phenol red, bromo-cresol purple etc.

6 Selective agents: chemicals, antimicrobial agents.

7 Gelling agent: usually agar.

There is often an overlap of functions of some media components, thus protein hydrolysates will
supply amino-nitrogen, energy, some metals/minerals and act as buffering agents. Phosphate
buffers are important suppliers of minerals and agar contributes metals.

1 Nutrients
Naegeli is credited with the earliest publications (1880/82) describing the requirements of micro-
organisms for a protein component which he called `peptone'.
The nutrient components of culture media are carefully selected to recover the required spectrum
of organisms in the sample e.g. coliforms or anaerobes. General purpose media such as blood
agar in its various forms will often contain mixtures of peptones to ensure that peptides of
sufficient variety are available for the great majority of organisms likely to be present. However,
more demanding organisms will require supplemental growth factors to be added and examples
of such requirements can be seen in media for Legionella species.

Most of the components used for the nutrition of micro-organisms are undefined and require
extensive testing with careful selection to ensure a reasonable degree of uniformity. Would it not
be better to use wholly defined peptides and amino-acids to produce a totally defined medium?
Whilst such media would improve uniformity, experience has shown that they lack good
performance as general purpose media. They would also be very expensive compared with
undefined media. The use of totally defined culture media is an understandable goal of most
microbiologists but defined media have yet to prove themselves equal in performance to
currently used complex mixtures of meat and plant protein hydrolysates.

2 Energy
The most common substance added to culture media as a source of energy to increase the rate of
growth of organisms is glucose. Other carbohydrates may be used as required.

Carbohydrates added to media at 5-10 grammes per litre are usually present as biochemical
substrates to detect the production of specific enzymes in the identification of organisms. It is
usual to add pH indicators to such formulations.

3 Essential Metals and Minerals

The inorganic essential components of culture media are many and can be divided on a semi-
quantitative basis:
Typical macro-components (gm/litre): Na, K, Cl , P, S, Ca, Mg, Fe.
Typical micro-components (mgm-microgm/litre): Zn, Mn, Br, B, Cu, Co, Mo, V, Sr, etc.
As previously mentioned, a formulation may not have specific metals and minerals listed in its
formulation. In such cases it is assumed that all the factors required are present in the
hydrolysates, buffers and agar components.

4 Buffering Agents
It is important that the pH of a culture medium is poised around the optimum necessary for
growth of the desired micro-organisms. The use of buffer compounds at specific pK values is
especially necessary when fermentable carbohydrates are added as energy sources.

Phosphates, acetates, citrates, zwitterion compounds and specific amino-acids are examples of
buffering agents that may be added to culture media.

A side effect of such compounds is their ability to chelate (or bind) divalent cations (Ca ++ and
Mg ++). Polyphosphate salts, sometimes present in sodium phosphate, are compounds which can
bind essential cations so firmly that they are made inaccessible to the micro-organisms.

The effect of these binding or chelating agents will be seen in diminished growth or failure to
grow at all, unless care has been taken to supplement the essential cations in the formulation.
Opacity forming in a medium, after heating or on standing at 50°C for several hours, is
commonly caused by phosphate interaction with metals. Such phosphate precipitates can very
effectively bind Fe and lower the available amount of this essential metal in the medium.

5 Indicator Substances
The addition of coloured indicator substances is a very effective way of detecting fermentation of
specific carbohydrates in a culture medium. Such compounds should change colour distinctly
and rapidly at critical pH values.

Most of the compounds used e.g. phenol red, bromo-cresol purple, fuchsin, etc., are toxic and it
is essential to use low concentrations of pre-screened batches/lots. Known sensitive strains of
micro-organisms are used in the screening tests.

6 Selective Agents
Chemicals or antimicrobials are added to culture media to make them selective for certain micro-
organisms. The selective agents are chosen and added at specific concentrations to suppress the
growth of unwanted organisms in a polymicrobial sample. It is, of course, essential to have
established that the selective agents, at the appropriate concentration, will allow uninhibited
growth of the desired organisms.

Common chemical selective agents are: bile salts, dye-stuffs, selenite, tetrathionate, tellurite and
azide. Antimicrobial agents are commonly used in mixtures when suppressing polymicrobial
contaminating flora. Antimicrobials are more specific in their selective action than the chemical
agents shown above. However, the critical weighing and heat-lability of most antimicrobials
demand special care and post-sterilisation addition.

7 Gelling Agents
Although gelatin is still used for a few specific media and carrageenans, alginates, silica gel and
polyacrylamides are sometimes used as gelling agents, the outstanding gel-forming substance
used in culture media is agar.

Hesse, a worker in Robert Koch's laboratory, is credited with its first use in culture media,
although Frau Hesse gave him the idea from its use in table-jellies in hot climates.

Its inertness to microbial action, the unique setting and melting temperatures (38°C and 84°C
respectively) the high gel strength which allows low concentrations of agar to be used, its clarity
and low toxicity have contributed to its wide popularity with microbiologists. Its ability to retain
its gel structure at 60°C makes agar of special value to culture media which have to be incubated
at this temperature to isolate thermophilic organisms.

Other Components
There are many other substances added to culture media for specific purposes e.g. growth factors
for fastidious organisms, eH-reducing compounds for anaerobic organisms (thioglycollate and
cysteine), whole blood to detect haemolytic enzymes and encourage the growth of organisms
which are vulnerable to oxidation products.

CULTURING METHODS.
There are several types of bacterial culture methods that are selected based on the agent being
cultured and the downstream use.

Broth culture
One method of bacterial culture is liquid culture, in which the desired bacteria are suspended in a
liquid nutrient medium, such as Luria Broth, in an upright flask. This allows a scientist to grow
up large amounts of bacteria for a variety of downstream applications.
Liquid cultures are ideal for preparation of an antimicrobial assay in which the experimenter
inoculates liquid broth with bacteria and lets it grow overnight (they may use a shaker for
uniform growth). Then they would take aliquots of the sample to test for the antimicrobial
activity of a specific drug or protein (antimicrobial peptides).
Liquid cultures of thecyanobacterium Synechococcus PCC 7002

As an alternative, the microbiologist may decide to use static liquid cultures. These cultures are
not shaken and they provide the microbes with an oxygen gradient.
Agar plates
Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of
agar-based growth medium. Once the growth medium in the petri dish is inoculated with the
desired bacteria, the plates are incubated at the optimal temperature for the growing of the
selected bacteria (for example, usually at 37 degrees Celsius, or the human body temperature, for
cultures from humans or animals, or lower for environmental cultures). After the desired level of
growth is achieved, agar plates can be stored upside down in a refrigerator for an extended
period of time to keep bacteria for future experiments.
Culture collections
Microbial culture collections focus on the acquisition, authentication, production, preservation,
catalogueing and distribution of viable cultures of standard reference microorganisms, cell lines
and other materials for research in microbial systematic.[Culture collection are also repositories
of type strains
ISOLATION MMMETHODS

1. Streak Plate Method:

This method is used most commonly to isolate pure cultures of bacteria. A small amount of
mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface
of the agar medium. The successive streaks “thin out” the inoculums sufficiently and the micro-
organisms are separated from each other.
It is usually advisable to streak out a second plate by the same loop/needle without reinoculation.
These plates are incubated to allow the growth of colonies. The key principle of this method is
that, by streaking, a dilution gradient is established across the face of the Petri plate as bacterial
cells are deposited on the agar surface.

2. Pour Plate Method:

This method involves plating of diluted samples mixed with melted agar medium (Fig. 16.14).
The main principle is to dilute the inoculum in successive tubes containing liquefied agar
medium so as to permit a thorough distribution of bacterial cells within the medium.

Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium
maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C). The
bacteria and the melted medium are mixed well.
The contents of each tube are poured into separate Petri plates, allowed to solidify, and then
incubated. When bacterial colonies develop, one finds that isolated colonies develop both within
the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated
colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure
purity.

Pour plate method has certain disadvantages as follows:

(i) The picking up of subsurface colonies needs digging them out of the agar medium thus
interfering with other colonies, and

(ii) The microbes being isolated must be able to withstand temporary exposure to the 42-45°
temperature of the liquid agar medium; therefore this technique proves unsuitable for the
isolation of psychrophilic microorganisms.

However, the pour plate method, in addition to its use in isolating pure cultures, is also used for
determining the number of viable bacterial cells present in a culture.

3. Spread Plate Method:

In this method (Fig. 16.15), the mixed culture or microorganisms is not diluted in the melted agar
medium (unlike the pour plate method); it is rather diluted in a series of tubes containing sterile
liquid, usually, water or physiological saline.

A drop of so diluted liquid from each tube is placed on the center of an agar plate and spread
evenly over the surface by means of a sterilized bent-glass-rod. The medium is now incubated.

When the colonies develop on the agar medium plates, it is found that there are some plates in
which well-isolated colonies grow. This happens as a result of separation of individual
microorganisms by spreading over the drop of diluted liquid on the medium of the plate.
The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In
contrast to pour plate method, only surface colonies develop in this method and the
microorganisms are not required to withstand the temperature of the melted agar medium.

4. Serial Dilution Method:

As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms
that have not yet been successfully cultivated on solid media and grow only in liquid media.

A microorganism that predominates in a mixed culture can be isolated in pure form by a series of
dilutions. The inoculum is subjected to serial dilution in a sterile liquid medium, and a large
number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution.

The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that
there are some tubes showing growth of only one individual microbe. For convenience, suppose
we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms (Fig.
16.16.), i.e., 100 microorganisms/ml of the liquid medium.

If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would
then have 100 microorganisms in 10 ml or 10 microorganisms/ml. If we add 1 ml of this
suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single
microorganism.

5. Single Cell Isolation Methods:

An individual cell of the required kind is picked out by this method from the mixed culture and is
permitted to grow.
6. Enrichment Culture Method:

Generally, it is used to isolate those microorganisms, which are present in relatively small
numbers or that have slow growth rates compared to the other species present in the mixed
culture.

The enrichment culture strategy provides a specially designed cultural environment by

incorporating a specific nutrient in the medium and by modifying the physical conditions of the
incubation. The medium of known composition and, specific condition of incubation favours the
growth of desired microorganisms but, is unsuitable for the growth of other types of
microorganisms.

CHAPTER NINE.
FOOD SAMPLING.
 Food sampling is a process used to check that a food is safe and that it does not contain
harmful contaminants, or that it contains only permitted additives at acceptable levels, or that it
contains the right levels of key ingredients and its label declarations are correct, or to know the levels
of nutrients present.
IMPORTANCE OF FOOD SAMPLING
 Samples are usually collected from a lot of food for random surveillance
 collection of data for a specific purpose
 monitoring/and to determine whether the food is unsatisfactory for any reason
 Food sampling allows us to gather information about the quality and safety of food
produced, handled and sold in the district.

CHAPTER TEN.
CONCEPT OF INDICATOR ORGANISMS.
 Microbiological indicator organisms can be used to monitor hygienic conditions in
food production. The presence of specific bacteria, yeasts or molds is an indicator of poor
hygiene and a potential microbiological contamination.

Total Coliforms, Fecal Coliforms, and E. Coli

The most basic test for bacterial contamination of a water supply is the test for total Coliforms
bacteria. Total Coliforms counts give a general indication of the sanitary condition of a water
supply.

A. Total Coliforms include bacteria that are found in the soil, in water that has been
influenced by surface water, and in human or animal waste.

B. Fecal Coliforms are the group of the total Coliforms that are considered to be present
specifically in the gut and feces of warm-blooded animals. Because the origins of fecal
Coliforms are more specific than the origins of the more general total Coliforms group of
bacteria, fecal Coliforms are considered a more accurate indication of animal or human
waste than the total Coliforms.

C. Escherichia coli (E. coli) is the major species in the fecal Coliforms group. Of the five
general groups of bacteria that comprise the total Coliforms, only E. coli is generally not
found growing and reproducing in the environment. Consequently, E. coli is considered
to be the species of Coliforms bacteria that is the best indicator of fecal pollution and the
possible presence of pathogens.

Indicator Tests
The following are some of the testing methods used to determine whether total coliform bacteria
are present in a sample of water:

 Membrane
 Filtration (membrane filter technique)

Requirements

 Water sample (ground or waste water)

 Membrane filter - The filter (cellulose ester membrane) used for this technique has pores
of 0.45 micrometers and measures about 47milimeters
 MI agar
 Incubator

Procedure
  Poor about 100 milliliters of the water sample through the filter
 Place the filter on the plate agar (on MI agar)
 Incubate for about 24 hours at 35 degrees (temperature)

For this technique, the filter membrane is used to filter and thus retain any coliform bacteria that
may be present in the sample.

After incubation, the bacteria (if present) will use the nutrients in the agar plate to grow.

If a blue color is observed, this indicates that the beta-glucuronidase enzyme of E. coli was
involved in breaking down Indoxyl-beta-D-glucuronide (IBDG) in MI agar and thus indicates the
presence of E. coli.

A fluorescence appearance, however, indicates that beta-galactosidase was involved in breaking

down 4-methylumbel-liferyl-β-D-galactopyranoside (MUGal) which is also present in the agar.

* For this technique, absorbent pads with lauryl tryptose broth can also be used. These are
transferred to either M-endo media or agar to grow the bacteria.

* Once bacteria are cultures, the colonies are then counted under the microscope.
Multiple Tube Fermentation Technique

(To determine the presence of rod shaped, facultative anaerobic, gram-negative coliform group
of bacteria that do not form spores)

Requirements

 Lauryl tryptose broth

 Glass tubes

Procedure

For this technique, the procedure involves three main phases that include:

Phase 1: Presumptive stage

 Add lauryl trypsone broth into several fermentation tubes

 Inoculate varying quantities of the sample (water sample) in to the tubes
 Incubate the tubes for between 24 and 48 hours at about 35 degrees Celsius (check every
24 hours for gas formation)

* Gas formation (bubbles) in the tubes is marked as a positive presumptive test

Phase 2: Confirmed state (using fermentation tubes with brilliant green lactose bile broth)
  For this step, only the samples that marked positive in the first phase (positive
presumptive test) are used
 To the fermentation tube with the bile broth, inoculate a sample of the medium from the
tubes that tested positive in the first phase (immediately after gas formation)
 Inoculate the tubes for about 48 hours at 35 degrees Celsius

* If gas is formed during this phase, it is an indication of positive confirmed test

Phase 3: Completed test

For this phase, requirements include samples with positive confirmed test, eosin methylene blue
plate, incubator, lauryl tryptose broth fermentation tube as well as nutrient agar slant.

Procedure

 Using a wire loop, scoop and streak the methylene blue plates with the sample (this
simply involved marking out lines of the sample on the methylene blue plate, violet red
agar plate or MacConkey agar using a wire loop)
 Incubate the plates for about 24 hours at 35 degrees Celsius
 Obtain a colony of the bacteria from the plate and transfer it to the fermentation tube
(lauryl tryptose broth) and nutrient agar slant
 Incubate the two (agar slants and fermentation tubes) for between 24 and 48 hours at 35
degrees Celsius to determine whether any gas is produced

 For the agar slant sample, staining is required (gram-staining) in order to view the sample
under the microscope. As for the fermentation tube, check for presence of gas

* The presence of gas in fermentation tubes indicates the presence of bacteria and this is marked
satisfactory completed test. If gram-negative, rod shaped (without spores) bacteria are identified
through microscopy, this is also indicative of the presence of the bacteria (total coliform group)

For the agar plates, the media used (culture media) inhibit the growth of gram positive bacteria
and only allow the test to determine their presence of gram-negative bacteria capable of
fermenting lactose.

Depending on the media used, the color of the agar plate will help indicate whether coliform are
present in the sample:

 MacConkey agar will turns pink and cloudy indicating the presence of coliforms that
ferment lactose
 Eosin methylene blue agar will show a metallic green sheen in the presence of coliforms
  Violet red agar will turn red or pink in color in the presence of coliform bacteria
MPN (Most Probable Number) Technique

Essentially, MPN is similar in principle to multiple tube fermentation technique. However, rather
than simply being used to determine the presence of the bacteria (particularly fecal coliform)
MPN is used to estimate bacterial concentration in water in order to determine whether the water
is safe for use in homes.

As with multiple tube fermentation technique, the technique involves three main steps
(presumptive test, confirmatory test and completed test). The sample is diluted in different tubes
of different sample concentration and inoculated in lactose broth. This technique makes it
possible to determine the amount of bacteria in different dilutions of the sample after they are
cultured.

The presence of the bacteria in the sample is indicated by the production of either gas or acid
(change in the color of the media or presence of bubbles). For instance, all the tubes with the
highest concentration of the sample may test positive for the bacteria while a few of the less
concentrated tubes (less sample concentration) may prove positive following the test.
MPN Index

The MPN index is used to show the number of bacteria in the water and thus help determine
whether the water is safe to drink.

This involves the following steps:

For instance, if there were three sets of broth tubes each with 5 tubes. Each set would be of
different concentration. The first set (with 5 tubes) may be the original, undiluted sample, the
second set (with 5 tubes) may be 10 to the negative 1 dilution (half the concentration of the
original) while the third set (also with 5 tubes) may be 10 to the negative 2 dilutions (half the
concentration of the second set).

Assuming that all of the tubes in set 1 are positive of the bacteria, 3 in the second set are positive
and only 1 in the third set is positive, then these results can be compared to the MPN table to
determine the MPN index (estimated number of coliforms in 100mL of water) and therefore
determine whether the water is safe for use.

* MPN index below 2 is considered safe for drinking.

MICROSCOPY.

Microscopy can be used to view E.coli coliform in wastewater, ground water or urine. Here,
microscopy can be used to view bacteria colonies or count individual bacterial cells.

Observing bacterial cells on a membrane

Requirements

 Membrane filter (with 0.45 microns pores)

 A pair of forceps
 A low power microscope
 Sample (water, urine, wastewater)
 Collecting apparatus
 A funnel
 Prepared agar plate

* Before any step is taken, it is important to ensure that all apparatus used are sterile. This helps
prevent contamination that can result in false results.

Procedure

 Place the funnel on the collecting apparatus (the collecting apparatus may be connected
to a vacuum system)
 Place the filter below the funnel so that it is between the funnel and the collecting
apparatus
 Pour the sample slowly into the funnel and apply a vacuum in the collecting apparatus
until the funnel is empty
 If some sample is still in the funnel, pour some sterile buffered dilution water (20-30ml)
to rinse it
 Apply the vacuum to empty the funnel

Using a pair of forceps, carefully remove the membrane filter and place it on the agar plate in a
Petri dish (MacConkey agar, Eosin methylene blue agar and Violet red agar)

 Cover the plate with a lid and invert the plate

 Incubate the plate for about 2 hours at about 35 decrees Celsius
 Using a low power microscope, observe the coliform colonies on the plate (using 10 to
15x magnification)
 Count the number of individual colonies on the filter

* To determine the number of colonies in 100ml of the sample, divide the number of colonies
counted with the milliliters of the sample used in the procedure and multiply the results with a
100. This will give the percentage of the coliform in the water/urine or wastewater.
Fluorescent in Situ Hybridization Technique (FISH)

FISH (fluorescent in situ hybridization) refers to a technique that uses fluorescent probes for the
purposes of detecting and comparing DNA sequences.
When it comes to detecting E.coli bacteria, this technique has the advantage of saving time
compared to the other techniques commonly used for detecting the presence of coliform.

Requirements

 0.40-um
 black polyester membrane filter
 A pad (soaked in 80 percent ethanol)
 Petri dish
 Hybridization buffer (50ul) with respective probes
 Hybridization chamber
 550 ul of washing
 buffer

Procedure

 Filter the sample using the 0.40 um membrane filter

 Transfer the filter the ethanol soaked pad and allow to stand for about 3minutes at room
temperature in a Petri dish
 Dry the filter membrane and filter at room temperature for about 3 minutes
 Place the membrane filter on the hybridization buffer (50ul) in the hybridization chamber
and incubate for about 90 minutes at 46 degree
 Place the membrane on the washing buffer soaked buffer (550ul)

* for this technique, epifluorescence microscopy is used to view the sample (with WIBA filter
block)
Observation
Depending on the type of coliform present in the sample, microscopy will show weak or high
fluorescence intensity from the fluorescent probes attached to the bacteria. This makes it possible
to count the number of individual bacteria in the specimen.

CHAPTER ELEVEN
CONTROL OF MICROORGANISM IN FOOD.
Non Sterilizing Methods to Control Microbial Growth

Many physical and chemical technologies are employed by our civilization to control the growth
of (certain) microbes, although sterility may not the desired end-point. Rather, preventing
spoilage of food or curing infectious disease might be the desired outcome.

Applications of Heat
The lethal temperature varies in microorganisms. The time required to kill depends on the
number of organisms, species, nature of the product being heated, pH, and temperature.
Autoclaving, which kills all microorganisms with heat, is commonly employed in canning,
bottling, and other sterile packaging procedures. This is an ultimate form of preservation against
microbes. But, there are some other uses of heat to control growth of microbes although it may
not kill all organisms present.

Boiling: 100o for 30 minutes (more time at high altitude). Kills everything except some
endospores. It also inactivates viruses. For the purposes of purifying drinking water, 100o for
five minutes is a "standard" in the mountains" though there have been some reports that Giardia
cysts can survive this process. Longer boiling might be recommended for Mississippi River
water the closer to the Gulf.

Pasteurization is the use of mild heat to reduce the number of microorganisms in a product or
food. In the case of pasteurization of milk, the time and temperature depend on killing potential
pathogens that are transmitted in milk, i.e., staphylococci, streptococci, Brucella
abortus and Mycobacterium tuberculosis. But pasteurization kills many spoilage organisms, as
well, and therefore increases the shelf life of milk especially at refrigeration temperatures (2°C).

Milk is usually pasteurized by heating, typically at 63°C for 30 minutes (batch method) or at
71°C for 15 seconds (flash method), to kill bacteria and extend the milk's usable life. The process
kills pathogens but leaves relatively benign microorganisms that can sour improperly stored
milk.

During the process of ultrapasteurization, also known as ultra high-temperature (UHT)

pasteurization, milk is heated to temperatures of 140 °C. In the direct method, the milk is brought
into contact with steam at 140°C for one or two seconds. A thin film of milk falls through a
chamber of high-pressure steam, heating the milk instantaneously. The milk is flash cooled by
application of a slight vacuum, which serves the dual purpose of removing excess water in the
milk from condensing steam. In the indirect method of ultrapasteurization, milk is heated in a
plate heat exchanger. It takes several seconds for the temperature of the milk to reach 140°C, and
it is during this time that the milk is scalded, invariably leading to a burned taste. If
ultrapasteurization is coupled with aseptic packaging, the result is a long shelf life and a product
that does not need refrigeration.

Low temperature (refrigeration and freezing): Most organisms grow very little or not at all at
0oC. Perishable foods are stored at low temperatues to slow rate of growth and consequent
spoilage (e.g. milk). Low temperatures are not bactericidal. Psychrotrophs, rather than true
psychrophiles, are the usual cause of food spoilage in refrigerated foods. Although a few
microbes will grow in supercooled solutions as low as minus 20oC, most foods are preserved
against microbial growth in the household freezer.

Drying (removal of H2O): Most microorganisms cannot grow at reduced water activity (Aw <
0.90). Drying is often used to preserve foods (e.g. fruits, grains, etc.). Methods involve removal
of water from product by heat, evaporation, freeze-drying, and addition of salt or sugar.
Irradiation (UV, x-ray, gamma radiation): destroys microorganisms as described under
"sterilization". Many spoilage organisms are readily killed by irradiation.

Control of microbial growth by chemical agents

Antimicrobial agents are chemicals that kill or inhibit the growth microorganisms.
Antimicrobial agents include chemical preservatives and antiseptics, as well as drugs used in the
treatment of infectious diseases of plants and animals. Antimicrobial agents may be of natural or
synthetic origin, and they may have a static or cidal effect on microorganisms.

Types of antimicrobial agents

Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous
membrane; should not be taken internally. Examples include alcohols, mercurials, silver nitrate,
iodine solution, alcohols, detergents.

Disinfectants: agents that kill microorganisms, but not necessarily their spores, but are not safe
for application to living tissues; they are used on inanimate objects such as tables, floors,
utensils, etc. Examples include hypochlorites, chlorine compounds, lye, copper sulfate,
quaternary ammonium compounds, formaldehyde and phenolic compounds.

Common antiseptics and disinfectants and their uses are summarized in Table 2. Note:
disinfectants and antiseptics are distinguished on the basis of whether they are safe for
application to mucous membranes. Often, safety depends on the concentration of the compound.

Preservatives: static agents used to inhibit the growth of microorganisms, most often in foods. If
eaten they should be nontoxic. Examples are calcium propionate, sodium benzoate,
formaldehyde, nitrate and sulfur dioxide. Table 3a and 3b are lists of common preservative and
their uses.

Salt - retards bacterial growth. Not good for blood pressure.

Nitrates - can be found in some cheeses, adds flavor, maintains pink color in cured meats and
prevents botulism in canned foods. Can cause adverse reactions in children, and potentially
carcinogenic.

Sulfur Dioxide and Sulfites - are used as preservatives and to prevent browning in alcoholic
beverages, fruit juices, soft drinks, dried fruits and vegetables. Sulfites prevent yeast growth and
also retard bacterial growth in wine. Sulfites may cause asthma and hyperactivity. They also
destroy vitamins.

Benzoic Acid and Sodium Benzoate - are used to preserve oyster sauce, fish sauce, ketchup, non-
alcoholic beverages, fruit juices, margarine, salads, confections, baked goods, cheeses, jams and
pickled products. They have also been found to cause hyperactivity.
Propionic Acid and Propionates - used in bread, chocolate products, and cheese for lasting
freshness.

Sorbic Acid and Sorbates - prevent mold formation in cheese and flour confectioneries

Table 3b. Common food preservatives and their uses

Effective
Preservative Uses
Concentration
Propionic acid and Antifungal agent in breads, cake,
0.32%
propionates Swiss cheeses
Sorbic acid and Antifungal agent in cheeses,
0.2%
sorbates jellies, syrups, cakes
Benzoic acid and Antifungal agent in margarine,
0.1%
benzoates cider, relishes, soft drinks
Sodium diacetate 0.32% Antifungal agent in breads
Antimicrobial agent in cheeses,
Lactic acid unknown buttermilk, yogurt and pickled
foods
Sulfur dioxide, Antimicrobial agent in dried
200-300 ppm
sulfites fruits, grapes, molasses
Antibacterial agent in cured
Sodium nitrite 200 ppm
meats, fish
Prevents microbial spoilage of
Sodium chloride unknown
meats, fish, etc.
Prevents microbial spoilage of
Sugar unknown preserves, jams, syrups, jellies,
etc.
Prevents microbial spoilage of
Wood smoke unknown
meats, fish, etc.

CHAPTER TWELVE
FOOD SPOILAGE.
 Food spoilage is the process where a food product becomes unsuitable to ingest by the
consumer. The cause of such a process is due to many outside factors as a side-effect of
the type of product it is, as well as how the product is packaged and stored.
 Food spoilage is the process of change in the physical and chemical properties of the
food so that it becomes unfit for consumption. Food spoilage is any undesirable change in
food.

SPOILAGE MICROORGANISMS IN FOODS.

  microorganisms that break down fats grow in sweet butter (unsalted butter) and cause a
type of spoilage called rancidity. Certain types of fungi and bacteria fall into this
category. Species of the Gram-negative bacterial rod Pseudomonas are major causes of
rancidity. The microorganisms break down the fats in butter to produce glycerol and
acids, both of which are responsible for the smell and taste of rancid butter.
 Another example occurs in meat, which is primarily protein. Bacteria able to digest
protein (proteolytic bacteria) break down the protein in meat and release odoriferous
products such as putrescine and cadaverine. Chemical products such as these result from
the incomplete utilization of the amino acids in the protein.
 Food spoilage can also result in a sour taste. If milk is kept too long, for example, it will
sour. In this case, bacteria that have survived pasteurization grow in the milk and produce
acid from the carbohydrate lactose in it. The spoilage will occur more rapidly if the milk
is held at room temperature than if refrigerated. The sour taste is due to the presence of
lactic acid, acetic acid, butyric acid, and other food acids.
CHAPTER THIRDTEEN.
FOOD INFECTIONS.

 A food borne infection is an inflammation of the stomach and bowels. The infection can
happen when you eat or drink something that is contaminated by a bacteria, virus or
parasite.
 Toxin is a poisonous substance produced within living cells or organisms; synthetic
toxicants created by artificial processes are thus excluded.
 Exotoxins are toxins, often proteins in nature, secreted from a living bacterium but also
released upon bacterial lysis.
 Endotoxin release when a bacterium is lysed or dead. After lysis of bacteria the cell wall
of gram negative (lps) bacteria works as a endotoxin. Endotoxin increases the
temperature of human body so it is fever producing toxin. so endotoxin testing is
necessary in all IM & IV fluid

An enterotoxin is a protein exotoxin released by a microorganism that targets the intestines.

SPECIFIC FOOD INFECTIONS

 Salmonellosis is a symptomatic infection caused by bacteria of the salmonella type.

[1]
The most common symptoms are diarrhea, fever, abdominal, and vomiting.
[1]
Symptoms typically occur between 12 hours and 36 hours after exposure, and last
from two to seven days.Occasionally more significant disease can result in
dehydration.
 Typhoid and paratyphoid fever are diseases of the intestinal tract caused by the
Salmonella Typhi and Salmonella Paratyphi bacteria. Typhoid and paratyphoid fever
are acquired through consumption of water or food contaminated by feces of an
acutely infected or convalescent person or a chronic, asymptomatic carrier. The
 symptoms of typhoid fever may be mild or severe and may include prolonged fever,
severe headache, malaise, constipation or diarrhoea, rose-coloured spots on the trunk
and an enlarged spleen.
 Cholera is an infectious disease that causes severe watery diarrhea, which can lead to
dehydration and even death if untreated. It is caused by eating food or drinking water
contaminated with a bacterium called Vibrio cholerae.
 Vibriosis infection is the illness produced by Vibrio bacteria is known as vibriosis. In
most cases, the illness results from eating contaminated food, such as raw or
undercooked shellfish from water that contains the bacteria. Exposing a wound to
contaminated water can cause a Vibrio infection of the skin.
 Shigellosis is an infectious disease caused by a group of bacteria called Shigella(shih-
GEHL-uh). Most who are infected with Shigella develop diarrhea, fever, and stomach
cramps starting a day or two after they are exposed to the bacteria. Shigellosis usually
resolves in 5 to 7 days. Some people who are infected may have no symptoms at all,
but may still pass the Shigella bacteria to others. The spread of Shigella can be
stopped by frequent and careful handwashing with soap and taking other hygiene
measures.
 Streptococcal infections are any type of infection caused by the group of bacteria
Streptococcus.
 Listeriosis is a food-borne infection caused by Listeria bacteria. Most commonly
contracted by eating improperly processed deli meats and unpasteurized milk
products. A person with listeriosis usually has fever and muscle aches, sometimes
preceded by diarrhea or other gastrointestinal symptoms. Almost everyone who is
diagnosed with listeriosis has “invasive” infection, in which the bacteria spread
beyond the gastrointestinal tract
 Campylobacter infection (campylobacteriosis) is a bacterial infection which most
commonly causes gastroenteritis (also known as 'gastro') but may also cause illness
affecting the entire body. Commonly found in raw or undercooked poultry meat.
 Enteric (typhoid) fever is a systemic disease characterized by fever and abdominal
pain caused by dissemination of Salmonella Typhi or Salmonella Paratyphi type A, B,
or C. Gastrointestinal (GI) symptoms include anorexia (55%), abdominal pain (30-
40%), nausea (18-24%), vomiting (18%), and diarrhea (22-28%) more commonly
than constipation (13-16%)
 Gastroenteritis, also known as infectious diarrhea, is inflammationof
the gastrointestinal tract—the stomach and small intestine.[8]Symptoms may
include diarrhea, vomiting and abdominal pain. Fever, lack of energy
and dehydration may also occur
 Taeniasis is a parasitic disease due to infection with tapeworms belonging to the
genus Taenia. The two most important human pathogens in the genus are Taenia
solium (the pork tapeworm) and Taenia saginata (the beef tapeworm). The third
species Taenia asiatica is found only in East Asia.
 Symptoms: Constipation; Weight loss; abdominal pain. nausea, diarrhoea or constipation
might arise and last until the tapeworm dies following treatment
 CHAPTER FOURTEEN
FOOD INTOXICATION.
 Foodborne intoxication is an illness caused by ingesting food containing toxins formed
by bacteria which resulted from the bacterial growth in the food item. The live
microorganism does not have to be consumed.

SPECIFIC FOOD INTOXICATION.

 Botulism is a serious illness caused by the botulinum toxin. The toxin causes paralysis.
Paralysis starts in the face and spreads to the limbs. If it reaches the breathing muscles,
respiratory failure can result
The toxin is produced by Clostridium botulinum (C. botulinum), a type of bacterium.

Signs and symptoms include nausea, vomiting, and diarrhea followed by constipation and
abdominal distention. There may be weakness and difficulty breathing. Symptoms
normally appear between 18 and 36 hours after consuming the

 Foodborne botulism. The harmful bacteria thrive and produce the toxin in
environments with little oxygen, such as in home-canned food.

 Wound botulism. If these bacteria get into a cut, they can cause a dangerous infection that
produces the toxin.

 Infant botulism. This most common form of botulism begins after Clostridium botulinum
bacterial spores grow in a baby's intestinal tract. It typically occurs in babies between the
ages of 2 months and 8 months.

 Staphylococcal food poisoning is one of the most frequent forms of food poisoning.
Staphylococcus aureus is the main enterotoxigenic microorganism of the Staphylococcus
genus, which produces the toxins responsible for the development of this form of food
poisoning
 Aflatoxins are produced by fungi of the Aspergillus genus (mainly A. flavus and
parasiticus). There are 5 types of aflatoxins. Aflatoxin B1 is the most potent hepatic
carcinogen known, particularly in patients with chronic viral hepatitis B. Fungi affect
plant cultures in tropical and warm regions (especially peanuts and corn), and aflatoxins
are produced in dangerous amounts under inadequate drying or storage conditions
 Staphylococcal intoxication, emetic poisoning of Bacillus cereus, botulism, toxigenic
molds, poisonous mushrooms, and biogenic amines.
CHAPTER FIFTEEN
 FOOD BORNE INTOXICATION BY MOULDS
o Aflatoxins are poisonous carcinogens that are produced by certain molds
(Aspergillus flavus and Aspergillus parasiticus) which grow in soil, decaying
vegetation, hay, and grains.
o Aflatoxicoses is a disease caused by aflatoxin consumption and acute
aflatoxicosis resulted in death.
CHAPTER SIXTEEN
ZOONOTIC DISEASE
 Zoonotic Diseases (also known as zoonoses) are caused by infections that spread between animals and
people.
HOW DO GERMS SPREAD BETWEEN ANIMALS AND PEOPLE?

 Direct contact: Coming into contact with the saliva, blood, urine, mucous, feces, or other body fluids of an
infected animal. Examples include petting or touching animals, and bites or scratches.
 Indirect contact: Coming into contact with areas where animals live and roam, or objects or surfaces that
have been contaminated with germs. Examples include aquarium tank water, pet habitats, chicken coops,
plants, and soil, as well as pet food and water dishes.
 Vector-borne: Being bitten by a tick, or an insect like a mosquito or a flea.
 Foodborne: Each year, 1 in 6 Americans get sick from eating contaminated food. Eating or drinking
something unsafe (such as unpasteurized milk, undercooked meat or eggs, or raw fruits and vegetables that
are contaminated with feces from an infected animal).

TYPES

Common zoonotic illnesses include:

Rabies

Rabies is a disease that affects the nervous system of mammals. It is usually caused by a virus
and is transmitted if an infected animal bites a person or other animal.

Rabies is almost always fatal once symptoms appear. However, rabies vaccines exist and are
commonly available.

Lyme disease and Rocky Mountain spotted fever

Lyme disease is transmitted through tick bites. Symptoms can range from mild to severe, but it
can be treated using antibiotics.

Dengue, malaria, and chikungunya

These are mosquito-borne diseases and are more common in certain areas, such as the Caribbean.
Symptoms include fever, vomiting, and headaches. It is vital to treat these conditions as soon as
possible, as they can be fatal.

Salmonella infection

Salmonella is often caused by handling reptiles or amphibians that carry Salmonella, or by

handling baby chicks or ducks.

The illness usually lasts for between 4 and 7 days, and symptoms include diarrhea, fever, and
abdominal cramps. People can usually recover without medical treatment, although conservative
measures are recommended.

E. coli infection

This infection is often caused by touching infected animals or handling contaminated food. Cows
also have E. coli germs on their udders.

Often associated with food poisoning, salmonella can cause vomiting, abdominal cramps, and
diarrhea. It is essential that infected people rest and drink plenty of fluids.

Psittacosis

Also known as ornithosis or parrot fever, psittacosis is a bacterial disease that most often affects
birds. Humans can get it from feathers, secretions, and droppings.

Symptoms include fever, headache, and dry cough. In serious cases, it may cause pneumonia and
require a hospital visit.

Other types

There are hundreds of Zoonotic diseases, but many are rare. Other well-known types include:

 anthrax
 avian influenza or bird flu
 bovine tuberculosis
 brucellosis
 cat scratch fever
 Ebola
 West Nile virus
 leprosy
 Zika fever
 trichinosis
 swine influenza
 histoplasmosis

What can you do to protect yourself and your family from Zoonotic diseases?
  Keep hands clean. Washing your hands right after being around animals, even if you didn’t touch any
animals, is one of the most important steps you can take to avoid getting sick and spreading germs to
others.
o Always wash your hands after being around animals, even if you didn’t touch the animals.
o Many germs are spread by not washing hands with soap and clean, running water.
o If clean, running water is not accessible, use soap and available water.
o If soap and water are unavailable, use an alcohol-based hand sanitizer that contains at least 60%
alcohol to clean hands. Because hand sanitizers do not eliminate all types of germs, it is important
to wash your hands as soon as soap and water are available.
 Know the simple things you can do to stay safe around your pets.
 Prevent bites from mosquitoes, ticks, and fleas.
 Learn more about ways to handle food safely—whether it’s for yourself or your family, your pet, or other
animals.
 Be aware of Zoonotic diseases both at home, away from home (such as at petting zoos or other animal
exhibits), in child care settings or schools and when you travel.
 Avoid bites and scratches from animals.

CHAPTER SEVENTEEN
PARASITES
A parasite is an organism that lives within or on a host. The host is another
organism.
Endoparasite

These live inside the host. They include heartworm, tapeworm, and flatworms. An intercellular
parasite lives in the spaces within the host's body, within the host's cells. They include bacteria
and viruses.

Endoparasites rely on a third organism, known as the vector, or carrier. The vector transmits the
endoparasite to the host. The mosquito is a vector for many parasites, including the protozoan
known as Plasmodium, which causes malaria.

Epiparasite

These feed on other parasites in a relationship known as hyperparasitism. A flea lives on a dog,
but the flea may have a protozoan in its digestive tract. The protozoan is the hyperparasite.

Types

There are three main types of parasites.

Protozoa: Examples include the single-celled organism known as Plasmodium. A protozoa can
only multiply, or divide, within the host.
Helminths: These are worm parasites. Schistosomiasis is caused by a helminth. Other examples
include roundworm, pinworm, trichina spiralis, tapeworm, and fluke.

Ectoparasites: These live on, rather than in their hosts. They include lice and fleas.

Symptoms

There are many types of parasite, and symptoms can vary widely. Sometimes these may
resemble the symptoms of other conditions, such as a hormone deficiency, pneumonia, or food
poisoning.

Some parasite-related problems, such as giardiasis and amebic dysentery, can cause abdominal pain.

Symptoms that might occur include:

 skin bumps or rashes

 weight loss, increased appetite, or both
 abdominal pain, diarrhea, and vomiting
 sleeping problems
 anemia
 aches and pains
 allergies
 weakness and general feeling unwell
 fever

However, parasites can pass on a wide variety of conditions, so symptoms are hard to predict.

Often there are no symptoms, or symptoms appear long after infection, but the parasite can still
be transmitted to another person, who may develop symptoms.

Human parasites

Many types of parasites can affect humans. Here are some examples of parasites and the diseases
they can cause.

Acanthamoebiasis

This tiny ameba can affect the eye, the skin, and the brain. It exists all over the world in water
and soil. Individuals can become infected if they clean contact lenses with tap water.

Babesiosis

This disease that comes from parasites that are spread by ticks. It affects the red blood cells. The
risk is highest in summer in the Northeast and upper Midwest of the United States.

Balantidiasis
This is passed on by Balatidium coli , a single-cell parasite that usually infects pigs but can, in
rare cases, cause intestinal infection in humans. It can be spread through direct contact with pigs
or by drinking contaminated water, usually in tropical regions.

Blastocystosis

This affects the intestines. The blastocystis enters humans through the fecal-oral route. A person
can get it by eating food or drink contaminated with human or animal feces where the parasite is
present.

Coccidiosis

This affects the intestines. Coccidia is passed on through the fecal-oral route. It is found around
the world. It can also affect dogs and cats, but these are different kinds. Dogs, cats, and humans
cannot normally infect each other.

Amoebiasis

This is caused by the parasite Entamoeba histolytica . It affects the intestines. It is more likely
in tropical regions and in areas with high population density and poor sanitation. It is transmitted
through the fecal-oral route.

Giardiasis

Giardia, or "beaver fever" affects the lumen of the small intestine. If humans ingest food or water
contaminated with feces, dormant cysts may infect the body.

Isosporiasis or cystosporiasis

This disease is caused by the Cystoisospora belli , previously known as Isospora belli . It
affects the epithelial cells of the small intestine. It exists worldwide and is both treatable and
preventable. It is passed on through the fecal-oral route.

Leishmaniasis

This is a disease that is passed on by parasites of the Leishmania family. It can affect the skin,
the viscera, or the mucous membranes of the nose, mouth, and throat. It can be fatal. The parasite
is transmitted by types of sandflies.

Primary amoebic meningoencephalitis (PAM)

This is passed on through a free-living ameba known as Naegleria fowleri . It affects the brain
and the nervous system, and it is nearly always fatal within 1 to 18 days. It is transmitted through
breathing in contaminated soil, swimming pools, and contaminated water, but not from drinking
water.
 Malaria

Different types of plasmodium affect the red blood cells. It exists in tropical regions and is
transmitted by the Anopheles mosquito.

Rhinosporidiosis

This is caused by Rhinosporidium seeberi . It mainly affects the mucous of the nose,
conjunctiva, and urethra. It is more common in India and Sri Lanka but can occur elsewhere.
Polyps result in nasal masses that need to be removed through surgery. Bathing in common
ponds can expose the nasal mucous to the parasite.

Toxoplasmosis

This is a parasitic pneumonia caused by the parasite Toxoplasma gondii . It affects the liver,
heart, eyes and brain. It occurs worldwide. People can become infected after ingesting raw or
undercooked pork, lamb, goat, or milk, or though contact with food or soil that is contaminated
with cat feces.

A person with a healthy immune system will not usually have symptoms, but it can pose a risk
during pregnancy and for those with a weakened immune system.

Trichomoniasis

Also known as "trich" this is a sexually transmitted infection (STI) caused by the parasite
Trichomonas vaginalis . It affects the female urogenital tract. It can exist in males, but usually
without symptoms.

Trypanomiasis (Sleeping sickness)

This is passed on when the tetse fly transmits a parasite of the Trypanosoma family. It affects the
central nervous system, blood, and lymph. It leads to changes in sleep behavior, among other
symptoms, and it is considered fatal without treatment. It can cross the placenta and infect a fetus
during pregnancy.

Chagas disease

This affects the blood, muscle, nerves, heart, esophagus and colon. It is transmitted through an
insect bite. Over 300,000 people in the U.S. have the parasite that can lead to this disease.

Worms

Worms, or helminth organisms, can affect humans and animals.

Anisakiasis: This is caused by worms that can invade the intestines or the stomach wall. The
worms are passed on through contaminated fresh or undercooked fish and squid.

Roundworms can be passed on by raccoons.

Roundworm: Ascariasis, or a roundworm infection, does not usually cause symptoms, but the
worm may be visible in feces. It enters the body through consuming contaminated food or drink.

Raccoon roundworm: Baylisascaris is passed on through raccoon stools. It can affect the brain,
lungs, liver, and intestines. It occurs in North America. People are advised not to keep raccoons
as pets for this reason.

Clonorchiasis: Also known as Chinese liver fluke disease, this affects the gall bladder. Humans
can become infected after ingesting raw or poorly processed or preserved freshwater fish.

Dioctophyme renalis infection: The giant kidney worm can move through the wall of the
stomach to the liver and eventually the kidney. Humans can become infected after eating the
eggs of the parasite in raw or undercooked freshwater fish.

Diphyllobothriasis tapeworm: This affects the intestines and blood. Humans can become
infected after eating raw fish that live wholly or partly in fresh water. Prevalence has increased in
some parts of the developed world, possibly due to the growing popularity of sushi, salted fillets,
ceviche, and other raw-fish dishes.

Guinea worm: This affects subcutaneous tissues and muscle and causes blisters and ulcers. The
worm may be visible in the blister. As the worms are shed or removed, they enter the soil or
water, and are passed on from there.

Hookworms can cause intestinal disease.

Hookworm: These can cause intestinal disease. They lay their eggs in soil and the larvae can
penetrate the skin of humans. Early symptoms include itching and a rash. They are most
common in damp places with poor sanitation.

Hymenolepiasis: Humans can become infected by ingesting material contaminated by rodents,

cockroaches, mealworms, and flour beetles.

Echinococcosis tapeworm: Cystic echinococcosis can lead to cysts in the liver and lungs, and
alveolar echinococcosis can cause a tumor in the liver. Humans can be infected after eating foods
contaminated by the feces of an infected animal, or from direct contact with an animal.
Enterobiasis pinworm: A pinworm, or threadworm, Enterobius vermicularis can live in the
colon and rectum of humans. The worm lays eggs around the anus while a person sleeps, leading
to itching. It spreads through the oral-fecal route.

Fasciolosis liver fluke: This affects the gall bladder and liver. It is common in countries where
cattle or sheep are reared, but rare in the U.S. It can affect the liver and the bile ducts and it
causes gastrointestinal symptoms. It passes from one mammal to another through snails. A
person may get it from eating watercress, for example.

Fasciolopsiasis intestinal fluke: This affects the intestines. It can also transmitted when
consuming contaminated water plants or water.

Gnathostomiasis: This causes swellings under the skin, and occasionally affects the liver, the
eyes, and the nervous system. It is rare, but it can be fatal. It occurs in Southeast Asia. It is
transmitted by eating freshwater fish, pigs, snails, frogs, and chicken.

Loa loa filariasis: Also known as loaisis, this is caused by the Loa loa worm, or African eye
worm. It causes itchy swellings on the body. It occurs mainly in Central and West Africa and is
transmitted through deerfly bites.

Mansonellosis: This is passed on through the bites of midges or blackflies. It affects the layers
under the surface of the skin, but it can enter the blood. It can lead to angioedema, swellings,
skin rash, fever, and joint problems. It is present in Africa and Cental America.

River blindness: Caused by a worm known as Onchocerca volvulus , this affects the eyes,
skin, and other body tissues. It is found near fast flowing water. It is transmitted through the bite
of a blackfly. It occurs in South America, but 90 percent of cases are in Africa.

Lung fluke: Also known as paragonimiasis, this affects the lungs, causing symptoms similar to
those of tuberculosis (TB). However, it can reach the central nervous system, leading to
meningitis. It is transmitted when eating undercooked or raw freshwater crabs, crayfishes, and
other crustaceans. It is most common in parts of Asia.

Schistosomiasis, bilharzia, or snail fever: There are different types of schistosomiasis. They
can affect the skin and internal organs. It results from exposure to fresh water that has snails in it
that are infected with the blood fluke, or trematode worm. The worms are not found in the U.S.
but they are common worldwide.

Sparganosis: Humans can become infected if they eat foods tainted with dog or cat feces that
contains the larvae of a tapeworm of the Spirometra family. It can lead to a migrating abscess
under the skin. It is rare.

Strongyloidiasis: This can lead to severe and possibly fatal immunodeficiency. The parasite
penetrates through the skin and affects the lungs, skin, and intestines. It is passed on through
direct contact with contaminated soil. It most occurs in tropical and subtropical regions.
Different types of tapeworm can affect the intestines, the liver, or the lungs.

Beef and pork tapeworms: Taeniasis is caused by tapeworms of the taenia family. They affect
the intestines. They are passed on by eating undercooked beef or pork.

Toxocariasis: A roundworm transmits this infection from animals to humans. It affects the eyes,
brain, and liver. It is caused by accidentally swallowing the eggs of the parasite, for example,
when young children play with soil. Nearly 14 percent of people in the U.S. have antibodies,
suggesting that millions have been exposed. Most never have symptoms.

Trichinosis: This is caused by the roundworm of the Trichinella family. Infection can lead to
intestinal symptoms, fever, and muscle aches. It is passed on by eating undercooked meat.

Whipworm: Also known as trichuriasis, whipworms live in the large intestine. Eggs are passed
in feces. It is common all over the world. Humans can become infected when ingesting the eggs,
for example on unwashed fruit or vegetables.

Elephantiasis lymphatic filariasis: This is transmitted through mosquito bites. The adult worms
live in the lymph system. Infection can lead to lyphedema and elephantiasis, in which swelling
can cause disfigurement and disability. In the Americas, it is passed on by the Culex
quinquefasciatus mosquito.

Ringworm is sometimes mistaken for a worm, but it is not a worm. It is a fungal infection.

Ectoparasites

These are parasites that live on the outside of the body, such as fleas.

Bed bugs are ectoparasites: They live on the outside of the body.

Bedbug: These can affect the skin and vision. They are found all over the world. Sharing
clothing and bedding can spread infection. They may be present in newly rented accommodation
and hotel rooms.

Body lice: These are common worldwide. Infection can spread through sexual activity, skin-to-
skin contact, and sharing bedding or clothing.

Crab lice: These affect the pubic area and eyelashes. They are common all over the world and
spread through sexual activity, skin-to-skin contact, and sharing bedding or clothing.

Demodex: These affect the eyebrow and eyelashes. They are common all over the world and can
spread through prolonged skin contact.
Scabies: This affects the skin. It is common all over the world and can spread through sexual
activity, skin-to-skin contact, and sharing bedding or clothing.

Screwworm: This is transmitted by a fly, and it affects skin and wounds. It is found in Central
America and North Africa.

Head lice: These live on the scalp and affect the hair follicles. They are common all over the
world and spread through head-to-head contact. A reaction to their saliva causes itching.

Parasites come in many shapes and sizes and can lead to a wide variety of symptoms and health
issues. Some parasites are treatable and others are not.

Prevention

To increase your chance of avoiding parasites:

 find out which kind are prevalent in your area or in locations you may travel
 take precautions, for example, using insect repellant in places where mosquitoes are common
 be careful to eat only well-cooked fish and meat
 when traveling, drink only water from bottles with a sealed top
 take care when bathing in fresh-water lakes or rivers

If you have any symptoms, see a doctor.

In the United States

According to the Centers for Disease Control and Prevention (CDC), the following parasitic
infections are common in the U.S.:

 neurocysticercosis
 Chagas disease
 toxocariasis
 toxoplasmosis
 trichomoniasis, or trich

GENERAL CLASSIFICATION OF MICROORGANISM.

 Microorganisms or microbes are microscopic organisms that exist as unicellular,
multicellular, or cell clusters. Microorganims are widespread in nature and are beneficial
to life, but some can cause serious harm. They can be divided into six major types:
bacteria, archaea, fungi, protozoa, algae, and viruses.

Bacteria

Bacteria are unicellular organisms. The cells are described as prokaryotic because they lack a
nucleus. They exist in four major shapes: bacillus (rod shape), coccus (spherical shape), spirilla
(spiral shape), and vibrio (curved shape). Most bacteria have a peptidoglycan cell wall; they
divide by binary fission; and they may possess flagella for motility. The difference in their cell
wall structure is a major feature used in classifying these organisms.

According to the way their cell wall structure stains, bacteria can be classified as either Gram-
positive or Gram-negative when using the Gram staining. Bacteria can be further divided based
on their response to gaseous oxygen into the following groups: aerobic (living in the presence of
oxygen), anaerobic (living without oxygen), and facultative anaerobes (can live in both
environments).

According to the way they obtain energy, bacteria are classified as heterotrophs or autotrophs.
Autotrophs make their own food by using the energy of sunlight or chemical reactions, in which
case they are called chemoautotrophs. Heterotrophs obtain their energy by consuming other
organisms. Bacteria that use decaying life forms as a source of energy are called saprophytes.

Archaea

Archaea or Archaebacteria differ from true bacteria in their cell wall structure and lack
peptidoglycans. They are prokaryotic cells with avidity to extreme environmental conditions.
Based on their habitat, all Archaeans can be divided into the following groups: methanogens
(methane-producing organisms), halophiles (archaeans that live in salty environments),
thermophiles (archaeans that live at extremely hot temperatures), and psychrophiles (cold-
temperature Archaeans). Archaeans use different energy sources like hydrogen gas, carbon
dioxide, and sulphur. Some of them use sunlight to make energy, but not the same way plants do.
They absorb sunlight using their membrane pigment, bacteriorhodopsin. This reacts with light,
leading to the formation of the energy molecule adenosine triphosphate (ATP).

Fungi

Fungi (mushroom, molds, and yeasts) are eukaryotic cells (with a true nucleus). Most fungi are
multicellular and their cell wall is composed of chitin. They obtain nutrients by absorbing
organic material from their environment (decomposers), through symbiotic relationships with
plants (symbionts), or harmful relationships with a host (parasites). They form characteristic
filamentous tubes called hyphae that help absorb material. The collection of hyphae is called
mycelium. Fungi reproduce by releasing spores.

Protozoa

Protozoa are unicellular aerobic eukaryotes. They have a nucleus, complex organelles, and
obtain nourishment by absorption or ingestion through specialized structures. They make up the
largest group of organisms in the world in terms of numbers, biomass, and diversity. Their cell
walls are made up of cellulose. Protozoa have been traditionally divided based on their mode of
locomotion: flagellates produce their own food and use their whip-like structure to propel
forward, ciliates have tiny hair that beat to produce movement, amoeboids have false feet or
pseudopodia used for feeding and locomotion, and sporozoans are non-motile. They also have
different means of nutrition, which groups them as autotrophs or heterotrophs.
Algae

Algae, also called cyanobacteria or blue-green algae, are unicellular or multicellular eukaryotes
that obtain nourishment by photosynthesis. They live in water, damp soil, and rocks and produce
oxygen and carbohydrates used by other organisms. It is believed that cyanobacteria are the
origins of green land plants.

Viruses

Viruses are noncellular entities that consist of a nucleic acid core (DNA or RNA) surrounded by
a protein coat. Although viruses are classified as microorganisms, they are not considered living
organisms. Viruses cannot reproduce outside a host cell and cannot metabolize on their own.
Viruses often infest prokaryotic and eukaryotic cells causing diseases.

Multicellular Animal Parasites

A group of eukaryotic organisms consisting of the flatworms and roundworms, which are
collectively referred to as the helminths. Although they are not microorganisms by definition,
since they are large enough to be easily seen with the naked eye, they live a part of their life
cycle in microscopic form. Since the parasitic helminths are of clinical importance, they are often
discussed along with the other groups of microbes.