Introductory Lecture:

General Microbiology:
General Microbiology includes physiology, biochemistry, control, growth,
morphology and anatomy of germs.

• Test/procedures take 3 minutes to 1 hour

• Pre-test takes more time (24 hours)
• Pre and post process of practical takes time
• Microbiology is not visible. It is micro.

General sizes of microorganisms:

• Bacteria — 10-6
• Virus — 10-9
• Prions — 10-10
• Fungus — 10-3-6
• Algae — 10-3-6
• These microorganisms are not visible with naked eye.
• Microbiology is a logic based or inference based study.
• Microbes are most enormous in the world.
• Level of microbes is 1012/ml or 1018/ml or 10 Trillion per ml in marshy water.
• Level of microbes in intestine is 1012/ml.
• Microbiology starts from microbiome.
• Microbiome decides the health of humans and animals or microbiome
effectively controls the health and disease status.
➢ If microbiome is normal, health is normal.
➢ If microbiome is not normal disease is caused.

SOP's in practicals:
• SOP's stands for "Standard operating procedure/protocol"
• Before each experiment, you should have SOP's.
• SOP's should be noted on practical notebook.

Introduction to microbiology:
Microbiology is the study of microbes.

Biosafety:
"Bio" means "life" and "safety" means "protection".

• Biosafety includes the safety of:

1. Product safety
2. Safety of life

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Product safety:
Product safety includes:
• Safety of test
• Safety of germs
Safety of life:

Safety of life includes:

• Safety of environment (lab, surroundings)

• Safety of personnel (Lab attendants, Lab assistants, supervisors, directors)
• Safety of surrounding populations (humans, animals, plants, faunna, flora)

Biosafety measurements in Microbiology Lab:

Following are the biosafety measurements in Microbiology lab:
i. Microbes cause diseases so you should avoid direct contact with microbes.
ii. Chemicals are used to identify germs, these chemicals are toxic. You should
avoid direct contact with chemicals.
iii. Instruments of microbiology lab are costly. They should not be mishandled.
iv. Protection should be given to surrounding humans. You should not spread
germs and chemicals.

Biosecurity:
Biosecurity means to provide security to personnel, building and infrastructure while
working. Infrastructure includes:

• Lock and key (guard,camera,thumb impression, retina scan)

• Equipment
• Chemicals
• Biological tests/Germs

Points:
➢ Biosafety is the protection of tests/germs in the lab.
➢ Biosecurity includes protection of tests/germs against notorious physical
factors.
➢ Typhoid XDR:
• Disease spread in Karachi
• Disease spread through flies/ not properly cooked meat.
• Extreme drug resistance is produced by microbes causing the disease.

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LECTURE NO.01:
Morphology of microorganisms:

• Bacteria
• Virus
• Fungus
• Algae

Bacteria:
Every bacterium has a unique shape.
Cocci (sing. Coccus):
Round shaped bacteria are called cocci.
Size:
Size ranges from 0.5 micrometer to 1 micrometer.
Example:

• Mycoplasma ( micrococcus)
• Staphylococcus (cocci)
Division of cocci:

There are four main divisions of cocci:

i. staphylococci:
• Reproduce three dimensionally
• Grape like clusters
• For example, Staphylococcus
ii. streptococci:
• Reproduce in one dimension/single plane
• Form chains
• For example, Streptococcus
iii. tetrad:
• When cocci divide in two dimensions ( length and width ) they form tetrad.
• For example, Tetrad
iv. sarcina (p.sarcinae):
• When cocci divides in three dimensions at the same time they form a cube
like structure called sarcina.

Bacilli (sing. Bacillus ):

• Staff-like/Rod like bacteria

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Size:

• Ranges from 3 micrometer to 10 micrometer in length.

• Ranges from 0.5 micrometer to 1 micrometer in width.

Division of bacillus:
There are three main divisions of bacillus:
i. streptobacillus:
• Long chains
• Divide by width
• For example, Bacillus
ii. Chinese letter formation:
• For example, Corynibacterium
iii. palisade:
• Stacks of coins
• Divide by length
• For example, Palisade
Sometimes bacteria have:
Filamentous / Branching shape:

• Like hairs

Size:

• More than 10 micrometer

• For example, Actinomycetes, Nocardia

Corkscrew:

• Spiral shaped
• For example, Treponema, Spirilla

Comma shaped/vibrio:

• For example, Vibrio fetus( humans )

• Campylobacter (animals)

Square shaped bacteria:

• For example, hollacrula

Star shaped bacteria:

• For example, Stella

Viruses:
According to morphology viruses are of five types:
i. Enveloped:

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 ➢ Icosahedral:
• Envelope is circular or disorganized.
• Size is 100nm
• For example Herpes viridae
• Some have size more than 100 nm (e.g. Orthomyxoviridae)
ii. Naked:
➢ Icosahedral (without envelope)
➢ Size is 30nm to 100nm
iii. Complex:
They are of two types:
i. Pox viruses:
• Large brick like
• Size is 300nm to 1500nm
ii. Bacteriophage:
• Size is 50nm
iv. Filamentous viruses:
• They have length in mm
• They have width in nm
• For example , Filonidae
v. Mega viruses:
• Live in insects and protozoans
• Not important for humans and animals
• Size is in micrometers
• For example, Megavirales

Fungus:
Fungus is of two types:
1. Unicellular
2. Multicellular
• Unicellular:
• Unicellular fungus is called yeast.
• They are round in shape
• Their size is 5 micrometer to 10 micrometer
• Overall range of size is 5 micrometer to 40 micrometer
• Maximum size is 40 micrometer
• For example, Saccharomyces
• Multicellular:
• They have mycelium ,aerial spores and aerial hyphae
• Size is more than 10 micrometer
• For example, Aspergillus
➢ Some fungi are dimorphic/ bimorphic because they can exist as both
unicellular and multicellular at different temperatures.

Algae:
• Diverse in size
• Used as food source

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• Photosynthetic
• They can cause poisoning but yeast cannot cause poisoning
• Size is 5 micrometer to 40 micrometer
• Blue Green algae have the size of 5 micrometer
• There is no algae of veterinary importance up till now.

LECTURE NO. 02:

Prokaryotes vs. Eukaryotes
Sr. No. Prokaryotes Eukaryotes

Term Before nucleus True nucleus

Primitive Modern

Groups Bacteria, Archeobacteria Algae, fungi, plants,

animals

Origin 3.5 billion years ago 2 billion years ago

Size 0.5 - 3 micrometer More than 5 micrometer

Cells Unicellular Unicellular/ Multicellular

Complexity Simple Complex

Nucleus location Free Center

• Free, attached to • Bounded in

cell membrane nucleus

• Circular • Linear

• One in bacteria, • Two or more than

two in some two in Eukaryotes
DNA
• Replication, • Replication and
transcription, translation takes
translation takes place in cytoplasm,
place in cytoplasm transcription takes
at the same time place in nucleus
but not at the same
• Cell membrane time
aids in genomic
process • No aid by cell
membrane in
genomic process

Nuclear membrane None Yes

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 Nucleolus Absent Present

Chromosomes Usually one More than one

Chromosome shape Circular Linear

Operon Individually
Genes Multiple genes for a single Single gene for single
process process

DNA bases (G+C ratio) 28 - 73 40

DNA wrapping Non histonic proteins Histone proteins

Absent Present

• Inclusion bodies (Nucleus, E.R, Golgi

are present. apparatus, lysosome,
Membrane based mitochondria etc)
organelles • Respiration takes
place at the cell
membrane

Ribosomes 70S (50S+30S) 80S (60S+40S)

Ribosomal region Cell membrane or free Endoplasmic reticulum

Mitochondria Absent Present

Chloroplast If present, chlorophyll is Present, chlorophyll within

scattered chloroplast

Microtubules/
cytoskeleton/ cell
movement Simple or absent Present

Cell membrane No sterols Sterols

Cell wall Peptidoglycan or muramic Starch/ Chitin

acid

Reproduction Simple/ binary fission Complex (2 gametes)

Basal metabolic rate High Low

Fimbrae / flagella/ cillia Simple Complex

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LECTURE NO.03:
Introduction to Microbiology
The word “Microbiology" is derived from Greek words:

MIKROS → small

BIOS → life

LOGIA → study

• It is the study of microorganisms either unicellular, multicellular or

acellular.
• Microbiology includes disciplines of Virology, Mycology,
Bacteriology etc.

Prokaryotic Vs. Eukaryotic Organisms:

• Prokaryotic microorganisms are conventionally classified as
lacking organelles and includes:
→Eubacteria
→Archaeobacteria
→Blue Green Algae

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 ➢ Eukaryotic microorganisms exhibit cell organelles and include:

→Fungi

→Protists

→Algae

Additional Points:

• Only 1% of microorganisms are studied by culturing.

• The microorganisms which can be grown in culture media are
studied by using Molecular Biology. Their molecular study includes
study of DNA, RNA & genes.

• Prions caused a disease called “Wasting Dear/Zombie Dear

disease in America.

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How Microbiologists Study Microbes:
Microbiologists traditionally relied on culture, staining and microscopy.
However, 1% of Microbes present in the environment are culturable.
That's why Microbiologists often rely on extraction or detection of
nucleic acids either DNA or RNA.

Viruses & Prions:

• Viruses are not always classified as
organisms as they have been identified
either as very simple microorganisms or very
complex molecules.
• Prions are never considered as
microorganisms and they have been
investigated by the virologists as the clinical
effects traced to them were originally
presumed due to chronic viral infections and
virologists identified them as “infectious
agents. Following are some infectious
→Mad cow disease in cattle.
→Scrapie disease in sheep.
→Wasting deer / Zombie deer disease in deers (America).

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 BRANCHES OF MICROBIOLOGY:
Two broad branches of Microbiology are as follows:
1. Pure Microbiology
2. Applied Microbiology
Pure Microbiology:
It includes the study of
Microbes only for the sake
of study. No benefits or
loses are studied. Pure
Microbiology has further
seven divisions:

1. Bacteriology:
Study of bacteria is called
Bacteriology.

2. Mycology:
Study of fungi is called Mycology.

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3. Phycology:
Study of algae is called Phycology.

4. Immunology:
Study of immune system is called
immunology.

5. Virology:
Study of viruses is called virology.

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6. Microbial Physiology:
Study of microbial cell functions is called Microbial Physiology. It
includes the study of microbial growth, metabolism & cell functions.

7. Microbial Cytology:
Study of microscopic & sub-microscopic details of microorganisms is
called Microbial Cytology.

• APPLIED MICROBIOLOGY:

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Study of microorganisms to determine benefits as
well as harmful effects of microorganisms.
Applied Microbiology is further divided into four
branches:
1.Veterinary Microbiology:
Study of microbes of Veterinary importance such as probiotics &
pathogens which are related to Veterinary medicine.

2.Pharmaceutical Microbiology:
Study of microorganisms which are related to the production of
antibiotics, enzymes, vitamins, biologics and other pharmaceutical
products & those microbes which cause pharmaceutical
contamination & spoilage.

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3. Microbial Biotechnology:
Manipulation of microorganisms at genetics & molecular level to
generate useful products is called Microbial Biotechnology e.g.
production of insulin from E.coli.

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 4. Food Microbiology:
Study of microorganisms causing food spoilage and food
borne illness. Use of microbes to produce food is called food
Microbiology.

• HISTORY OF MICROBIOLOGY:
Ancient History of Microbiology:
• Recent discovery of Mycobacterium DNA in the three thousand
years old Egyptian mummies reminds us that microorganisms
have been around for a much longer period of time.
• Infact, bacterium ancestors were the first living cells to
appear on the earth.

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Golden Age of Microbiology:
From 1857-1940 has been named Golden age of Microbiology. During
this period, rapid advancement spread hold mainly by Pasture & Robert
Koch which led to the establishment of
Microbiology as a science. During this era, salient achievements include;

➢ Discovery of immunity
➢ Discovery of disease causing agents

Koch's Postulates:
• Robert Koch a Germen physician, discovered the cause of Anthrax
(Bacillus anthracis) in 1870s.
• He devised a theory relating micro-organisms to every specific
disease. The POSTULATES of that theory are as under.
• Same pathogen must be present in every case of the disease.
• Pathogen must be isolated from the diseased host & grown in a
pure culture.
• Pathogen from the pure culture must cause the disease when it is
inoculated into susceptible lab. Animal.
• The same pathogen must be isolated from the inoculated animal
and it must be same as original organism.

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Vaccination:
Edward Jenner, a young British physician used scrapping of
cowpox blisters to vaccinate against smallpox.
• Edward Jenner is called the father of Vaccinology.

Fermentation:
• Microorganisms like yeast convert sugar into alcohol in the
absence of air. This process is called Fermentation.
• It is used to make wine & beer.
• In the presence of air bacteria converts alcohol into acetic acid or
vinegar.

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Pasteurization:
• It is the heat treatment of beverage & milk at 72°C for 30
minutes to kill microorganisms (which cause spoilage of liquid
in food) without compromising on its quality.
• Now a days, it is only used for milk products.

Germ Theory of Disease:

• Microorganisms are the cause of diseases. In 1860's Joseph Lister
used phenol, carbolic acid as a disinfectant & antiseptic solution.
• This practice reduced the incidence of infection & death. Other
surgeons readily adopted it.
• Lister's technique was the earliest medical attempt to control
infection caused by microorganisms.

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LECTURE NO.04:
SIZE OF BACTERIA:
Many sizes and most bacteria range from:

• 0.2 to 2.0 μm in diameter

• 2 to 8 μm in length
• Overall average size 1-10 μm .

SHAPE OF BACTERIA:
• Spherical / coccus (plural: cocci; means rounded)
• Rod Shaped / bacillus (plural: bacilli)
• Spiral

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Cocci:
Cocci are usually round but can be oval or elongated. When they divide
to reproduce, they remain attached to each other.

CLASSIFICATION ACCORDING TO PLANE OF DIVISION:

Dipplococci:
Those cocci that remain in pairs after dividing are called diplococci.

Streptococci:
Those cocci that divide and remain attached in chain like patterns are
called streptococci.

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Tetrad:
Those cocci that divide into two planes and remain in groups of four are
known as tetrads.

Sarcinae:
Those cocci that divide in three planes and remain attached in cube like
groups of eight is called sarcina.

Staphylococci:
Those cocci that divide in multiple planes and form grape like clusters or
broad sheets are called staphylococci.

BACILLI:
Bacilli are the rod like bacteria e.g. Bacillus anthracis

Single bacillus:
Most bacilli appear as single rods, called single bacilli.

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Diplobacilli:
Those bacilli which appear in pairs after division are called Diplobacilli.

Streptobacilli:
Those bacilli that are found in chains in single plane are called
Streptobacilli.

MONOMORPHIC VS PLEOMORPHIC:
• Bacteria that maintain a single shape are called monomorphic or
the bacteria having same shape.
• Some bacteria can have many shapes known as pleomorphic. e.g.:
Corynebacterium pyogenese.
• Some bacteria occur in the shape of a star e.g. Stella.
• Some bacteria occur in the shape of rectangle e.g. Haloarcula.
• Some bacteria also appear triangular in shape.

FUNCTIONAL ANATOMY OF BACTERIA:

Cell Wall:
Acts as an antigen, provide protection and rigidity

Cell membrane:
Serve as a barrier through which materials enter and exit the cell

Capsule:

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Acts as an antigen, has feeding importance, sticking features and cause
disease

Mesosomes:
Has role in metabolism

FUNCTIONAL ANATOMY OF BACTERIA:

Fimbrae:
Helps in motility, jerky movement, has sticking feature and acts as an
antigen

Pilus:
Helps in reproduction (conjugation)

Ribosomes:
Protein formation

Nucleoid:
Transcription and translation
FUNCTIONAL ANATOMY OF BACTERIA:
Chromosomes:
Hereditary material

Droplets:
Helpful in storage

Flagella:
Act as an antigen and helps in motility

Plasmid:
Have special features of resistance and infection.

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LECTURE NO.05:
CULTURE MEDIA / GROWTH MEDIA
A nutrient material prepared for the growth of microorganisms in a laboratory is
called a culture medium.

• Solid / Semi-solid media known as Nutrient Agar

• Liquid media known as Nutrient Broth

INOCULUM:
Microbes that are introduced into a culture medium to initiate growth are called an
inoculum

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PROPERTIES OF CULTURE MEDIA
1. It must contain the right nutrients for the specific microorganism we want to
grow.i.e. MacConkey Agar contains bile salt which is required for the growth of E.
coli.

2. It should also contain sufficient moisture.

3. The pH of media must be properly, adjusted as per requirement of microorganism

to be grown e.g. pH 7.0-7.2 for bacteria and 5.5-5.6 for fungi.

4. Suitable level of oxygen or no oxygen at all.

5. The medium must initially be sterile that means it must initially contain no living
microorganisms.

Most of these media, which are available from commercial sources, have premixed
components and require only the addition of water and then sterilization.

AGAR & ITS PROPERTIES

When it is desirable to grow bacteria on a solid medium, a solidifying agent such as
agar is added in a medium. Agar is a complex polysaccharide derived from marine
algae.

Properties of Agar

➢ Only a few microbes can degrade agar, so it remains solid.

• Agar liquefies at about 100°C (the boiling point of water) and at sea level
remains liquid until the temperature drops to about 40-45 degree celcius
• Agar media are usually contained in test tubes or petri dishes. The test tubes
are called slants.

When they are allowed to grow

microaerofiles with the tube held at an angle
so that a large surface area for growth is
available. It has two portions

• Slant
• Butt

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CHEMICALLY DEFINED MEDIA
Chemically defined media is that media whose exact chemical composition is known.

• Organisms that require many growth factors are described as fastidious.

• Organism of this type such as Lactobacillus are sometimes used in test that
determines the concentration of a particular vitamin in a substance.

Composition of Staphylococcus Medium 110

COMPLEX MEDIA

These media are made up of nutrients including extracts from yeast, meat, plants or
digests of protein from these and other sources.

Composition of nutrient agar

REDUCING MEDIA
These media contains ingredients such as sodium thioglycolate that chemically
combine with dissolved oxygen and deplete the oxygen in the culture medium.

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SELECTIVE MEDIA

Such type of media which suppress the growth of unwanted bacteria and
encourages the growth of desired microbes. e.g;

• Bismuth sulphite agar is one medium used to isolate the gram’negative

Salmonella typhi from faeces.

Salmonella typhi (GROWTH ON BISMUTH SULPHITE AGAR)

➢ Sabouraud’s dextrose agar which has a pH of 5.6 is used to isolate fungi that
outgrows most bacteria at this pH.

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DIFFERENTIAL MEDIA

Such type of media which makes it easier to distinguish colonies of the desired
organisms from other colonies growing on the same plate.

• On blood agar, some bacteria show alpha haemolysis while other shows beta
haemolysis.

• On manitol salt agar, Staphylococcus aureus produces yellow colonies while

Staphylococcus epidermis
produces pink colonies

pH indicator phenol red

In Basic pH, color is RED

In Acidic pH, color is Yellow

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On Eosin Methylene Blue (EMB) agar, E. coli produces black centered colonies with
metallic sheen while Enterobacter aerogenes gives dark centered colonies.

MACCONKEY AGAR IS A SELECTIVE AS WELL AS DIFFERENTIAL MEDIA. IT IS

SELECTIVE AS IT ONLY ALLOWS ENTERIC BACTERIA TO GROW AND DIFFERETIAL AS E.
coli GIVES PINK COLOURED COLONIES ON IT.

ENRICHMENT CULTURE MEDIA

• It is usually liquid and provides

nutrients and environmental
conditions that favor the growth of a
particular microbe but not others. It is
also a selective medium but

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designated to increase very small numbers of the desired type of organism to
detachable levels e.g.;

Selenite F broth for the enrichment of Salmonella Species

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LECTURE NO.06:
Transformation in bacteria
Transformation:

• Genes are transferred from one bacterium to another as naked”DNA in

solution.
• First demonstrated over 70 years ago
• Not understood at that time
• Study of this phenomenon eventually led to the conclusion that DNA is the
genetic material

Griffith’s Experiment:
The initial experiment on transformation was performed by Frederick Griffith in
England in 1928 while he was working with two strains of Streptococcus pneumoniae.

• One, a virulent strain, has a polysaccharide capsule that prevents

phagocytosis. The bacteria grow and cause pneumonia.
• The other, an avirulent strain, lacks the capsule and does not cause disease.

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Purpose of Griffith’s Experiments:
• Griffith was interested in determining whether injections of heat-killed
bacteria of the encapsulated strain could be used to vaccinate mice against
pneumonia.

➢ Prepare Killed Vaccine

First Experiment & Result:

• Injections of living encapsulated

bacteria killed the mouse

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Second Experiment & Result:

• Mice were injected with live non-encapsulated bacteria

and remained healthy

Third Experiment & Result:

• Dead encapsulated bacteria did not kill the mouse.

Fourth Experiment & Result:

➢ The dead encapsulated bacteria were mixed with live non-

encapsulated bacteria and injected into the mice, the mice
died. In the blood of the dead mice, Griffith found living,
encapsulated bacteria.
• Hereditary material (genes) from the dead bacteria had
entered the live cells and changed them genetically so that
their progeny were encapsulated and therefore virulent.

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Bacterial Transformation in Broth:
Subsequent investigations based on Griffith’s research revealed that bacterial
transformation could be carried out without mice. A broth was inoculated with live
non-encapsulated bacteria. Dead encapsulated bacteria were then added to the
broth. After incubation, the culture was found to contain living bacteria that were
encapsulated and virulent. The non-encapsulated bacteria had been transformed;
they had acquired a new hereditary trait by incorporating genes from the killed
encapsulated bacteria.

Components which caused Transformation:

Crucial experiments were performed in the United States by

• Oswald T. Avery and his associates

• Colin M. MacLeod
• Maclyn McCarty
• After years of research, they
announced in 1944 that the
component responsible for
transforming harmless S.
pneumoniae into virulent strains
was DNA.
• Their results provided one of
the conclusive indications that
DNA was indeed the career of genetic information.

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Mechanism of Genetic Transformation in Bacteria:

• After death and cell lysis, bacteria, release their DNA into the environment.
• Other bacteria take up fragments of DNA and
integrate them into their own chromosomes by
recombination.
• A protein called Rec A binds to the cell’s DNA and
then to donor DNA causing the exchange of
strands.
• A recipient cell with this new combination of
genes is a kind of hybrid, or recombinant cell.
• All the descendants of such a recombinant cell
will be identical to it.
• Transformation occurs naturally among very few
genera of bacteria, including Bacillus,
Haemophilus, Neisseria, Acinetobacter and
certain strains of the genera Streptococcus and
Staphylococcus.

Competent and Competence:

• When a recipient cell is in a physiological state in which it can take up the
donor DNA, it is said to be competent. Competence results from alterations
in the cell wall that makes it permeable to large DNA molecules.

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39
Lecture NO.07
Conjugation in Bacteria:
• Bacterial conjugation is the transfer of genetic material between bacterial
cells by direct cell-to-cell contact or by a bridge-like connection between two
cells.
• Conjugation is mediated by one kind of plasmid that replicates independently
from the chromosomal DNA.
➢ The plasmids responsible for conjugation are transmissible between cells
during conjugation.

Conjugation Vs. Transformation:

Conjugation differs from transformation in two major ways.

1. Conjugation requires direct cell-to-cell contact.

2. The conjugating cells must generally be of opposite mating type

• Donor cells must carry the plasmid

• Recipient cells usually do not carry that specific plasmid.

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Conjugation in Gram Negative Bacteria:
In gram-negative bacteria, the plasmid carries genes that code for the synthesis of
sex pilli that contact the recipient and help bring the two cells into direct contact.

Conjugation in Gram Positive Bacteria:

Gram-positive bacterial cells produce sticky surface molecules that cause cells to
come into direct contact with each other (mating bridge).

Plasmid Replication in Conjugation:

In the process of conjugation, the plasmid is replicated during the transfer of a

single-stranded copy of the plasmid DNA to the recipient, where the complementary
strand is synthesized.

1. Process of conjugation:

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In E.coli, the F factor (fertility factor) was the first plasmid observed to be
transferred between cells during conjugation.

Donors carrying F factors (F+ cells) transfer the plasmid to recipients (F- cells), which
become F+ cells as a result.

2. Process of conjugation:

In some cells carrying F factors, the factor integrates into the chromosome,
converting the F+ cell to an Hfr cell (high frequency of recombination).

3. Process of conjugation:

• When conjugation occurs between an Hfr cell and an F- cell, the Hfr cell’s
chromosome (with its integrated F factor) replicates, and a parental strand of
the chromosome is transferred to the recipient cell.
• Replication of the Hfr chromosome begins in the middle of the integrated F
factor, and a small piece of the F factor leads the chromosomal genes into
the F- cell. Usually, the chromosome breaks before it is completely
transferred.
• Once within the recipient cell, donor DNA can recombine with the recipient’s
DNA. (Donor DNA that is not integrated is degraded.)

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 • Therefore, by conjugation with an Hfr cell, an F- cell may acquire new
versions of chromosomal genes. However, it remains an F- cell because it
did not receive a complete F factor during conjugation.

Application of Conjugation for Mapping Gene Location:

• Conjugation is used to map the location of genes on a bacterial chromosome

➢ The genes for the synthesis of threonine (thr) and leucine (leu) are first,
reading clockwise from 0.

Their locations were determined by conjugation experiments. Assume that

conjugation is allowed for only 1 minute between an Hfr strain that is his +, pro+, thr+,
and leu+, and an F- strain that is his-, pro-, thr-, and leu-.

If the F- acquired the ability to synthesize threonine, then the thr gene is located
early in the chromosome, between 0 and 1 minute. If after 2 minutes the F- cell now
becomes thr+ and leu+, the order of these two genes on the chromosome must be
thr, leu.

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LECTURE NO.08:
TRANSDUCTION IN BACTERIA:
TRANSDUCTION:
Transfer of bacterial DNA from a donor cell to a recipient cell inside a virus that
infects bacteria (Bacteriophage) is called Transduction.

BACTERIOPHAGE:
These are group of viruses involved in transfer of
genetic material from one bacterium to the other.
The carrier phage is called transducer or vector.

HISTORY OF TRANSDUCTION:

• Transduction was first described by Joshua Lederberg & Norton Zinder in

1952. Bacteriophage are widely used as vectors in recombinant DNA
Technology.

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GENERALIZED TRANSDUCTION:

• Generalized transduction is mediated by phages where any DNA segment can

be transferred by the virus and may not integrate the segment to the
bacterial chromosome.
• Here a portion of the donor bacterial DNA accidently get enclosed in a capsid.
• Upon lysis and further infection of the virus particle to another bacterium,
the genetic material of the donor is released and recombination occurs
between the injected DNA segments and homologous part of the recipient
chromosome, forming a rDNA.
• All genes contained within a bacterium infected by a generalized transducing
phage are equally likely to be packaged in a phage coat and transferred.

SPECIALIZED TRANSDUCTION:

• Only certain bacterial genes are transferred by specialized transduction.

• In one type of specialized transduction, the phage codes for certain toxins
produced by their bacterial hosts, such as diphtheria toxin for
Corynebacterium diphtheriae and erythrogenic toxin for Streptococcus
pyogenes, and Shiga toxin for E. coli O157:H7 are transferred by specialized
transduction.

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COMPLETE TRANSDUCTION:

• In complete transduction, the transduced DNA fragment gets integrated

within the recipient bacterial chromosomes, forming a recombinant
chromosome.
• This DNA fragment replicates along with recipient bacterial chromosome
replication and passed on to the daughter cells.

ABORTIVE TRANSDUCTION:

• In abortive transduction, the transduced DNA fragment may not get

integrated within the recipient bacterial chromosome, and remains in the
cytoplasm as free particle.
• These DNA fragments cannot undergo replication.

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LECTURE NO.09:
Fungal Spores:
Fungal Reproduction:

• Filamentous fungi can reproduce asexually by fragmentation of their hyphae.

• Both sexual and asexual reproduction in fungi occurs by the formation of
spores.
• Fungi are usually identified by spore type.

Bacterial Endospore Vs. Fungal Spore:

• Fungi reproduce by producing cells called spores.

• While these fungal spores are somewhat resistant to destruction they are not
usually pathogenic to humans.
• Certain bacteria can produce a thick walled spore structure which allows
them to survive adverse environmental conditions for prolonged periods of
time.
• The bacterial spore is more properly called an endospore because its function
is to protect the bacterial DNA from destruction by conditions or substances
in the environment that destroy non-endospore forming bacteria.

Fungal Spores Types:

• Spores are formed from aerial hyphae in a number of different ways,

depending on the species.

47
There are two types of fungal Spores
• Asexual Spores
• Sexual Spores

1. Asexual spores:
These are formed by the hyphae of one organism.
When these spores germinate, they become organisms that are genetically
identical to the parent.
• Asexual spores are produced by an individual fungus through mitosis and
subsequent cell division; there is no fusion of the nuclei of cells.
• Two types of asexual spores are produced by fungi.
• Sporangiospore
• Conidiospore

1) Sporangiospore:
It is formed within a sporangium, or sac, at the end of an aerial hypha called a
sporangiophore. The sporangium can contain hundreds of sporangiospores. Such
spores are produced by Rhizopus.

48
2) Conidiospore:
Conidiospore, or conidium (plural: conidia), a unicellular or multicellular spore that
is not enclosed in a sac. Conidia are produced in a chain at the end of a conidiophore.
Such spores are produced by Penicillium and Aspergillus

Arthroconidia:

Conidia formed by the fragmentation of a septate hypha into single, slightly

thickened cells are called arthroconidia

49
Blastoconidia:
Another type of conidium, blastoconidia, are formed from the buds of its parent cell.
Such spores are found in some yeasts, such as Candida albicans and Cryptococcus.

Chlamydoconidium:
A chlamydoconidium is a thick-walled spore formed by rounding and enlargement
within a hyphal segment. A fungus that produces chlamydoconidia is the yeast
Candida albicans.

50
Sexual spores:

• It results from the fusion of nuclei from two opposite mating strains of the
same species of fungus.
• Sexual spores require two different mating strains and so are made less
frequently than asexual spores.
• Organisms that grow from sexual spores will have genetic characteristics of
both parental strains.
• A fungal sexual spore results from sexual reproduction, which consists of
three phases:

1. Plasmogamy: A haploid nucleus of a donor cell (+) penetrates the cytoplasm of a

recipient cell (-).

2. Karyogamy: The (+) and (-) nuclei fuse to form a diploid zygote nucleus.

3. Meiosis: The diploid nucleus gives rise to haploid nuclei (sexual spores), some of
which may be genetic recombinants

51
52
LECTURE NO.10:
"Viral Replication"
Introduction:

• Replication of virus is very complicated process.

• Viruses never reproduce by division.
• They are replicated by a process in which all components of virus are
produced separately and are assembled into intact virons.
• For replication of virus host is necessary
• Visuses are host specific.
• Host may be a bacteria, plant or an animal.
• Replication of viruses is studied for first time by experimenting on
bacteriophage of the T series [T2 , T4 and T6].

There are 2 types of life cycle commonly seen in viruses They are:

i] Lytic Cycle

ii] Lysogenic Cycle

Key steps in the Viral Replication Cycle:

• Attachment
• Penetration (Entry)
• Uncoating
• Genome replication
• Assembly

53
 • Maturation
• Release

1. Attachment:

• Virus are host specific and enters into the host or target cell
• This event is electrostatic, does not require any cellular or metabolic energy
• Virus exhibits cellular tropism
HIV T lymphocytes, macrophages

Rabies Muscle, neurons

Hepatitis A, B, C Liver (hepatocytes)

➢ Virus has host range and it may be narrow or broad

• Rabies virus is an example for broad range virus
• HIV is an example for broad range virus
• Viruses use receptors and antireceptors for attachment and entry into host
cell.
• Cellular receptors and antireceptors are mostly protein but sometimes they
may be glycoprotein, carbohydrates or lipids.
• The presence of virus specific receptors is necessary.
• For example HIV- CD4 receptor, Rabies-Acetylcholine, phospholpids.

54
2) Penetration [entry]:
Penetration is energy dependent process. Virus may penetrate into host by:

• Endocytosis
• Translocation
• Fusion

3) Uncoating:

• Refers to the removal or degradation of capsid (uncoating), there by

releasing the genome into host cell.
• The virus genome is transported to the site where transcription/replication
can begin.
• In some there is no degradation of capsid as capsid proteins play a role in
viral transcription and replication

4) Genome replication:

• Viral genetic material or genome is multiplied within the host

• Simultaneously viral structural proteins like capsids are synthesised
• Type of genetic material varies from virus to virus
• With respect to this all viruses are divided into seven groups by Dr. David
Baltimor in 1971
• Dr. David Baltimor shared “ NOBEL PRIZE “with Renato Dulbecco, Howard
Martin Temin in 1975 for their work on "interaction between tumor viruses
and the genetic material of the cell".

Seven groups as follows:

• Double stranded DNA

• Single stranded DNA
• Double standard RNA
• Single stranded (+)ve sense RNA

55
 • Single stranded (-)ve sense RNA
• Single stranded (+)ve sense RNA with DNA intermediate
• Double stranded DNA with RNA intermediate

1.Double stranded DNA:

Replication of genome of double stranded DNA virus

Example:Poxvirus, Herpes virus.

II. Single stranded DNA:

Replication of genome of single stranded DNAvirus

Example: Pircovirus, Parvovirus

56
III. Double stranded RNA:

Replication of genome of double stranded RNA virus

Example: Reoviruses, Orbibiruses

IV. Single stranded (+)ve sense RNA:

Replication of genome of +sense single stranded RNA virus

Example: Toga virus & Hepatitis E virus.

V. Single stranded (-)ve sense RNA:

Replication of genome of -sense single stranded RNA virus

Example: Rabies, Paramyxoviruses etc.

57
vi. Single stranded (+)ve sense RNA with DNA intermediate:

Replication of genome of single stranded (+)ve sense RNA virus with DNA
intermediate

Example: Retrovirus

VII. Double stranded DNA with RNA intermediate:

Replication of genome of double stranded DNA virus with RNA intermediate

Example: Hepadnaviruses

5)Assembly:

• Involves the collection of all components necessary for formation of viron.

• It takes place at a particular site in the cell.
• For example in pox viruses assembly occurs in the cytoplasm; in adeno virus it
occurs in nucleus.

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6) Maturation:

➢ Maturation is the stage of life cycle at which the virus becomes infectious.
• It involves structural change in virus particles.
• For some viruses maturation occurs only after release of viurs particle from
the cell.

7) Release:

• Newly formed viruses are released to outside of the cell either by lysis (as in
bacteriophage) or by budding(as in paramyxovirus, retrovirus)
• Generally non enveloped viruses release by cell lysis which results in the
death of host cell
• Release of virus by budding may or may not kill cell

Conclusion:
In general terms, virus replication involves three broad stages carried out by all types
of virus; the initiation of infection, replication and expression of the genome, and,
finally, release of mature virions from the infected cell. At a detailed level, there are
many differences in the replication processes of different viruses which are imposed
by the biology of the host cell and the nature of the virus genome. It is possible to
derive an overview of virus replication and the common stages which, in one form or
another, are followed by all viruses.

59
LECTURE NO.11:
General Characteristics of Viruses:
Virus:

• The word virus, is a Latin word means poison.

• In 1935, Wendell Stanley, an American chemist, isolated tobacco mosaic virus,
making it possible for the first time to carry out chemical and structural
studies on a purified virus. At about the same time, the invention of the
electron microscope made it possible to see viruses.

Viral Size:
➢ Viruses range from 20 to 1000 nm in length.
• Different viruses vary considerably in size.
• Although most are quite a bit smaller than
bacteria, some of the larger viruses. For
Example: Adenovirus 90 nm, Poliovirus 30
nm.

General Properties of Virus:

• Viruses are inert/crystalline outside the

body of host as soon as they come into the
host they become living.
• They are obligatory intracellular parasites
as, they absolutely require living host cells
in order to multiply.

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Distinctive features of Viruses:

• Contain a protein coat (sometimes itself enclosed by an envelope of lipids,

proteins, and carbohydrates) that surrounds the nucleic acid.
➢ Multiply inside living cells by using the synthesizing machinery of the cell.
• Cause the synthesis of specialized structures that can transfer the nucleic
acid to other cells.
➢ Virus contains a single type of nucleic acid, either DNA or RNA. (Both in a
single virus not present)
• Viruses have few or no enzymes of their own for metabolism.
• They lack enzymes for protein synthesis and ATP generation.
• To multiply, viruses must take over the metabolic machinery of the host cell.

Host Specificity

• Viruses are host specific.

• Most viruses are able to infect specific types of cells of only one host species

61
 • In rare cases, viruses cross the host-range barrier, thus expanding their host
range.
• The particular host range of a virus is determined by following factors;
• Virus ligand molecules which binds to certain receptors on host cell
• Availability within the potential host of cellular functions required for viral
multiplication.
• For the virus to infect the host cell, the outer surface of the virus must
chemically react with specific receptor sites on the surface of the cell.

Viral Structure:

• Viruses may be Hexagonal, Octagonal or have any other shape and have a 3D
structure.
• A virion is a complete, fully developed, infectious viral particle composed of
nucleic acid and surrounded by a protein coat outside of a host cell, and is a
vehicle of transmission from one host cell to another.

62
1. Nucleic Acid:

• A virus can have either DNA or RNA—but never both. The nucleic acid of a
virus can be single-stranded or double-stranded. There are viruses with the
following possibilities as their genome.
• double-stranded DNA (dsDNA)
• Single-stranded DNA (ssDNA)
• Double-stranded RNA (dsRNA)
• Single-stranded RNA (ssRNA)
• +ve sense (Positive Sense)
• –ve sense (Negative Sense)
• Depending on the virus, nucleic acid can be linear or circular.
• In most of the RNA viruses (such as the influenza virus) the nucleic acid is in
several separate segments and called segmented genome.

2. Capsid and Envelope:

• The nucleic acid of a virus is protected by a protein coat called the capsid.
• Each capsid is composed of protein subunits called capsomere.
➢ In some viruses, the proteins composing the capsomeres are of a single type.
• In other viruses, several types of protein may be present in a particular virus.
• In some viruses, the capsid is covered by an envelope which usually consists
of some combination of lipids, proteins, and carbohydrates which is derived
from the host cell membrane.
➢ Some viruses are not covered by an envelope are known as Naked Virus /
non-enveloped viruses.
➢ The capsid of a non-enveloped virus has following functions;

63
• protects the nucleic acid from nuclease enzymes in biological fluids
• promotes the viral attachment to susceptible host cells

• Depending on the virus, envelopes may or may not be covered by spikes.

• Spikes are carbohydrate-protein complexes that project from the surface of
the envelope.
• Some viruses attach to host cells by means of spikes.
• Spikes are such a reliable characteristic of some viruses that it can be used as
a means of identification.
• The ability of certain viruses, such as the influenza virus to clump red blood
cells is associated with spikes.
• Such viruses bind to red blood cells and form bridges between them. The
resulting clumping is called Hemagglutination.

64
65
LECTURE NO.12:
BACTERIAL GROWTH & MULTIPLICATION
Binary Fission:

• Normally bacteria reproduce by binary fission.

• It is asexual reproduction by a separation of the body into two new bodies. In
the process of binary fission an organism duplicates its genetic material and
then divides into two parts, with each new organism receiving one copy of
DNA.

Budding:

• A few bacterial species reproduce by budding; they form a small initial

outgrowth (a bud) that enlarges until its size approaches to the parent cell
and then it separates.

66
Generation Time:
➢ Time period required for a cell to divide is called the generation time. Some
bacteria like E. coli require 20 minutes for doubling under favorable condition
while others like Mycobacterium tuberculosis requires 24 hours for doubling
under favorable condition.

LOGARITHMIC PRESENTATION OF BACTERIAL POPULATION:

A few bacteria are inoculated into a liquid growth medium and the population is
counted at intervals, it is possible to plot bacterial growth curve that shows the
growth of cells over time.

PHASES OF GROWTH:

• Lag Phase
• Log Phase
• Stationary Phase

67
 • Death Phase

LAG PHASE:

• The period of little or no cell division is called the lag phase and it can last
for 1 hour or several days. The microbial population is undergoing a period
of intense metabolic activity involving synthesis of enzymes and various
molecules.

68
LOG PHASE / EXPONENTIAL GROWTH PHASE:

• In this period, eventually cells begin to divide and enter a period of growth or
logarithmic increase called the log phase or exponential growth phase.
• Cellular reproduction is most active during this period and as the generation
time is constant, logarithmic plot of growth during the log phase is a straight
line. The log phase is the time when cells are most active metabolically and is
referred for industrial purposes where a product is needed to be produced
efficiently.

Stationary PHASE:

• If the exponential growth continues unchecked, a large no. of cell could arise.
• For example, a single bacterium weighing 9.5x10 -11 gm / cell dividing every
20 minute for 24 hours can theoretically produce a huge mass of population
but in reality, this does not happen.
• In this period, the growth rates slow down and the no. of microbial cell death
balances the no. of new cell and the population stabilizes. The metabolic
activities of individual cell surviving also slow at this stage.

69
DEATH PHASE / DECLINE PHASE:

• The no of deaths eventually exceeds the no. of new cells formed and the
population enters the death phase or logarithmic decline phase. This phase
continues until the population is diminished to a tiny fraction of the no. of
cells in the previous phase or the population dies out entirely. Many bacterial
cells often undergo involution during this phase, meaning that their
morphology changes dramatically and makes them difficult to identify.

70
LECTURE NO.13:

Replication / Multiplication of Viruses

DNA Viruses, RNA Viruses & Retroviruses

Multiplication of Animal Viruses:

• For replication, a virus needs live host cells but it must stop synthesis of host
proteins, so that viral genes are translated.
• Research indicates that viruses use several mechanisms to inhibit expression
of host cell genes. Early proteins translated from the viral genome may block
transcription, existing mRNA, or in progress translation.

Attachment:

• Animal viruses have attachment sites that attach to complementary receptor

sites on the host cell’s surface.
• The receptor sites of animal cells are proteins and glycoproteins of the
plasma membrane. Moreover, animal viruses don’t possess appendages like
the tail fibers of some bacteriophages.
• The attachment sites of animal viruses are distributed over the surface of the
virus, and the sites themselves vary from one group of viruses to another.
• In adenoviruses, which are icosahedral viruses, the attachment sites are small
fibers at the corners of the icosahedron.
• In many of the enveloped viruses, such as influenza virus, the attachment
sites are spikes located on the surface of the envelope. As soon as one spike
attaches to a host receptor, additional receptor sites on the same cell
migrate to the virus. Attachment is completed when many sites are bound

71
 • Receptor sites are proteins of the host cell. The proteins have normal
functions for the host and are hijacked by the virus.
• Understanding the nature of attachment can lead to the development of
drugs that prevent viral infections. Monoclonal antibodies that combine with
a virus’s attachment site or the cell's receptor site may soon be used to treat
some viral infections.

Entry:
Following attachment, entry occurs.

• Many viruses enter into eukaryotic cells by receptor-mediated endocytosis.

• A cell’s plasma membrane continuously folds inward to form vesicles.
These vesicles contain elements that originate outside the cell and are
brought into the interior of the cell to be digested.
• If a virion attaches to the plasma membrane of a potential host cell, the host
cell will enfold the virion and form a vesicle.
➢ Enveloped viruses can enter by an alternative method called fusion, in which
the viral envelope fuses with the plasma membrane and releases the capsid
into the cell’s cytoplasm.

72
Uncoating:

• Viruses disappear during the eclipse period of an infection because they are
taken apart inside the cell.
• Uncoating is the separation of the viral nucleic acid from its protein coat.
• This process varies with the type of virus. Some animal viruses accomplish
uncoating by the action of lysosomal enzymes of the host cell. These
enzymes degrade the proteins of the viral capsid.
• The uncoating of poxviruses is completed by a specific enzyme encoded by
the viral DNA and synthesized soon after infection.
• Uncoating of influenza virus occurs at the lower pH in a vesicle.
• Uncoating of togaviruses occurs at ribosomes in the host cytoplasm.

Biosynthesis of DNA Viruses:

• Generally, DNA-containing viruses replicate their DNA in the nucleus of the

host cell by using viral enzymes, and they synthesize their capsid and other
proteins in the cytoplasm by using host cell enzymes.
• Then the proteins migrate into the nucleus and are joined with the newly
synthesized DNA to form virions.
• These virions are transported along the endoplasmic reticulum to the host
cell’s membrane for release.
• Herpesviruses, Papovaviruses, Adenoviruses, and Hepadnaviruses all follow
this pattern of biosynthesis.
• Poxviruses are an exception because all of their components are synthesized
in the cytoplasm.

An example of multiplication of a DNA virus,the sequence of events in papovavirus

is under:

73
Biosynthesis of RNA Viruses:

• The multiplication of RNA viruses is essentially the same as that of DNA

viruses, except RNA viruses
multiply in the host cell’s cytoplasm.
• Several different mRNA formation
mechanisms occur among different
groups of RNA viruses.
• The major differences among the
multiplication processes lie in how
mRNA and viral RNA are produced.
• These viruses have RNA-dependent
RNA polymerase. This enzyme isn’t
encoded in any cell’s genome. Viral
genes cause the enzyme to be
made by a host cell. This enzyme
catalyzes the synthesis of another
strand of RNA, which is
complementary in base sequence
to the original infecting strand.
• Once viral RNA and viral proteins
are synthesized, maturation occurs
by similar means among all animal
viruses.

Biosynthesis of Retroviruses:

• These viruses carry reverse transcriptase, which uses the viral RNA as a
template to produce complementary double-stranded DNA.
• This enzyme also degrades the original viral RNA.
• The name retrovirus is derived from the first letters of reverse transcriptase.
• The viral DNA is then integrated into a host cell chromosome as a provirus.
• Unlike a prophage, the provirus never comes out of the chromosome. As a
provirus, HIV is protected from the host’s immune system and antiviral drugs.
• Sometimes the provirus simply remains in a latent state and replicates when
the DNA of the host cell replicates.
➢ In other cases, the provirus is expressed and produces new viruses, which
may infect adjacent cells. Mutagens such as gamma radiation can induce
expression of a provirus. In oncogenic retroviruses, the provirus can also
convert the host cell into a tumor cell.

74
Maturation & Release:

• The first step in viral maturation is the

assembly of the protein capsid; this
assembly is usually a spontaneous
process.
• The capsids of many animal viruses are
enclosed by an envelope consisting of
protein, lipid, and carbohydrate.
Examples of such viruses include
orthomyxoviruses and paramyxoviruses.
• The envelope protein is encoded by the
viral genes and is incorporated into the
plasma membrane of the host cell.
• The envelope lipid and carbohydrate
are encoded by host cell genes and are
present in the plasma membrane. The

75
 envelope actually develops around the capsid by a process called budding.
• After the sequence of attachment, entry, uncoating, and biosynthesis of viral
nucleic acid and protein, the assembled capsid containing nucleic acid pushes
through the plasma membrane. As a result, a portion of the plasma
membrane, now the envelope, adheres to the virus. This extrusion of a virus
from a host cell is one method of release. Budding doesn’t kill the host cell
immediately, and in some cases the host cell survives.
• Non-enveloped viruses are released through ruptures in the host cell plasma
membrane. In contrast to budding, this type of release usually results in the
death of the host cell immediately.

76
LECTURE NO.14:
Requirements for microbial growth
Microorganisms need both physical and chemical requirements for their growth.

PHYSICAL REQUIREMENTS
1. SURFACE

• Microorganisms does not grow in air, they need certain medium for
their growth. These medium may be liquid or solid.

• In liquid medium bacteria can grow at very rapid rate up to 1012-14 ,

and if the liquid media is mixed regularly or agitated the number can
reach up to 1016-18. This is because in liquid medium nutrients are
easily dissolved and readily available.

• In solid medium bacterial growth rate is slow as compared to liquid

medium however if the nutrients are provided is solid medium bacteria
can grow up to 106-8. Most commonly used solidifying agent is agar
agar.

3. TEMPERATURE

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On the basis of temperature, bacteria are divided into 3 classes:

PSYCHROPHILES (COLD LOVING):

These bacteria live in temperature range of -10 to 25 degree centigrade’s.

Psychrophillic microbes grow at -10 degree centigrade while other bacteria grow at 4
to 25 degree centigrade pschrotrophs. These include staphylococcus, streptococcus,
bacillus cereus and moulds etc. These microbes mostly cause the spoilage of food.
GLYCOLS.

MESOPHILES (MODERATE TEMPERATURE LOVING):

78
These microbes range from 25 to 40 degree centigrade. Most of these bacteria live at
37 degree centigrade. About 99 percent of disease causing bacteria are included in
this group. These bacteria do not cause as much spoilage of food as psychrophiles

THERMOPHILES ( HEAT LOVING):

These bacteria live at 100 degree centigrade or above 100 degree centigrade. The
bacteria living above 100 degree centigrade are called extreme thermophiles. These
are not pathogenic bacteria. These bacteria are found in volcanic, thermal springs,
geezers etc.The application of these bacteria and their products are in PCR TAQ
Polymerase is isolated from thermus aquaticus.

STEROLS

3.HUMIDITY& LIGHT:

79
Bacteria grow at humid environment of humidity 40-60 percent. If bacteria are grown
in dry environment they will start to die.

UV light is bad for growth of bacteria. UV light will change the structure of DNA and
cause mutation. UV light will make loops in DNA THYMIDINE DIMERS

Simple light is necessary for some bacteria i.e. photosynthetic bacteria which need
light to synthesize their food. Some bacteria are not affected by light but they behave
different in light and dark medium.

OSMOLARITY
ISOTONIC

➢ Microorganisms obtain almost all their nutrients in solution from the

surrounding water. Thus, they require water for growth, and their
composition is 80-90 percent water. If the bacteria are grown in hypertonic
environment the water inside the cell will start to move outside the cell and
bacterial cell shrink and will die.

80
 ➢ If the bacteria are grown in hypotonic environment the water will start to
move inside the cell and bacterial cell will swell and burst.

➢ Different bacteria live in different osmotic pressure. Some organisms,

called extreme halophiles, have adapted so well to high salt concentrations
that they actually require them for growth.

➢ Obligate halophiles are organisms from such saline waters as the Dead Sea
often require nearly 30 percent salt.

➢ Facultative halophiles, which do not require high salt concentrations but

are able to grow at salt concentrations up to 2 percent, a concentrating that
inhibits the growth of many organisms. A few species of facultative
halophiles can tolerate even 15 percent salt.

ACID AND BASE REQUIREMENTS

➢ Acid loving bacteria, Milk spoilage, Food spoilage, Sulphur loving bacteria,
extreme Acetinobacter, Lactobacillus, Fungus acid loving.

➢ 6.5-7.5 normal range of PH for most bacreia

➢ Base loving bacteria Disease causing bacteria

CHEMICAL REQUIREMENTS CHNOPS

CARBON

➢ Besides water, one of the most important requirements for microbial growth is
carbon. It is needed for all the organic compounds that make up a living cell.

➢ Half the dry weight of a typical bacterial cell is carbon.

➢ Chemoheterotrophs get most of their carbon from the source of their energy-
organic materials such as proteins, carbohydrates, and lipids.

81
 ➢ Photoautotrophs derive their carbon from carbon dioxide.

NITROGEN, SULPHUR AND PHOSPHOROUS

➢ In addition to carbon microorganisms need other elements to synthesize

cellular material. For example, protein synthesis requires considerable
amounts of nitrogen as well as some sulphur. The synthesis of DNA and RNA
also require nitrogen and some phosphorous as does the synthesis of ATP,
these molecules are important for the storage and transfer of chemical energy
within the cell.

➢ Organisms use nitrogen primarily to form the amino group of the amino acids
of proteins. Many bacteria meet this requirement by decomposing protein

82
 containing material. Some bacteria use nitrogen from ammonium ions NH4 +
and some bacteria use direct nitrogen from environment. This process is called
nitrogen fixation.

➢ Sulphur is used to synthesize sulphur containing amino acids and vitamins

such as thiamine and biotin. Important natural sources of sulphur include
sulphate ion SO4 -2 , hydrogen sulphide, and the sulphur containing amino
acids.

➢ Phosphorous is essential for the synthesis of nucleic acids and phospholipids

of cell membranes. A source of phosphorous is phosphate ion PO43-

TRACE ELEMENTS

➢ POTASSIUM, SODIUM, CHLORIDE, MAGNESIUM AND CALCIUM are

also elements that microorganisms require often as cofactor for enzymes.

➢ Microbes require very small amounts of other mineral elements such as iron,
copper, molybdenum and zinc. These are referred to as trace elements. Most
are essential for the functions of certain enzymes and usually act as cofactors.

83
OXYGEN

Oxygen is needed for the breakdown of food but some bacteria can live without
oxygen. However the energy extract is greater if the bacteria use oxygen. There are
some bacteria that live in oxygen environment and others without oxygen.

Toxic forms of oxygen are,

1. Singlet Oxygen 1O2- SOD

O2- + O2- + 2H+ → H2O2 + O2

2. Super oxide free Radical O2- SOD

3. H2O2 Catalase

2 H2O2 → 2H2O + O2

4. OH-

These forms of oxygen are present in the lysosomes of macrophage which engulf the
pathogen peroxidase like catalase but not O2

H2O2 + 2 H+ → 2H2O

84
CLASSIFICATION OF MICROBES ON THE BASIS OF OXYGEN
DEMAND:

85
86
LECTURE NO.15:
Appendages external to bacteria:

1. Glycocalyx:

Unique substance used in bacteria. It is of two types:

• Capsule
• Slime
• Capsule:
• Thin layer
• General term
• Slime:
• Thick layer

Capsule Slime

All bacteria (99% of bacteria) Streptococcus

Composition is Lipopolysaccharide

Lipopolysaccharide gives 2 characters:

If it is present, bacteria forms

mucoid/wet colonies.

If capsule is not present, bacteria forms

dull / dry / rough colonies.

Rough strain:

Strains that do no have capsule.

Bacillus has protenacious capsule and

always form dull colonies.

Functions of Glycocalyx:

1. Protection against macrophages/immune organs.

2. Pathogenicity (disease causing ability)

3. Acts as an antigen (K antigen/capsular antigen which is unique for every bacteria).

Typing is done on the basis of K-antigen.

87
4. Used as identifier

5. Colonization (due to gummy nature)

6. Bacteria can use it for its own nutrition.

7. Food source for bacteria itself.

Point:

➢ Capsular antigen causes more diseases as compared to non-capsular antigens.

88
LECTURE NO.16:
Vaccination:

Vaccination/vaccine is an idea or thought to enhance the immunity of an organism.

Types of vaccines:

1. Wild Type Vaccines

2. Attenuated/Weakened Vaccines

3. Killed Vaccines

4. Toxin based Vaccines/Anti-toxins/Toxoids

5. Recombinant Vaccines

1. Wild Type Vaccines:

• Replicate in cells
• Do not cause disease

When they replicate:

• Mammary cells are formed.

• Immune cells are formed.
• Antibodies activate.
• Interferons are involved.
• Cytotoxic reactions take place.
• Inflammation occurs.
➢ All of them provide solid immunity.

Example: NDV vaccine, IBDV vaccine, Polio vaccine.

2. Attenuated/Weakened Vaccines:

• They require extraordinary media.

For example:

➢ Rabies virus, TB virus.

If we give a long time passage or short time passage to Rabies virus (passes from one
media to another), it loses its pathogenicity & gets weakened or attenuated.

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3. Killed Vaccines:

• Antibodies are formed against receptors.

• Can be injected IV / IM / sub-cutaneous

For example:

• FMDV (Foot & month disease virus)

• Capsid remains outside. It acts on RNA & destroys it.
• HS ( Hemorrhagic septicemia)

Point: Measles & Mumps are preventable diseases.

4. Toxin based Vaccines/Anti-toxins/Toxoids:

• Virus & bacteria are not important. Their toxins are important.

Example: Tetanus.

5. Recombinant Vaccines:

It can involve:

• Subunits
• Whole organism
• DNA/RNA (Genomic Vaccines)

Point: Wild Type Vaccines & Attenuated / Weakened Vaccines are Live Vaccines.

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LECTURE NO.17:
Quiz # 01: What is the mode of action of Viral Vaccines?

Viral Vaccines are of different shapes:

1. Live Vaccine:

• Virus with genome & peplomers

• Causes infection.
• When causes infection, it replicates in nucleus or cytoplasm.

After replication, there are 3 methods by which live Vaccines work:

1. Antibodies formation

• Vaccines bind to future viruses.

• Cannot attach the host so cannot cause disease.

2. Interferons:

• Antibodies ask neighbor cells to produce Interferons

• Block the replication of viruses.

3. T-Helper Activation:

• Activates T-cytotoxic.
• Tc destroys the cell containing the virus.

2. Fragments / Sub-units Vaccines:

• Fragments go to APC.
• APC present fragments to B-cells, B- cells secrete plasma cells , plasma cells
release Antibodies.
• Recombinant subunits
• Most Vaccines are in this form.

3. Empty Capsid:

• Empty capsid works by one method i.e. production of Antibodies.

• Empty capsid is engulfed by APC (Macrophage), present it to B- cells , B-cells
will form plasma cells & plasma cells will form Antibodies.
• Replication is not necessary.

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Working of Antibodies:

Antibodies work by covering the virus so that it does not enter the host. It is the best
way of working.

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LECTURE NO.18:
Quiz#02: What are the various routes used in vaccines?

Live Vaccines:

1. Intra oral (e.g ND,IBD,Polio)

2. Intra Nasal (given because there can be some drug/chlorine in water or water is
heated)

3. Eye drops

Killed Vaccines:

1. Injection (IM/Sub-cutaneous)

2. Alum precipitate/Gel based Vaccine (Sub-cutaneous/does not work in IM)

3. Bone Marrow

4. DNA / RNA are injected (In future)

5. Intra-food (In future)

Point: Killed Vaccines will be Sub-cutaneous either empty capsid or fragments.

Quiz#03: What are the causes of success / failure of vaccines?

LIVE VACCINES:

Following are the causes of success/failure of live Vaccines:

1. Adulteration (Addition of drug/disinfectant)

2. Fake / Fraud (Fake vaccine)

3. Dilution (Addition of water)

4. Tempered vaccine (Physically tempered e.g exposed to sunlight/heat, seal broken)

5. Expired Vaccine

6. High temperature

7. Untrained Vaccinator

8. Wrong injection site / area

9. Time of vaccination

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10. Age of animal

11. Infection / Parasitic Load (If animal is sick / has parasites , vaccines will not work

Deworming should be done first)

12. Food (The one to be vaccinated should not be undernourished/should have

ample food)

13. Immune depressed (The one to be vaccinated should not be immune depressant
otherwise vaccine will not work.

1-4 ... Production / Vaccination faults

7-9 ... Faults of staff

10-13... Faults of animal

Killed Vaccine:

1. Dilution

2. Toxins

3. Proteolytic Enzymes

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LECTURE NO.19:
Quiz#04: Write a note on flagella?

• Flagella (Plural), Flagellum (Singular)

Life forms of bacteria having flagella:

1. Prokaryotic:

• Eubacteria (Flagellum)
• Archaebacteria (Archaellum)

2. Eukaryotic:

• Flagella of eukaryotic bacteria is belly dancer.

• Literal meaning of flagella is "whip"
• Always move counter-clockwise
• One protein ---- changes charge
• Clutch protein ----- works as clutch
• These rings are classically present in gram -ve bacteria.
• Flagella does not require ATP for movement. It requires protein motive force
(Na+,H+)
• M-ring is embedded in cell membrane.
• RPM reaches 1000.

Functions of Flagella:

1. Main function is movement.

2. Acts as antigen (Antigen H)

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3. Chemotactic Response

4. Help bacteria in causing disease.

• When it is to grow, a protein comes in hollow then attaches at tip.

• It grows form tip.
• In some bacteria, flagellin is 7.
• Most bacteria have 11 flagellin.

Mechanism of Movement:

• Run - Tumble run

• Flagella rotate (Bacteria move forward)
• In unfavorable conditions (Flagella stop , bacteria rotate backward)
• Thousand times more traversing than its body length.
• Efficient method but speed is slow.

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LECTURE NO.20:
Growth of Viruses:

• Viruses cannot be grown on artificial media (Agar/Broth)

• Viruses are thus grown in lab animals (Rats, pigs , ginny pigs , horses)
• 7-8 days embryonated eggs were used.
• For egg yolk (Thick & long syringe)
• After removing the syringe seal the egg with hot wax.
• For egg, incubate at 99-100°F for 3 days till 10th or 11th day.
• Before 11th day, (usually on 10th day, chilling is done)
• Through chilling all vessels of embryo shrink, this is important to prevent
embryo from bleeding. If bleeding takes place, antibodies will act against
them. Because of chilling, antigen-antibody reaction will not take place.
• Then cut the sac with the help of scissor.

Growth in Cells:

• Embryo cells
• Neonatal cells
• Foetal cells
• Stem cells
• Bone Marrow cells
• Grown in minimum essential
media (MEM)
• Baby Hamster kidney cell line.
• Tissue culture/cell culture (To
form artificial/lab meat)

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