MicrobiologyPharm D Notes For Spiral Binding

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Chapter 1. Introduction to the Science of microbiology. Major divisions

of microbial world and relationship among them.
History of Microbiology :-
Microbiology might have been associated with life since time immemorial and the discovery of
Mycobacterium tuberculosis DNA in 3000 years old Egyptian mummies supported this belief.
However the science of microbiology is not more than few hundred years old.
The existence of the microbial world was unknown until the invention of microscopes because
the microorganisms are too small to be seen clearly by the naked eye. With the help of a
relatively crude microscope , Robert Hooke in 1665 reported to the world that life’s smallest
structural units were little boxes or cells as he observed in a thin slice of cork. The existence
of microorganisms was actually proven in 1677 when Antoni Van Leeuwenhoek ( 1632- 1723)
in Holland saw them for the first time . He used a single lens microscope with magnification
approaching 300 times , and discovered a wholly new , previous invisible world. In his
letters published by royal Society in London, Leeuwenhoek described ‘ animalcules’ ( now
described as bacteria and protozoa), observed their motility and hence proved that they were
live.
Most of these letters were published in the proceedings of the Royal society and therefore became
quickly and widely disseminated. Although Leeuwenhoek is named as the Father of Microbiology
yet he kept his methods secret. It took another 150 years for others to have access to microbes until
the development of a compound( multi lens) microscope .
The spontaneous generation of life ( ie. That fully formed creatures like bees, frog and other
animals sprang (jump) from fertile mud , decaying carcasses , warm rain or fog and the like was
an obvious concept because organic matter generally decomposes faster outside the body and it
was assumed that the causative agents from maggots(larva) to microbes , were constantly arising
by spontaneous generation. The belief in the spontaneous formation of living beings from non-
living matter is known as the doctrine of spontaneous generation or abiogenesis , and has had a
long existence.
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An Italian physician , Franceesco Redi showed in 1665 that the maggots , which develop in
putrefying meat ( decomposing ) are the larval stages of flies and will never appear if the meat is
protected by placing it in a vessel closed with fine gauze so that flies are unable to deposit
their eggs on it. Redi destroyed the myth that maggots develop spontaneously from meat. He
successfully defeated the doctrine of abiogenesis through his several experiments, with respect
to the forms of life then known.
In 1775 Lavoisier discovered oxygen and the relation between air and life but this renewed the
controversy about abiogenesis. The problem was resolved by Spallanzani ( 1729- 1799) by
showing that an infusion of meat would remain clear indefinitely if boiled and properly sealed.
Spallanzani’s experiments were not accepted on the argument that boiling and sealing of meat
infusion ( complete absence or limited supply of oxygen) dragged the vegetative force and
prevented spontaneous generation. As a counter Schroeder & Von Dusch reproduced
Spallanzani’s experiment but replaced sealed glass with the cotton plug ( used even today to
exclude air borne contaminants . Thus the objection to Spallanzani’s experiment was no more
sustainable.
In 1858, the German Scientist Rudolf Virchow challenged the concept of biogenesis from
living cells . The controversy was not however settled because some investigators failed to
reproduce the alleged stability of dust free sterilized organic infusions. It was that crucial time
when Louis Pasteur emerged on the scene ( 1822-1895) . He showed that boiled medium could
remain clear in a swan neck flask, open to the air through a sinuous horizontal tube in which
dust particles would settle as air re-entered the cooling vessel.
Pasteur’s Swan necked flask used in experiments disproving spontaneous generation. Pasteur
also demonstrated that in the relatively dust free atmosphere of a quiet cellar or of a mountain
top , sealed flasks could be opened and then resealed with a good chance of escaping
contamination.
Pasteur’s experiments were a public sensation and gave a mortal blow to the doctrine of
spontaneous generation. Unfortunately Pasteur’s work was also not considered decisive
because the accusations of technical in competence did not really explain why his opponents ‘
boiled infusions’ stubbornly refused to remain clear. The vital difference was in fact , due to
their infusion of hay , against yeast extract used by Pasteur. John Tyndall ( Also known for
Tyndall effect ) conducted the real decisive experiments who became involved in this
biological problem because of his interest in atmospheric dust . After bringing a bale of hay

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into his laboratory Tyndall could no longer achieve sterility in the same room by boiling . He
showed that the hay had contaminated his laboratory with an incredible kind of living
organism, which could survive boiling . Fortunately in the same year (1877) Ferdinand Cohn
demonstrated the resistant forms as small refractive spores, A special stage in the life cycle of
the hay bacillus. Tyndall also developed the ingenious method of sterilization ,now often called
Tyndallisation.
With the development of adequate microscopes in 1830’s several investigators concluded that
the microscopic globules found in the sediment of wine were alive and that their metabolic
activities caused the alcoholic fermentation. Yeast being non motile , it was doubted that they
were live. Liebig the Father of biochemistry , considered fermentation as a purely chemical
process attributable to a self –perpetuating instability of the grape juice up on exposure to air.
He also insisted that yeasts were a by product of the fermentation. Mean while Pasteur
developed interest in fermentation through his discovery of optical isomerism as a property of
certain fermentation products. Eg. Tartaric acid. In 1857 Pasture showed that alcoholic
fermentation was due to smaller rods ( lacto bacillus). Though his experiments focusing
microbial metabolism Pasteur showed that life is possible without air. Thus Pasteur eventually
overturned Liebig’s authority.
Based on his experiments in fermentation Pasteur made a most fruitful suggestion that specific
microbes might also be causes of specific diseases in man. He also developed the procedure of
gentle heating ( Pasteurization) to prevent the spoilage of beer& wine by contaminating
microbes. This process of vital importance to dairy industry is now employed to prevent milk
borne diseases of man.
Pasteur made another valuable contribution to microbiology by developing an attenuated

bacterial vaccine against Anthrax by growing the organism at elevated temperature.
Investigators those days were confronted with yet another controversy. Because they were
dealing with mixed cultures, transfer of the same culture to different media yielded different
appearance ( pleomorphism). Pasteur’s demonstration of microbial specificity established the
victory of monomorphism, which emphasizes the fixed properties of each organism .
After Pasteur microbiology remained for most of the Century, largely an applied and
descriptive field dominated by medical microbiology.
Germ theory of disease – Infection is a measure class of diseases. The control over microbial
diseases and over microbial pollution in the environment can be reckoned as the greatest
achievement of medical science one constraint was realized because many communicable
diseases are not contagious ( spread by contact) but transmitted indirectly by air , water, food or
insects. Leading physicians such as William Harvey, Hippocrates and Galen ascribed epidemics
to miasma ie. Poisonous vapours created by the influence of planetary conjunctions or by
disturbances arising within the earth. Germ theory of disease developed slowly. John Hunter, a
renowned surgeon in 18th century boldly inoculated himself with pus from a gonorrhoea patient
and acquired syphilis. Villemin transmitted tuberculosis from humans to guinea pigs in 1865. In
direct transmission was recognised in 1804s, when Semmelweis in Vienna & Oliver Wendell
Homes in Boston blamed obstetricians moving with unwashed hands from one patient to another
for the prevalence of puerperal sepsis in hospitals. The insulted physicians obviously refuted
those pioneers. In 1769 Jenner introduced vaccination against small pox with material from
lesions of a similar , milder disease of Cattle. John Snow was successful in terminating a

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localised epidemic of cholera in London in 1854 by closing the source of contaminated water
for the neighbourhood. In 1860s John Lister introduced antiseptic surgery by demonstrating the
value of phenol as a disinfectant , as well as of cleanliness in preventing serious wound
infections.
The first ecological agent to be recognised was fungi as it was larger than bacteria . Bassi in
1836 attributes a fungus for a disease of silkworms and Schonlein in 1839 blamed another
fungus for a human skin disease . In 1850 Davaine saw rod shaped bodies in the blood of
sheep with anthrax . He later transmitted the disease by inoculating as little as 106 bacilli per
ml of blood . However it was Koch who provided unequivocal proof that microbes were the
real cause , by infecting animals with organisms isolated in pure cultures . Lister & Koch
pioneered the pure culture technique . Koch perfected the techniques of identification that are
used today including the use of solid media , on which individual cells give rise to separate
colonies and the use of stains. Koch’s genius methods led to the identification of tubercle
bacillus in 1882. He then went on to formulate the postulates, popularly known as Koch’s
postulate s for distinguishing a pathogenic from an adventitious microbe.
1) The organism is regularly found in the lesions of the disease.

2) It can be isolated in pure culture on artificial media.
3) Inoculation of this culture produces a similar disease in experimental animals .
4) The organism can be recovered from the lesions in these animals.
Koch’s methodology opens the gates of the ‘golden era’ of medical microbiology. Many
important pathogens . eg. The cholera vibrio, Staphylo coccus , Strepto coccus, meningo
coccus, Gonococcus and tetanus bacilli, were further identified between 1879 and 1889 by
many German scientists.
The first virus to be recognised as filterable & was a plant pathogen , Tobacco Mosaic virus(
TMV) discovered independently by Ivanowski in 1892 in Russia & by Beijernick in 1899 in
Holland.
Loffler & Frosch demonstrated first filterable animal virus for foot and mouth disease of
Cattle in 1898.
In 1900 the U.S. Army Commission under Walter Reed demonstrated first filterable
human virus for Yellow fever.
During 1916 to 1917 bacteriophage (Virus that infect bacteria) were discovered by Twort
in England & by d’Herelle in France.
The period from 1857-1914 is considered as the golden age of microbiology because most
significant advances made during this period lead to the establishment of microbiology as a
science.
Paul Ehrlich a German physician popularly known for his concept of “ magic bullet”( a
bullet that could hunt down& destroy a pathogen with out harming the infected host),
discovered Salvarsan in 1910. This chemotherapeutic agent an arsenic derivative
effective against syphilis , dates next to quinine, which had been known in Europe as an
extract from the bark of a south American tree. In 1928 Alexander Fleming discovered
Penicillin , an Antibiotic produced by a fungus . Many synthetic drugs including Sulphas

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were discovered in late 1930s. Two more antibiotics Gramicidin & Tyrocidin were
discovered in 1939 by Rudolf Dubos. A large number of chemotherapeutic agents
have been discovered since then. The growing problem of drug resistance is a major
challenge before the scientists of today.
Introduction to Microbiology.
Microbiology is the study of living organisms of microscopic size, which include bacteria, fungi,
algae, protozoa and the infectious agents at the borderline of life that are called viruses. It is
concerned with their form, structure, reproduction, physiology, metabolism and classification. It
includes the study of their distribution in nature, their relationship to each other and to other
living organisms, their effects on human beings and on other animals and plants, their abilities to
make physical and chemical changes in our environment, and their reactions to physical and
chemical agents.
Microorganisms are closely associated with the health and welfare of human beings. Some
microorganisms are beneficial and others are detrimental (Harm or damage). For eg.
Microorganisms are involved in the making of yogurt, cheese and wine, in the production of
Penicillin, interferon, ( Class of glycoprotein produced by the body in response to a viral
infection, which inhibit the multiplication of viruses in protected cells and are generally non
specific in their action) alcohol and in the processing of domestic and industrial wastes.
Microorganisms can cause disease, spoil food and deteriorate materials like iron pipes, glass
lenses and wood pilings.
Most microorganisms are unicellular. In unicellular organisms all the life processes are
performed by a single cell. In the higher forms of life organisms are composed of many cells that
are arranged in tissues and organs to perform specific functions . Regardless of the complexity
of an organism, the cell is the basic structural unit of life. The word cell was first used by Robert
Hooke (1635- 1703), in his descriptions (1665) of the fine structure of cork and other plant
materials.
In 1835 Schleiden and Schwann put forward the cell theory stating that plant or animal body is
ultimately made up of minute cells and concluded that the “ Cell is the structural unit of
life”.
Protoplasm ( Greek proto “ first” plasm, “formed substance” introduced to characterize the
living material of a cell is a colloidal organic complex consisting largely of protein, lipids and
nucleic acids. These substances are enclosed by membranes or cell wall and the protoplasm
always contains nuclei or an equivalent nuclear substance. Developments in electron microscope
techniques have mad\e it possible to reveal the complex intracellular organisation.
All biological systems have the following characteristics in common.
1. The ability to reproduce. 2. The ability to ingest or assimilate food substances and metabolise
them for energy and growth. 3. The ability to excrete waste products. 4. The ability to react to
changes in their environment – sometimes called irritability and 5. Susceptibility to mutation
(Any change that alters the sequence of bases in DNA, changing the genetic material). In the
study of microbiology we encounter “organisms” which may represent the border line of life.
They are viruses, which are simpler in structure and composition than single cells. Viruses are
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obligate ( bind to do) parasites , that is , they are obligated to grow with in an appropriate host
cell – plant , animal or microbe. They cannot multiply outside a host cell. However when a
virus enters an appropriate living cell, it is able to direct the synthesis of hundreds of identical
viruses, using the cell’s energy and biochemical machinery. A virus is made up of substances
unique to life ( nucleic acids- ie chemicals that make up genetic material) and proteins
( complex nitrogenous substances found in various forms in animals and plants).
Microorganisms are exceptionally attractive models for studying fundamental life processes.
They can be grown conveniently (suitably) in test tubes or flasks, thus requiring less space and
maintenance than larger plants and animals. They grow rapidly and reproduce at an unusually
high rate; some species of bacteria undergo almost 100 generations in a 24 hr period. The
metabolic processes of microorganisms follow patterns that occur among higher plants and
animals. For eg. Yeasts ( fungus) utilize glucose in essentially the same manner as cells of
mammalian tissue , the same system of enzymes is present in these diverse organisms. The
energy liberated during the break down of glucose is trapped and made available for the work
to be performed by the cells , whether , they be bacteria , yeast, protozoa. The mechanism by
which organisms (or their cells ) utilize energy is fundamentally the same throughout the
biological world. Plants are characterized by their ability to use radiant energy, whereas animals
require chemical substances as their fuel. In this respect some microorganisms are like plants,
others like animals and some have the unique ability of using either radiant energy or chemical
energy and thus are like both plants and animals. Furthermore some microorganisms , the
bacteria in particular , are able to utilize a great variety of chemical substances as their energy
source – ranging from simple inorganic substances to complex organic substances . In
microbiology we can study organisms in great detail and observe their life processes while they
are actively metabolizing, growing reproducing, aging and dying. By modifying their
environment we can alter metabolic activities , regulate growth , even change some details of
their genetic pattern all without destroying the organisms. For eg. Bacteriophages which are
viruses that infect and reproduce in bacteria , demonstrate the complete sequence of host
parasite reactions and provide a model by which virus – host cell reactions can be postulated for
infections in higher plants and animals.
Microorganisms have wider range of physiological and biochemical potentialities than all other
organisms combined. eg. Some bacteria are able to utilize atmospheric nitrogen for the
synthesis of proteins and other complex organic nitrogenous compounds, other species require
inorganic or organic nitrogen compounds as the initial building blocks for their nitrogenous
constituents . Some microorganisms synthesis all their vitamins, while others need to be
furnished vitamins. By reviewing the nutritional requirements of a large collection of
microorganisms, it is possible to arrange them from those with the simplest to those with the
most complex requirements.
There is no field of human endeavour ( attempt) , whether it be in Industry or agriculture , or in

the preparation of food or in connection with problems of shelter or clothing , or in the
conservation of human or animal health and combating of disease , where the microbe does not
play an important and often dominant role .
Waksman ( 1952) was awarded the Nobel prize in physiology or Medicine for the part he
played in the discovery of the antibiotic Streptomycin , which is produced by a soil bacteria.
In Biology as in any other field , classification means the orderly arrangement of units
under study into groups of larger units. Present day classification in biology was established
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by the work of Carolus Linnaeus ( 1707- 1778), a Swedish botanist . His books on the
classification of plants and animals are considered to be the beginning of modern botanical
and zoological nomenclature
, a system of naming plants and animals .
Until the 18th century, the classification of living organisms placed all organisms into one of two
kingdoms, plants and animals. In microbiology we study some organisms that are
predominantly plant like , others that are animal like and some that share characteristics
common to both plants and animals. E.H. Haeckel (1866) a German zoologist suggested a third
kingdom Protista that include unicellular microorganisms that are typically neither plants nor
animals .The Protista, include bacteria, algae, fungi and protozoa. Viruses are not cellular
organisms and therefore are not classified as Protista. Bacteria are referred to as lower protists ,
the other algae , fungi and protozoa are called higher protists.
Haeckel’s kingdom Protista left some questions unanswered. for eg. What criteria could be used
to distinguish a bacterium from yeast or certain microscopic algae. Satisfactory criteria were
unavailable until late in the 1940s when more definitive observation of Internal cell structure was
made possible with the aid of the powerful magnification provided by electron microscopy. It
was discovered that in some cells, for eg. The nuclear substance was not enclosed by a nuclear
membrane. In other cells, such as typical algae and fungi,
ound structures in all others ( fungi, algae and protozoa ) was a discovery of fundamental
significance .( Protozoa- are referred to as animal like protists, they are motile. Eg. Amoeba)
1. Enlist the differences between prokaryotic and eukaryotic organisms. Give examples for
each
. 5 marks.Repeated.
Examples for eukaryotes –Fungi, protozoa, plants and animals.
Examples for procaryotics- Bacteria and cyanobacteria (blue green algae).

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Features distinguishing Prokaryotic from Eukaryotic cells.

Feature Prokaryotic Eukaryotic cells
Group where found as unit of Bacteria Algae, fungi, protozoa, plants
structure. and animals.
Size range of organism 1-2 by 1-4 μm or less. Greater than 5 μm in width or
diameter.
enetic system location Nucleoid (RNA surrounded by Nucleus (It is membrane
a protein shell), chromatin body enclosed organelle found in
( nucleoprotein in the eukaryotic cells . It contains
interphase nucleus where the most of the cell’s genetic
chromosomes are uncoiled) or material), mitochondria
Nuclear material. (cellular power houses because
they generate most of the cell’s
supply of ATP used as a source
of chemical energy) ,
Chloroplasts( Their main roles
is conducting photosynthesis.
Where chloroplasts capture the
energy from sunlight & store it
in the energy storage
molecules ATP).
Structure of Nucleus Not bounded by nuclear Bounded by nuclear membrane,
membrane, one circular more than one chromosome.
chromosome (Chromosome is
an organised structure of DNA
, protein & RNA found in cells)
.
Chromosomes does not contain Chromosomes have histones
histones , no mitotic division. (histones are highly alkaline
proteins found in eukaryotic cell
that package & order the DNA
into structural units called
nucleosomes), mitotic nuclear
division ( Mitosis is the process
by which a cell has previously
replicated each of its
chromosomes , separates the
chromosomes in its cell nucleus
into 2 identical sets of
chromosomes).
Nucleolus absent functionally Nucleolus present.( Round
related genes may be clustered. structure located in the nucleus
of eukaryotic cells that is
involved in ribosomal RNA
synthesis and ribosome
formation)

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(Ribosome- The site of protein

synthesis in a cell, composed of
RNA and protein)
Cytoplasmic nature and Absent Absent

Structures
Gas vacuoles (freely permeable Can be present Absent
to gas present in bacterial cells.)
Mesosomes (folds in the
plasma membrane, they have Present Absent
functions like DNA replication
& cell division, Mesosomes are
now considered as not true cell
structures)
Ribosomes 70S’ distributed in the 80 S’ arrayed on membrane as in

cytoplasm. endoplasmic reticulum.
Mitochondria Absent Present
Chloroplasts , Absent May be present.
Golgi structures, Absent Present
Endoplasmic reticulum Absent Present
Membrane bound vacuoles. Absent Present
(Vacuoles with tonoplast as
the membrane which is found in
plant cells).
Outer cell structures Generally do not contain Sterols present, do not carry
Cytoplasmic membrane. sterols, contain part of out respiration and
respiratory and in some photosynthesis.
photosynthetic machinery .
Cell wall Peptidoglycan (murein or Absence of Peptidoglycan.
mucopeptide) as component.
Locomotor organelles. Simple fibril. Multi fibrillated with “ 9+2”
microtubules.
Pili Present Absent
Fimbriae Present Cilia
Storage compounds Poly β hydroxyl butyrate often Poly β hydroxyl butyrate
present. absent.
S’ refers to the sedimentation coefficient of a particle in the ultra centrifuge.
Bacteria are prokaryotic microorganisms . The Eucaryotic microorganisms include the

protozoa , fungi and algae. (Plant and animal cells are Eucaryotic).
A more recent and comprehensive system of classification ‘A’ more recent and comprehensive
system of classification , the five kingdom system was proposed by R. H. Whittaker (1969), this

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system of classification is based on three levels of cellular organization which evolved to

accumulate three principal modes of nutrition , photosynthesis, absorption and ingestion (The
process by which food is taken in to the alimentary canal , it involves chewing & swallowing).
The procaryotes are included in the kingdom Monera, they lack the ingestive mode of nutrition .
Unicellular eukaryotic microorganisms are placed in the kingdom Protista.
The nutritional mode of microalgae is photosynthetic , the mode of nutrition of the protozoa is
ingestive and the mode of nutrition in some other protists is absorptive, with some overlap to the
photosynthetic and ingestive models . The multicellular and multinucleate eukaryotic
organisms are found in the kingdoms.
Plantae ( multicellular green plants and higher algae ), Animalia ( multicellular animals) and
Fungi
( multinucleate higher fungi). Their diversified nutritional modes lead to a more diversified
cellular organization . Microorganisms are found in three of the five kingdoms. Monera
( bacteria and cyano bacteria ), Protista ( microalgae and protozoa), and Fungi ( Yeasts and
molds).
1.Contribution of Louis Pasteur.2 marks.?Repeated.

Louis Pasteur was a French chemist & microbiologist who is well known for his discoveries of
the principles of vaccination, microbial fermentation & pasteurization. He is remembered for his
remarkable breakthrough in the causes & prevention of diseases , and his discoveries have saved
count less lives ever since. Pasteur reduced morality from puerperal fever & created the first
vaccines for Rabies & Anthrax. Pasteur is best known for the general public for his invention in
the technique of treating milk & wine to stop bacterial contamination, a process now called
pasteurization .

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2.Koch’s postulates?. 2 marks.Repeated.

a. The organism is regularly found in the lesions of the disease.
b. It can be isolated in pure culture on artificial media.
c. Inoculation of this culture produces a similar disease in experimental animals .
d. The organism can be recovered from the lesions in these animals.
Koch’s methodology opens the gates of the ‘golden era’ of medical microbiology. Many
important pathogens . eg. The cholera vibrio, Staphylo coccus , Strepto coccus, meningo coccus,
Gonococcus and tetanus bacilli, were further identified between 1879 and 1889 by many
German scientists.
3..Alexander Flemming?. 2 marks.Sir Alexander Flemming was a Scottish biologist ,
pharmacologist & botanist. His best known discoveries are the enzyme lysozyme in 1923 & the
antibiotic substance Penicillin from the mould Penicillium notatum in 1928. He investigated it’s
anti bacterial effect on many organisms and noticed that it affect bacteria such as Staphylo cocci &
many other gram+ ve pathogens that cause scarlet fever, pneumonia, meningitis & Diphtheria . It
also affected Neisseria gonorrhoea which cause Gonorrhoea.

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Chapter 2. Different methods of Classification of microbes and study of

bacteria, Fungi, Virus, Rickettsiae, Spirochetes etc. :-
Bergey’s manual of systemic Bacteriology places all bacteria in the kingdom Procaryotae
which in turn is divided into 4 divisions as follows .
Division 1- Gracilicutes.
Procaryotes with a complex cell wall structure characteristic of Gram –ve Bacteria.
Division 2- Firmicutes.
Procryotes with a cell wall structure characteristic of Gram + ve bacteria.
Division 3 – Tenericutes.
Prokaryotes that lack a cell wall .
Division 4- Mendosicutes.
Procaryotes that show evidence of earlier phylogenetic origin than those bacteria included in
Division 1 &2.
Bergey’s manual is the international standard for bacterial taxonomy.
Morphologically three different bacteria are recognised .
1. Spherical.
2. Cylindrical or rod shaped.
3. Spiral.
1. Spherical( Cocci, singular – coccus):- These are the smallest bacteria, more or less
spherical in shape . I
Micrococcus :- when they occur singly eg. Micro coccus .
Diplococci:- when predominantly in pairs. A coccus is divided in one plane in to two

cocci that remain attached in pairs.
Strepto cocci:- ( Greek – streptos, twisted). When they live in chains.

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Staphylo cocci:- When they are found in a group .
2. Cylindrical or rod shaped :- Bacillus, singular – bacilli, Bacillus meaning – stick.
Bacillus:- When only one rod shaped structure represents the bacterium.
Single bacillus.
Diplo bacillus :- When they occur in pairs.
Strepto bacillus:- These are found in
chains.
1. Spiral Bacteria:- Spiral bacteria have one or more twists , they are never straight.
Vibrio or comma:- They are comma (,) shaped which cause
chlorea. Spirilla:- These are longer rigid rods with several
curves or coils.
Spirochetes:- Unlike the spirilla, which use flagella to move,
spirochetes move by means of an axial filament , which is
contained under an external flexible sheath.

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Structure of Bacterial cell:-
Staining :- The cytoplasm of bacteria lacks colour & therefore staining techniques greatly
facilitate the study of microorganisms by imparting them colour. Any stain is applied over a
smear
. For making the smear, first the glass slides & cover slips are thoroughly cleaned with hot
soap water to render them grease free. The slide is passed through the burner’s flame several
times. A drop of water is put on the slide& rubbed with a small bit of solid culture. A uniform
thin, roughly circular smear is prepared , air dried & fixed by passing several times over the
burner at a temp. tolerable to human body. The purpose is to fix the bacteria by killing them
without sacrificing any of the internal or external structures, except motility which is obviously
lost.
Simple staining:- In simple staining only one dye is used. A smear of the sample is fixed on a
clear glass slide & any simple aniline dye solution is applied by flooding the smear with it for
about a minute. Then it is washed off with gentle stream of cool water dried by pressing
between two pieces of filter paper& finally examined under a microscope . Bacteria generally
take up basic dye such as methylene blue, crystal violet, safrarin, Eosin Y & basic fuchsin. This
is because most bacteria react towards stains as though they are composed of acidic
components; nucleia acid, acidic polysaccharides , proteins etc.
Gram’s staining :-
Developed by Christian Gram in 1884 in Denmark, Gram staining is one of the most
fundamental & most widely used technique for the differentiation & identification of bacteria .
It is not only reveals the shape & size of the bacteria but also enables them to be
differentiated immediately into two categories . Gram +ve & gram-ve hence it is also known
as differential staining.
In this procedure the cells are stained with a basic dye (crystal violet), treated with an iodine –
potassium iodide mixture to fix the stain, washed with alcohol or acetone (decolorizer) , &
counter stained with a polar dye of a different colour ( eg. Safranin).
All bacteria take up the initial blue- purple stain but only gram+ve ones retain it during the
subsequent steps. Gram –ve bacteria takes up the counter stain & appear as red objects in
contrast with violet cells of gram-ve bacteria.
The mechanism of gram staining is related to the properties of cell wall. The gram +ve
organisms have a much thicker cell wall, which retains the dye iodine complex by preventing it’s
elution. In ageing cultures the cell wall deteriorates & hence gram+ ve cells often become gram
–ve. It is believed that crystal violet & iodine form a chemical complex in the bacterial
cytoplasm. Since gram-ve bacteria have a high lipid content in their cell walls, alcohol

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dissolves the lipid & permits crystal violet iodine complex to leak out the cytoplasm . Gram
+ve bacteria trap less of the complex because of their considerably less peptidoglycan
content.
Pharmaceutically important principal bacterial groups according to the organisation of

Bergey’s Manual.
1. Spirochetes: Leeuwenhoek described Spirochetes from saliva & tooth scrapings as one of the
1st Microorganisms as early as in 1600s. These bacteria are Gram(-)ve , typically coiled &
actively motile by means of two or more axial filaments (thread like structure). They exhibit
helical morphology & constitute a number of important pathogenic bacteria. Eg. Treponema
pallidum , the cause of Syphillis , members of the genus Borrelia & Leptospira.
2. Aerobic/ Microaerophilic (describing microorganism that grow best at very low oxygen
concentration), motile, helical, vibroid Gram (-) ve bacteria.
These gram –ve bacteria have helical morphology & motility is due to flagella & no axial
filaments are present unlike spirochetes. Most spiral bacteria are harm less aquatic organisms but
some are pathogenic. Eg. Helicobacter pylori causes ulcers in humans , campylo bacter jejuni
causes outbreaks of food borne intestinal diseases.
3. Gram(-) ve aerobic rods and cocci:- This group includes Gram –ve bacteria of medical ,
industrial & environmental importance.eg. Pseudomonas aeruginosa is pharmaceutically
important. It can infect the urinary tract, burns & wounds & cause septicaemia ( ie. Wide spread
destruction of tissues due to absorption of disease causing bacteria or their toxins from the
blood stream. The term is also used loosely for any form of blood poisoning. Pseudomonas are
able to grow on minute traces of unusual carbon sources , such as soap residues or cap linear
adhesives found in a solution.
They are resistant to most antibiotics and can grow in some antiseptic solutions eg. Quarternary
ammonium compounds . These features of Pseudomonas are of great concern in pharmaceutical
industry & hospitals. The bacteria in this group also fall in some other genera including
Neisseria gonorrhoeae( Gonorrhea- venereal disease caused by the bacterium Neisseria
gonorrhoeae that affects the genital mucous membrane of either sex. Symptoms develop about a
week after infection & include pain on passing urine).
Brucella ( Cause of brucellosis)- Malta fever, Mediterranean fever – a chronic disease of farm
animals caused by bacteria of the genus Brucella, which can be transmitted to man either by
contact with an infected animal or by drinking non-pasteurized contaminated milk. Symptoms
include headache, sickness, loss of appetite & weakness, progressing to chronic fever & swelling
of lymph nodes.
4. Facultative anaerobic Gram- ve rods:-
Facultative an organism that is not restricted to one way of life. A Facultative parasite can live
either as a parasite or in different conditions , as a non-parasite able to survive without a host.
Enterobacteriaceae- Also known as enterics these bacteria inhabit the intestinal tracts of human
beings & animals. Eg. Escherichia, Salmonella, Shigella, Klebsiella, Serratia, Proteus, Entero
bacter. All enteric bacteria produce proteins called bacteriocins which cause the lysis of closely
related species of bacteria.

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Vibrionaceae- Mostly found in aquatic habitats , this is a family of Gram –ve Facultative
anaerobic rods, many of which are curved, vibrio ( eg. Vibrio cholera) the cause of cholera is
the most important genus in the family.
Pasteurellaceae- This family includes Pasteurella & hemophilus. The genus Pasteurella is also
known as a pathogen of domestic animals causing septicaemia in cattle, fowl cholera in chickens
& pneumonia in several types of animals. Hemophilus is responsible for several important
diseases . The organisms commonly inhabit the mucuos membrane of the upper respiratory
tract , mouth, vagina & intestinal tract.
5. Anaerobic , Gram(-ve) , straight , curved & helical rods.
Bacteroids are a group of non-endospore forming (endospore- the resting stage of certain
bacteria particularly species of the genera Bacillus & clostridium. In adverse conditions the
nucleus & cytoplasm with in the normal vegetative stage of the bacterium can become
enclosed with in a tough protective coat, allowing the cell to survive. On return of favourable
conditions the spore changes back to the vegetative form), non-motile organism which live in
the human intestinal tract but some also reside in anaerobic habitats such as gingival crevices (
gingival - the gum , the layer of dense connective tissue & overlying mucous membrane that
covers the alveolar bone & necks of the teeth & extends a short distance into each socket). The
microbes of another genus , Fusobacterium are long & slender, with pointed ends, most often
found in the gingival crevice & may be responsible for many dental abscesses ( pus
formation)).
6. Dissimulatory Sulpahte/ Sulphur reducing Bacteria- These bacteria are found in

anaerobic muds & sediments as well as in the intestinal tracts of humans & animals. (Anaerobic
– any organisms especially a microbe , that is able to live & grow in the absence of free
oxygen).
As obligately (describing an organism that is restricted to one particular way of life eg. parasite
cannot exist without a host) anaerobic organisms they use oxidised forms of sulphur (Sulphates)
rather than oxygen as electron acceptors. Their means of energy production are physiologically
(the science of the functioning of living organism & their component parts) unique &
ecologically ( the study of the relationship between man, plants & animals & the environment)
crucial. Desulphovibrio is the best known genus.
7. Anaerobic gram-ve Cocci:- They are non-motile, non-endospore forming anaerobic

organisms occurring typically in pairs, singly in chains or clusters. Members of the genus
Veillonella occur as part of the normal flora of the mouth & are components of dental plaque ( a
raised circular patch of skin or mucous membrane resulting from local damage , usually due to
infection).
Rickettsias & Chlamydias:- both can reproduce only within a host cell & thus resemble viruses
in being obligate intracellular parasites . But due to their morphological & biochemical aspects
they resemble bacteria & are therefore classified as such.
Rickettsias are gram –ve 0.8 to 2.0μm, rod shaped or Cocco bacilli, divide by binary fission (a
method of asexual reproduction in which the body of the protozoan or bacterium splits into
equal parts). The resulting products of the fission grow into complete organisms , usually
cultivated in the yolk sac of chicken embryos , transmitted to humans by insects & ticks are

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responsible for a number of diseases known as the spotted fever group.
Rocky Mountain spotted fever:- caused by R. rickettsia are transmitted by ticks ( a blood sucking
parasite belonging to the order of arthropods that also include the mites). Ticks bites can cause
serious skin lesions ( a zone of tissue with impaired functions as a result of damage by disease or
wounding. Apart from direct physical injury, egs. of primary lesions include abscesses, ulcers &
tumours) & occasionally paralysis.
Endemic murine typhus :-(Endemic occurring frequently in a particular region or population

applied to diseases that are generally or constantly found among people in a particular area.
Typhus- characterized by a severe headache , widespread rash, prolonged high fever).
Endemic murine typhus caused by R. Typhi & transmitted by rat fleas & epidemic typhus caused
by Rickettsia prowazekki & transmitted by lice, together constitute spotted fever group .
Chlamydias are gram (-) ve Coccoid bacteria ( 0.2- 1.5μm), transmitted to humans by
interpersonal contact or by air borne respiratory routes- do not require insects or ticks for their
transmission . There are only three species of Chlamydias – chlamydia – psittaci is the
causative agent for psittacosis (ie . parrot disease – an infectious disease of parrot , it can be
transmitted to man & cause headache, bleeding from nose, shivering, fever & complications
involving the lungs).C. pneumonia is the cause of mild form of pneumonia especially present
in young adults.
( Pneumonia- Inflammation of the lung caused by bacteria , in which the air sacs ( alveoli) fill
up with pus so that air is excluded & the lung becomes solid).
C. trachomatis is the causative agent of trachoma & seems to be the primary causative agent of
nongonococcal urethritis.
(Trachoma- a chronic contagious eye disease . The conjunctiva of the eye lids becomes inflamed
leading to discharge of pus.
Conjunctiva- the delicate mucous membrane that covers the front of the eye lines & inside of the
eyelids.
Urethritis – Inflammation of the Urethra. Urethra- The tube that conducts urine from the bladder
to the exterior .
8. Mycoplasmas:- Most species of mycoplasma are aerobes or facultative anaerobes , don’t

form cell wall hence highly pleomorphic ( the condition in which an individual assume a number
of different forms during it’s life cycle eg. Malarial parasite) produce filaments that resemble
fungi, very small ( 0.1- 0.25μm), can be grown on artificial media providing sterols & other
nutritional or physical requirements. M. pneumonia is the most important human pathogen
among mycoplasma & is the causative organism of typical pneumonia.
9.Gram + Cocci- These are the members of the genera Staphylococcus & Strepto coccus.
Staphylococci occur typically in garge like clusters& are facultative anaerobics. Strepto cocci
typically appear in chains , do not use oxygen , a few may be obligately anaerobic & are
primarily pathogenic.
11. Endospore forming Gram+ ve rods & cocci:-

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These are gram+ve microbes which could be strict aerobes, facultative anaerobes, obligate
anaerobes, Bacillus ( eg. B. anthracis causing anthrax). Anthrax – An acute infections disease
of farm animals caused by the bacterium Bacillus anthracis, which can be transmitted to man
by contact with animal hair, hide or excrement. In man the disease attacks either the lungs ,
causing pneumonia or the skin , producing severe ulceration and clostridium ( eg. Cl. Tetani
causing tetanus). Ulcer- a break in the skin or in the mucous membrane. Tetanus – an acute
infectious disease , affecting the nervous system caused by the bacterium clostridium tetani.
Infections occurs by contamination of the wounds bacterial spores. Bacteria multiply at the site
of infection & produce a toxin that irritates nerves so that they cause spasmodic contraction
of muscles. Cl.botulism causing botulism ( a serious form of food poisoning from foods
containing the toxin produced by the bacterium clostridium botulism. The toxin selectively
affects the central nervous system, in fatal cases, death is often caused by heart & lung failure
resulting from a malfunction of the cardiac & respiratory centres of the brain.
Clostridium perfringens, clostridium welchii causing gas gangrene are the two important rod
shaped genera ( gas gangrene – death & decay of wound tissue infected by the soil bacterium
clostridium welchii. Toxins produced by the bacterium cause putrefactive decay of connective
tissues with the generation of gas. (Putrefaction- the process whereby proteins are decomposed
by bacteria . This is accompanied by the formation of amines ie . such as putrescine &
cadaverine , having a strong & very unpleasant smell).
12. Regular nonsporing Gram+ve rods:- These are represented by the genus Lacto bacillus .
Lactobacilli find industrial use in the production of pickles , yogurt, butter milk. They are
aerotolerant & produce lactic acid from simple carbohydrates. In humans they are located in the
oral cavity , intestinal tract & vagina ( lower part of the female reproductive tract).
13. Irregular nonsporing gram +ve rods:-- The organisms in this group may be aerobic ,
anaerobic , or pleomorphic & are represented in a group called Corynebacteria eg. C.
diphtheriae, the causative agent of diphtheria.( Diphtheria – an acute highly contagious infection
caused by the bacterium Coryne bacteria, Diphtheriae generally affecting the throat but
occasionally other mucous membranes & the skin. The disease is spread by direct contact with
a patient or carrier or by contaminated food. After an incubation period of 2- 6 days a sore
throat, weakness & mild fever develop. Later a soft gray membrane forms across the throat ,
constricting the air passages & causing difficulty in breathing & swallowing. Bacteria multiply
at the site of infection & release a toxin into the blood stream, which damages heart & nerves.
The genus consists of anaerobes found in the mouth & throat of humans & animals.
Actinomyces Israeli is the causative agent of actinomycosis (a tissue destroying disease).
Actinomycosis- a non-contagious disease caused by the bacterium Actinomyces Israelii , which

most commonly affects the jaw but may also affects the lungs , brain or intestine . The bacterium
is normally present in the mouth but it may become pathogenic when the tooth is extracted ,
causing the slow formation of abscesses & ulcers.
14. Mycobacteria :- They often exhibit filamentous growth ( myco indicates fungus like) are
aerobic , non-endospore forming rods . Most important pathogens of this genus are M.
tuberculosis , the causative agent of Tuberculosis & M. leprae that cause leprosy.
Tuberculosis- An infectious disease caused by the bacillus mycobacterium tuberculosis,

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characterized by the formation of nodular lesions in the tissue. Lesions- a zone of tissue with
impaired function as a result of damage by disease or wounding. Apart from direct physical
injury, egs. of primary lesions include abscesses, ulcers & tumours , secondary lesions ie crust
and scars). Nodule- small swelling or aggregation of cells.
Leprosy – A chronic disease caused by the bacterium mycobacterium leprae, that affects the skin
mucous membrane & nerves. Lepromatous leprosy is a contagious steadily progressive form of
the disease characterized by the development of widely distributed lumps on the skin,
thickening of the skin & nerves & in serious cases by severe numbness of the skin, muscle
weakness, paralysis leading to disfigurement and deformity.
15. Nocardioforms- Nocardia is the best known genus in this group that resembles actinomyces
but are aerobic, often acid- fast, common in soil. Nocardia asteroids occasionally causes
pulmonary nocardiosis & cause a destructive infection of the feet or hands.
16. Gliding sheathed & budding and or appendaged bacteria:-
Appendaged bacteria are an unusual group found in low nutrient aquatic environments such as
lakes & are linked taxonomically by the presence of protrusions such as stalks and buds.
Caulobacter has stalks that anchor the organisms to surfaces, reproduces by binary fission with
out resulting in two identical cells.
17. Chemoautotropic bacteria :- Endowed with great synthesizing ability they are autotrops
capable of using inorganic chemicals as energy sources & CO2 as the only source of Carbon. The
genera Nitrobacter and Nitromonas use reduced nitrogenous compounds as the energy source.
Thiobacillus & other sulphur oxidizing bacteria are capable of obtaining energy by oxidizing the
reduced forms of Sulphur.
18. Archaea:- Microbes with highly unusual morphology and environment niches (comfortable
to different environment) are included in this group because of their related tRNA sequences .
Extreme halopathic bacteria (bacteria that survive in very high concentrations of salt) .
Prominent among the archaea include Halobacterium & Halococcus. Other archaea thrive (grow
rich) in acidic, sulphur rich hot springs (eg. Sulpholobus).
19. Phototropic bacteria :- These are the purple bacteria, the green bacteria & the
cyanobacteria , all are prokaroytes that use light as the source of energy . Purple & green bacteria
are generally anaerobic. Purple nonsulphur & green non sulphur bacteria use organic compounds
for the photosynthetic reduction of CO2.
20. Cyanobacteria :- These are unicellular forms that divide by simple binary fission ,
essentially aerobic carry out oxygen producing photosynthesis & are capable of fixing
nitrogen.
21. Actinomycetes :- Actinomycetes are common inhabitants of soil. Being filamentous

bacteria , actinomycetes superficially resemble filamentous fungi in morphology but the
filaments of actinomycetes consist of prokaryotic cells having diameters much smaller than
those of moulds. One genus Frankia causes nitrogen fixing nodules to form in alder tree roots.
1.Spirochaetes?. 2 marks.Repeated in 4 question papers.

Leeuwenhoek described Spirochetes from saliva & tooth scrapings as one of the 1st

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Microorganisms as early as in 1600s. These bacteria are Gram(-)ve , typically coiled &
actively motile by means of two or more axial filaments. They exhibit helical morphology &
constitute a number of important pathogenic bacteria. Eg. Treponema pallidum , the cause of
Syphillis , members of the genus Borrelia & Leptospira. Spirochetes have a unique structure that
is responsible for the motility. The spirochete cell has a central protoplasmic cylinder bounded
by a plasma membrane & a typical gram –ve cell wall. Unlike the other bacilli, this cylinder is
enveloped by an outer sheath composed of glycosaminoglycans. Between the cell wall& outer
sheath are located multiple periplasmic flagella that do not protrude from the cell but are
oriented axially. Bundles of these endoflagella, called axial filaments, span the entire length of
the cell and anchored at both ends. Although the mechanisms are not totally clear, it is likely
that these axial periplasmic flagella rotate like the external flagella of other motile bacteria,
there by propelling the cell in a cork screw like manner.
2.Discuss classification of Microorganisms. 5 marks.?

Bacteria are the smallest , unicellular organisms measuring about 0.75 μm to 4 μm in diameter,
equipped with all the machinery for growth & self-replication at the expense of food stuffs .
Formerly classified with the fungi, bacteria were considered as primitive members of the plant
kingdom, but they are now called prokaryotes. They are ubiquitous (in an indefinite number of
places at the same time) & occur in water, air, soil, animals & plants as well as under extremely
adverse conditions . Only a few species of bacteria cause disease, often due to the cell’s ability
to produce specific poisons and toxins. The toxin produced by Clostridium botulism is one of
the most poisonous substance.
Morphologically three different bacteria are recognised .
1. Spherical.
2. Cylindrical or rod shaped.
3. Spiral.
1. Spherical( Cocci, singular – coccus):- These are the smallest bacteria, more or less spherical in
shape . In Greek kokkos means berry. Incompletely separated cocci may appear in a number of
different patterns depending up on the planes in which they divide.
Micrococcus :- ( Greek – mikros – small, kokkos –berry):- when they occur singly eg. Micro
coccus . These are usually Gram(+ ve) , non flagellated , spherical cells , which are facultative
parasites or saprophytes.
Diplococci:- when predominantly in pairs. Eg. Diplo coccus pneumonia . A coccus is divided in
one plane in to two cocci that remain attached in pairs.

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Strepto cocci:- ( Greek – streptos, twisted). When they live in chains. Strepto coccus lactis.
Although this organisms also divides into one plane, yet the cocci arrange themselves in chains.
Staphylo cocci:- ( Greek- Staphyle- bunch of grapes). When they are found in a group . eg.
Staphylo coccus aureus.
Tetrads( Tetra coccus)- When they divide into two planes & live in groups of four.
Sarcinae:- When they divide in three planes & remain attached in cube like groups of eight,
Sarcina.
2.Cylindrical or rod shaped :- Bacillus , singular – bacilli, It is derived from Greek word
Bacillus meaning – stick. The number of rod shaped bacteria is far greater than that of
spherical organisms due to the fact that the ratio of surface area to volume is higher in
rods.
Bacillus:- When only one rod shaped structure represents the bacterium.
Single bacillus.
Diplo bacillus :- When they occur in pairs.
Strepto bacillus:- These are found in chains. Eg. Bacillus anthracis.
3.Spiral Bacteria:- Spiral bacteria have one or more twists , they are never straight.
Vibrio or comma:- They are comma (,) shaped which cause chlorea.
Spirilla:- Greek –spira, coil. These are longer rigid rods with several curves or coils. They have a
helical shape, like a cork screw & fairly rigid bodies. Eg. spirillum ruprem.
Spirochetes:- Unlike the spirilla, which use flagella to move, spirochetes move by means of an
axial filament , which is contained under an external flexible sheath.

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In addition to these three basic shapes , the star shaped cells ( genus stella )
Square , flat cells ( halophilic archae) of the genus Haloarcula and triangular cells are also
known.
Q. Draw a neat labelled diagram of Bacteria. 2 marks.?
Structure of Bacterial cell:-
Being prokaryotes, bacteria are simpler than animal cells & lack a membrane bound nucleus ,
an extensive endoplasmic reticulum & mitochondria . Bacteria have a more complex surface
structure, with a rigid cell wall surrounding the cytoplasmic membrane. The membrane provides
the osmotic barrier & the active transport require to maintain an appropriate intracellular
concentration of specific ions & metabolites. The cell wall protects the cells against osmotic
rupture in dilute media & against mechanical damage. The wall is also responsible for bacterial
shapes, their major division into Gram+ve & Gram- ve organisms. The cell wall & membrane are
often referred together as the cell envelope, where as the more optional capsule, flagella & pili

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are referred to as appendages.
External Structures:-
Q. Describe in brief the composition & structure of bacterial cell wall – 5 marks.
Cell Wall :- The cell wall protects the bacterium from the harmful environmental effects. The
presence of a cell wall may be demonstrated by plasmolysis ie. Exposure to a hypertonic
solution, which causes the protoplast to contract& shrink away from the wall. The walls of
Gram+ve organisms are relatively thick ( 15- 80 nm) as compared to Gram-ve organisms. The
bacterial cell wall is composed of a macromolecular network of peptidoglycan ( also called as
murein). Peptidoglycan consists of a repeating disaccharide attached by polypeptides to form a
lattice (net work) that protects the entire cell. The disaccharide portion is composed of
monosaccharides called N-acetyl glucosamine (NAG ) & N- acetyl muramic acid ( NAM) which
are related to Glucose. Alternating NAM & NAG molecules are linked in rows of 10-65 sugars to
form a carbohydrate back bone . Adjacent rows are linked by polypeptides .Attached to the N-
acetyl muramic acid is a peptide chain of 3-5 amino acids. Gram –ve cells have a much thinner
peptidoglycan layer. Penicillin interferes with the final linking of the Peptidoglycan rows by
peptide cross bridges which greatly weakens the cell wall & the cell under goes lysis.
NAG& NAM joined as in peptidoglycan through β -1,4 linkage.

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Q. Formation of bacterial spore & capsule – 5 marks.
Capsules :- The term glycocalyx (sugar coat) generally implies substances that surround cells.
The bacterial glycocalyx is a viscous , gelatinous polymer , external to the cell wall &
composed of polysaccharide, polypeptide or both. Mostly it is made inside the cell & excreted to
the cell surface. If the substance is organised firmly attached to the cell wall, the glycocalyx is
described as a capsule. Capsules are optional bacterial structures, not essential to the life of a cell.
The presence or absence of capsules is genetically controlled. Negative or background staining
(suspension in Indian ink easily demonstrates them. In-ve staining the capsule is not stained but
is visible because of the space it occupies around the bacterial cell. (Acidic dyes are Nigrosin,
Eosin,Indian Ink. When an acidic dye is added , the bacteria form a complex , with the cations of
the dye which are colourless & the anion of the acidic dye which is coloured gives a coloured
back ground.) Capsules often protect pathogenic bacteria from phagocytosis by the cells of the
host.( Phagocytosis – the engulfment & digestion of bacteria & other foreign particles by a cell).
Capsules vary in thickness from a fraction of micrometre to 10μm or more. Distinction between
cell wall & capsule is not always clear. Sometimes wall & capsule appear into each other.Some
organisms may lack a definite capsule nevertheless they may possess a very thin but distinctive
molecular layer on the cell surface . These very thin layers have been called microcapsules,
sheaths or envelope.
Flagella :- Flagella ( singular flagellum, meaning whip) are long , slender, thin hair like
cytoplasmic appendages, which are responsible for the motility of bacteria. They are long (3µm to
12μm) filamentous appendages (additional thing), readily visualised by dark field microscopy,
(use of a special condenser results in oblique illumination of the specimen. Light does not enter
the objective directly but only when scattered by bacteria which appear brightly illuminated
against a dark back ground) but too thin ( 12 to 25 nm) to be seen by ordinary microscopy unless
specially stained . A flagellum is attached to the cell wall, the cell membrane or both by a basal
body, which is a complex molecular machine that rotates the flagellum like the screw propeller.
Q. Distribution pattern of Flagella ? 2 marks.

On the basis of Flagella bacteria are divided into following types.
1) Atrichous:- Flagellum is absent in these bacteria & hence they are non- motile

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.eg. Pasturella.
2. Monotrichous:- When a single polar flagellum is found. Eg .Pseudomonas,
Thiobacillus
3.Amphitrichous:- Either single or clusters at both cell poles. eg. Nitrosomonas.
4) Lophotrichous:- When two or more flagella are found on one end of the cell
eg. vibrio.
5) Peritrichous:- When flagella are found on entire body of the bacterium

eg. Escherichia, clostridium.
Q. Ultra structure of Flagella ? 2

marks.
A flagellum has three basic parts.

Filament is the long outermost region having a constant diameter & contains the globular protein
flagellin molecular weight ranging from 20,000 to 40,000 arranged in several chains that
intertwine & form a helix around a hollow core. Flagellar proteins serve to identify certain
pathogenic bacteria. The filament is attached to a slightly wider hook, consisting of different
protein. The third component is the basal body which anchors the flagellum to the cell wall &

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plasma membrane . The basal body is composed of a small central rod inserted into a series of
rings.
Q. Write the principle and procedure involved in flagella staining- 5 marks.
Flagella staining of Bacteria:-
Reagents:-
Flagella
mordent,
Ziehl’s carbol
fuchsin, Carbol
fuchsin,
Dichromate
solution, Alcohol
95%
Procedure:-
1. Take a grease free slide by dipping it in dichromate solution , washed with water and
rinsed in 95% alcohol. The slide is dried & passed through flame & allowed to cool.
2. Make suspension of a given culture in sterile distilled water .
3. Incubate at room temperature for 10-20 minutes.
4. Put a loopful of a suspension at one end of a slide.
5. Tilt the slide & allow the drop to spread to form a thin film on it.
6. Allow the film to air dry & cover it with flagella mordent for 10minutes.
7. Wash the slide with distilled water.
8. Flood the slide with carbol fuchsin for 5 minutes.
9. Wash again with distilled water, air dry it & observe under oil immersion
objective. Result:- The cell will appear pink & the outer coat bearing pink stained
flagella . Alternate methods of Flagella staining:-

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Gray staining method:-

Reagents:-
Solution A :-
Tannic acid (20%) – 2 ml Potassium alum (saturated aqueous) -5ml
Mercuric chloride (saturated aqueous)-2ml
Basic fuchsin (3%) in 95% in ethanol-0.4ml

Add the fuchsin dye to the outer ingredients & filter the solution before use.
Solution B:-
Ziehl carbol fuchsin
Basic fuchsin - 0.3gm
Ethanol -10ml
Phenol -5.0 gm
Distilled water -95ml
Procedure :
1. Grow any flagellated bacteria on a suitable broth.

2. Centrifuge it to remove the medium & to obtain desirable pellets.
3. Re-suspend the pellets in 10% formalin to produce a light, faint turbidity.
4. Take a loopful of suspension on a slide & prepare a smear.
5. Flood the smear with solution A for 6 minutes & wash with distilled water.
6. Place a piece of blotting paper from slide over the fill & flood the slide with solution B
for 4 minutes.
7. Remove the paper from slide, wash again, blot dry & observe under oil immersion
objective.
Result:- Flagella will appear red.
Pili and Fimbriae:-

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Many gram-ve bacteria contain hair like appendages pili or fimbriae given off from the surface.
Although the two terms are used interchangeably, yet they can be distinguished. Fimbriae can be
evenly distributed over the entire surface of the cell or they occur at the poles of the bacterial
cell. They enable a cell to adhere to surfaces, including of other cells. Their number may vary
from a few to several hundred per cell. Pili are usually longer than fimbriae & number only one or
two per cell. They join bacterial cells in preparation for the transfer of DNA from one cell to
another & are therefore also known as sex pili.
They are shorter, thinner& straighter than flagella. Pili consists of a protein pilin arranged
helically around a central core. They are non motile. Pili are responsible for haemagglutination
(sticking together) in bacteria & also for intercellular adhesiveness giving rise to clumping
( sticking together). When pili are mechanically removed cells rapidly form them again.
Internal structures:-
Q. Bacterial cell membrane – 2mark.
Cytoplasmic ( Plasma ) membrane :- The plasma membrane of prokaryotes is a distinct &
separable structure consisting primarily of phospholipids & proteins. Prokaryotic plasma
membrane less rigid than Eukaryotic membranes due to the lack of sterols. Both prokaryotic &
Eukaryotic plasma membranes appear like two layered structures. The phospholipid molecules
are arranged in two parallel rows, called a phospholipid bilayer. Phospholipid & protein
molecules move quite freely within the membrane surface & this movement may be responsible
for many functions performed by the plasma membrane. The membrane has three main functions.
1. It maintains a favourable intracellular osmotic pressure for the cell. It behaves like an
osmotic barrier due to it’s selective permeability.
2. The plasma membrane houses a number of specific active transport systems. Each
transport system is specific for a given compound.
3. Plasma membranes of bacteria contain enzymes capable of catalysing the chemical reactions that
break down nutrients & ATP, there by producing energy. Thus plasma membrane is the site for
many of the key enzymatic reactions involved in energy metabolism. Many antimicrobial agents
exert their effects on plasma membranes of bacteria. A group of antibiotics, called polymyxins
cause leakage of intracellular contents & kill cells.
Cytoplasm:- It is non-homogenous, thick, aqueous, semitransparent & elastic substance. Referring
to the substance of the cell inside the plasma membrane, cytoplasm is about 89% water & contains
primary proteins, carbohydrates, lipids, inorganic ions, and many low molecular weight compounds.
DNA, ribosomes & reserve deposits called inclusions are the major structures in the cytoplasm.

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Mesosomes:- Electron micrographs of Gram-ve bacteria show one or more large , irregular
convoluted (involuntary) invaginations(folding) of the cytoplasmic membrane called mesosomes.
They are smaller in gram –ve cells. Mesosomes are considered to be folds in the plasma membrane,
which develop by the process employed for preparing specimens for electron microscopy. The
mesosomes have two functions. participation in DNA replication & cell division, function in
secretion. Mesosomes are now considered as not true cell structures.
Ribosomes:-The cytoplasm of all Eukaryotic & Prokaryotic bacteria is richly populated with
ribosomes which are roughly spherical , densely stained objects, measuring about 18 nm in
diameter. Depending on their origin, ribosomes differ with respect to their size & shape. Each
ribosome consists of a small subunit & a large subunit. Each subunit consists of protein & rRNA.
Prokaryotic ribosomes are called 70S ribosomes & eukaryotic ribosomes are called as 80S
ribosomes ( S refers to sedimentation coefficient). Ribosomes function as the sites of protein
synthesis. Several antibiotics (eg. Streptomycin, gentamycin, chloramphenicol & erythromycin)
exert their action by inhibiting protein synthesis in ribosomes.
Nuclear Body (Nucleoid):- The nuclear region in bacteria is referred to as nuclear body or
nucleoid. It contains a single long circular molecule of double stranded DNA, the bacterial
chromosomes, which is the cell’s genetic information. The nucleoid can be spherical , elongated or
dumb bell shaped. The Eukaryotic nucleus is enclosed within a definite nuclear membrane &
exhibits mitotic cycles but prokaryotic nucleoid is not membrane enclosed & does not exhibit
either mitotic or meiotic phenomena .In addition to chromosome, bacteria often contain extra
chromosomal genetic material each called a plasmid. Plasmids are small, circular double stranded
DNA molecules distinct from the cellular chromosomes. In general, plasmids carry genes that are
not essential for host cell growth. While the chromosomes carries all the necessary genes. Plasmids
can be transferred from one bacterium to another. Plasmid DNA is used for gene manipulation in
biotechnology.
Inclusions:- The cytoplasm of many Eukaryotic & prokaryotic microorganisms store up reserve
food deposits known as inclusions. These may be lipids( Polyβ- hydroxyl butyric acid),
polysaccharides ( glycogen & starch), metachromatic granules or volutin ( reserve of inorganic
phosphate), sulphur granules, gas vacuoles etc. Such inclusions diminish or disappear when food is
scarce but are very conspicuous when extra cellular food is abundant.
Spores ( Endospores):- Under conditions of depleted (empty) supply of nutrients , certain gram+
ve rods such as those of the genera Bacillus ( aerobic) & Clostridium ( anaerobic) , and a few
sarcinae & actinomycetes form highly resistant , dehydrated , specialized resting cells called
endospores or simply spores. They are internal to the bacterial cell membrane & the surrounding
mother cell is known as sporangium. Bacterial spores are adapted for prolonged survival under
adverse conditions such as extreme heat, dehydration, freezing toxic chemicals& radiation. True
endospores are found only in gram +ve bacteria. The process of endospore formation is known as
sporulation & may take several hours in a vegetative cell. In the first stage, a newly replicated
bacterial chromosome & a small portion of cytoplasm are isolated by an in growth of the plasma

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membrane called a spore septum & remains entirely enclosed within the original cell. Thick layers
of peptidoglycan are laid down between the two membrane layers. Then a thick spore coat of
protein forms around the outside membrane. This coat is responsible for the resistance of
endospores to many harsh chemicals. Endospores can remain dormant for thousands of years. An
endospore returns to it’s vegetative state by a process called germination; which has three distinct
stages.1) activation 2) germination 3) Out growth or post germination development. Spores can be
detected by staining with a specially prepared stain (Schaeffer – Fulton ndospore strain) along with
heat.

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Q. Differences between gram + ve and gram – ve organisms.2
marks. Q.Differentiate gram+ve & gram- ve cell wall. 5 marks.
Difference between Gram+ve and Gram-ve species.
Gram +ve organisms:- Gram +ve bacteria have a thick multilayered peptidoglycan cell wall
that is exterior to the membrane. The peptidoglycan in most gram+ve species is covalently linked
to teichoic acid which is essentially a polymer of substituted glycerol units linked by
phosphodiester bonds (For some teichoic acid variants, other alcohols such as ribitol, may
substitute for glycerol). All gram+ve species also have teichoic acid in their membranes,
where it is covalently linked to glycolipid. The teichoic acids are major cell surface antigens.
Gram –ve organisms:- Gram –ve bacteria have two membranes – an outer membrane & an
inner (cytoplasmic) membrane. Their peptidoglycan layer is located between the two
membranes and periplasmic space. The periplasmic space also contains enzymes & various
other substances. In contrast to gram +ve cells, the peptidoglycan layer at gram –ve cells is thin
and the cells are consequently more susceptible to physical damage. The outer membrane is

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distinguished by the presence of various embedded lipo polysaccharides. The polysaccharide

portion (o- polysaccharide ) is antigenic and can therefore be used to identify different strains
and species. The lipid portion (called lipid A ) is toxic to humans & animals. Lipid A because it
is an integral part of the membrane is called an endotoxin, as opposed to exotoxins, which are
secreted substances.
Q. Examples of Gram +ve organisms . 2 marks.
Examples of Gram+ve bacteria Gram-ve bacteria
Bacillus anthracis ( Anthrax) Bordetella pertussis ( whooping cough).

Clostridium tetani ( tetanus) Escherichia coli (Urinary infections).
Clostridium botulinum ( Botulism) Haemophilus influenza.(Meningitis).
Coryne bacterium diphtheria( Diphtheria) Klebsiella pneumonia.
Staphylo coccus aureus ( Boils, sepitcemia). Neisseria gonorrhoeae.
Strepto coccus pyrogenes( scarlet fever, Salmonella typhi ( Typhoid fever).
rheumatic fever, septicaemia).
Strepto coccus pneumonia ( Pneumonia) Vibrio cholera ( cholera).
Mycobacterium leprae ( leprosy) & mycobacterium tuberculosis ( tuberculosis) are gram
variable ie. Either +ve or –ve.
Distinguishing characteristics of Gram(+ve) and Gram(-ve ) Bacteria.
Characteristics Gram +ve Gram –ve

Susceptibility to Marked Much less
sulphonamide & penicillin
Susceptibilityto Tetracycline, Slight Marked.
Streptomycin&
Chloramphenicol.
Susceptibility to anionic Marked Much less.
detergents.
Susceptibility to lysis by Slight Marked.
Complement
Resistance to sodium azide. Marked Much less.
Toxins produced. Primarily exotoxins. Primarily endotoxins.
Outer membrane Absent Present.
Lipid & lipoprotein content. Low High
Periplasmic space Absent Present
Teichoic acids Present Absent
Peptidoglycan layer Multi layered ( thick) Single layered ( thin)
Resistance to physical Marked Much less.
disruption.
Growth & Reproduction of Bacteria:-
Bacteria differ widely in their nutritional requirements. Some bacteria can synthesis all their
requirements from the simplest elements but others need a ready made supply of some of the organic
compounds necessary for growth. The growth requirements of different bacteria are provided in
different types of culture media.

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Nutritional Requirements:- Microorganisms are extra ordinary diverse in their specific nutritional
requirements. Most of the microorganisms require the following major materials .
Water :- It is the major essential nutrient as it accounts for about 80-90 % of the total weight of the
cells. In addition to hydrogen & Oxygen, which can be derived metabolically from water, other
elements required include Carbon, nitrogen, phosphorous & sulphur. These four elements accounts
for nearly 95% of the dry cell weight. In addition , microorganisms generally require potassium,
magnesium, calcium, Iron, manganese, cobalt, copper, molybdenum & zinc. All the metallic
elements are easily supplied in the form of their respective soluble salts mainly chlorides &
sulphates. Micronutrients or trace elements required for the growth of bacteria include manganese,
cobalt, copper, nickel, molybdenum & zinc. This explains why the culture media are usually
prepared in tap water, rather than in distilled water ( tap water contains most of the trace minerals as
impurity). Some biological groups may have specific mineral requirements eg. Diatoms & certain
algae- silica, photosynthetic bacteria , cyanobacteria & certain marine bacteria – sodium. Carbon:-
Bacteria that derive energy from the oxidation of inorganic compounds & photosynthetic organisms
use oxidized form of carbon as CO2 as the principal source of cellular carbon. All other organisms
obtain carbon largely from organic nutrients. Certain bacteria of the Pseudomonas group can use any
one of organic compounds as their sole source of carbon & energy. On the contrary the methane
oxidising bacteria can use only two organic substrates, methane & methanol. Similarly some
cellulose decomposing bacteria can use only cellulose for the same purpose.Most microorganisms
that depend on organic carbon sources also require CO 2 as a nutrient in very small amount, which is
utilized in a few biosynthetic reactions. The organisms, which can thrive on an entirely inorganic
diet, using CO2 or carbonates as a sole source of carbon are designated as autotrops (auto – self,
trophe- nourshing). The organisms which can not use CO2 as a sole source of carbon ( glucose or
amino acids) , in addition to minerals, are designated as heterotrophs ( hetero- other). In fact some
bacteria can live either autotrophically or heterotropically , for eg. some soil inhabiting bacteria of
genus Hydrogenomonas & others of the family Athiorhodaceae. Nitrogen & Sulphur:- These
elements must be in reduced form for organic combinations.eg. Nitrogen as in amino acids or
sulphur as in sulphydryl compounds. Most photosynthetic as well as many non-photo synthetic
bacteria & fungi assimilate nitrogen & sulphur in the oxidised inorganic state, as nitrates &
sulphates. The requirements of these two elements can also be met by organic nutrients containing
them in reduced organic combination. Eg. amino acids or peptones. Some bacteria can also utilise
the most abundant natural nitrogen source, N2 . This process of nitrogen assimilation (the
absorption and digestion of food or nutrients by the body or any biological system) is known as
nitrogen fixation , which involves a preliminary reduction of N2 to ammonia.
Oxygen:- Microorganisms that are dependent on aerobic molecular oxygen (O2 ) for their growth.
Such organisms are called as obligate or strict aerobes. Eg. Clostridium species. Organisms that are
able to grow either in the presence or absence of molecular oxygen are called as facultative
anaerobes. eg. E.coli . Obligate aerobes that grow best at partial pressure of oxygen considerably
below 0.2atm are termed microaerophilic. Aerotolerant anaerobes cannot use oxygen for their
growth but tolerate it fairly well.
Organic growth factors :- Essential organic compounds that an organism cannot itself synthesis from
simple organic sources, but require as a precursor or constituent of it’s organic cell material; are
known as organic growth factors.
Growth factors are of 3 types.

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1) Amino acids required as constituents of proteins .
2) Purines & pyrimidine required as constituents of nucleic acid.
3) Vitamins forming parts of the prosthetic groups or active centres of certain enzymes.
Energy :- With respect to energy source, organisms that are able to use light as an energy source,
are designated as phototrophs, and organisms which are dependent on a chemical source are termed
chemotrophs .
 Based on these considerations microorganisms can be divided into four nutritional

categories.
1.
Photoautotrophs:- Organisms using light as the energy source & CO2
as the principal carbon source eg. higher plants , algae & photosynthetic bacteria.
2.
Photoheterotrophs:- Organisms using light as the energy source & an organic
compound as the principal carbon source. Eg. Certain purple& green bacteria.
3.
Chemoautotrophs:- Organisms using a chemical energy source & CO2
as the principal carbon source. Because of their ability to grow in strictly mineral
media in the absence of light , these organisms are also called chemolithotrophs ( litho-
rock or stone).
4.
Chemoheterotrophs:- Organisms using a chemical energy source & an
organic substance as the principal carbon source. Chemoheterotrophs ( also known as
chemo organotrophs ) are the commonest of all the nutritional types.
Temperature :- The favourable temperature for the growth of

most microorganisms is the human body temperature ie. 370C.
On the basis of their preferred temperature range microorganisms are classified into
three groups.
1) Psychrophiles (Cold loving).
2) Mesophiles ( Moderate temperature loving).
3) Thermophiles ( Hot loving).
 Temperature range for Bacterial Growth:-
Growth Temperature ( 0 c)
Group Minimum Maximum Optimum
Psychrophiles
Obligate 5 & below 19-22 15-18
Facultative 5 & below 30-35 25-30
Mesophiles 10-15 35-47 30-45
Thermophiles 40-45 60-85 55-75

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Minimum growth temperature- refers to the lowest temperature at which the organisms grows.
Maximum growth temperature –refers to the highest temperature at which organisms grows.
Optimum growth temperature – Is the temperature at which the rate of multiplication is most rapid.
Mesophiles having an optimum growth temperature of 30- 450 C are include most of the common
spoilage & disease producing organisms.
Osmotic pressure:- For their survival & growth microorganisms prefer isosmotic ( same osmotic
pressure as that inside bacterial cell) rather than hyper osmotic ( higher osmotic pressure than that
inside bacterial cell) environment media). Both higher & lower osmotic pressure environments are
used for killing or inhibiting the bacterial growth.
pH:- Most bacteria grow best in a narrow pH range between 6.5 & 7.5 . ie. That is near neutral.
Growth:- Bacteria reproduce by binary fission ( an asexual process). Bacterial growth is the result of
a balanced increase in the mass of cellular constituents & structures, energy for the biosynthetic
processes being supplied from ATP. Cell division is initiated when the increase in cellular
constituents & structures reaches a critical mass. After the division is complete, bacteria grow &
attain the features unique to each species. The interval of time until the completion of
the next division is known as generation time. The generation time is a determining factor in
the duration of time that passes before disease symptoms appear in an infected individual . The
generation may be as short as 20 minutes for Escherichia coli or 30 minutes for Staphylococcus
aureus to very long eg. 18 hrs to Mycobacterium tuberculosis or 33 hrs for Treponema pallidum.
Bacterial growth rate is also expressed in terms of doubling time, which is also known as the mean
generation time ( MGT).
Bacterial growth cycle:-

Q. Bacterial growth cycle- 5 marks.( repeated).
Q. Write the different phases of bacterial growth curve. Give the reasons for each
phase. 5 marks.
Q. What is Lag time – 5 marks.
When dormant bacteria are inoculated ( transferred) into fresh medium, they exhibit a typical growth
curve.

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1.Lag phase:- This phase corresponds to a period of adaptation with active macromolecular
synthesis. Some organisms may die from the shock of transfer but the biochemical activity in the
remaining bacteria is intense. The number of bacteria in the lag phase remains stable, balanced by
early reproduction in some cells & death in others.
2.Log (Exponential) Phase:- After the bacteria have adapted to new environment ( favourable
growth conditions like nutrient medium & aeration) the cell division proceeds at the log (
logarithmic ) rate. The number of bacteria doubles with each generation time. Because the number of
bacteria increases exponentially( more and more rapidly), the logarithmic phase is also known as
the exponential phase. If logarithms of the actual numbers are used for the curve as shown in Fig. the
doubling time or mean generation time can be calculated from the slope of this straight line.
Reproduction & growth are at their highest rates during the logarithmic phase.
3.Stationary phase:- The reproduction & death rates equalise & hence the
bacterial population remains same. During stationary phase the nutrients become scarce,
waste products accumulate & oxygen or water etc. are in short supply. These conditions
lead the growth curve into the last phase of the cycle.
4.Decline Phase:- After a period in the stationary phase , the number of bacteria starts declining
because the reproduction occurs at the lowest rate while death of bacteria occurs at the highest
rate. The number of cells dying exceeds the number of new cells formed.
These phases can also be identified in the bacterial diseases in human. Beginning the history with
the entry of the bacteria into human respiratory tract during the lag phase, scavenging white blood
cells may engulf & destroy some bacteria. During the log phase the bacterial population reaches a
level high enough to cause tissue damage. Coughing or fever may occur& fluid may enter the lungs
if the air sacs are damaged. Treatment of the infection by administering antibiotics leads to a
stationary phase followed by a decline phase characterised by destruction of bacteria & curing of
symptoms.
Reproduction :-Binary fission :- Under ordinary conditions of growth most of the microorganisms
multiply by the asexual process of cell fission which results in division of the cell into two or more
vegetative cells. Most bacteria multiply by transverse binary fission ie. Division into two equal
cells . The circular chromosomes divide into two or more vegetative cells. The circular
chromosomes divides into two identical circles , which segregate at opposite ends of the cell.
Simultaneously , the cell wall is laid down in the middle of the cell, which finally grows to produce
two new cells each with it’s own wall & nucleus . Each of the two cells will be exact copy of the
original cell from which they arose & no new genetic material is received & none lost.

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Reproduction involving genetic exchange:-

Earlier it was believed that among bacteria multiplication is entirely asexual. But now it is known
that there are three methods of reproduction involving genetic exchange between pairs of cells,
exhibiting sexual reproduction . Genetic material may be transferred between bacteria in several
ways, all involving a donor cell that gives a portion of the total DNA to a recipient cell. The
recipient cell that incorporates donor DNA into its own DNA is called a recombinant .
Q. Explain the genetic exchange in Bacteria Transformation – 5 marks. Q.Discuss

transformation & transduction . 5 marks.
Q.Transduction 2
marks. Q.Binary fission
- 2 marks.
Transformation:- In 1928 Griffith found that a culture of Diplococcus pneumoniae deficient in
capsular material could be made to produce normal capsulated cells by the addition of cell free
filtrate from a culture in which a normal capsulated strain had been growing . Sixteen years later it
was established that DNA was the material in the culture filtrate that was responsible for the re-
establishment of capsulated cells. It involves autolysis of the culture with loss of genetic
material.
During the process of transformation genes are transferred from one bacterium to another as
naked DNA in solution. Transformation occurs naturally among few genera of bacteria, including
Bacillus , Hemophilus , Neisseria, Acinetobacter & certain strains of the genera Staphylococcus &
Streptococcus.
Bacterial Transformation
Transformation is the process of DNA uptake by the bacteria from the surrounding environment.
The cells that have the ability to uptake DNA are known as competent cells .. This process was first
reported in Streptococcus pneumonia by Griffith.
Bacterial Competence
Not all bacteria are capable of taking up DNA from the surrounding environment. Such bacteria are
made artificially competent. This is achieved by using chemicals and electrical pulses.
Chemicals- The cells are chilled and made permeable in the presence of calcium phosphate. They
are then incubated with the DNA and provided with a heat shock treatment that causes the DNA
to enter the cells.
Electroporation- The bacterial cells are subjected to electrical pulses to make them permeable and
cause the DNA to enter into the cells.

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Q. Conjugation -2 marks ( repeated).
Conjugation :- Conjugation is a natural process found in certain bacterial genera & involves the
active passage of genetic material from one cell to another by means of the sex pili. It is
mediated by one kind of Plasmid , a circular piece of DNA that replicates independently from the
cell’s chromosome. Bacteria which are able to affect transfer contain in their genetic make up a
fertility factor & are designated F+ strains ( F stands for fertility factors). These are able to transfer
part or whole of their genetic material to F- strains. There are various conjugal plasmids carried
by various bacterial species. Conjugation is carried out in several steps:
Mating pair formation

Conjugal DNA synthesis
DNA transfer
Maturation
Mechanism of Bacterial Conjugation
Bacterial conjugation involves the following steps:
Pilus Formation .The donor cells (F+ cells) form a sex pilus and begin contact with an F- recipient
cell. Physical Contact between Donor and Recipient Cell.The pilus forms a conjugation tube and
enables direct contact between the donor and the recipient cells.
Transfer of F-Plasmid
The F-factor opens at the origin of replication. One strand is cut at the origin of replication, and the
5’ end enters the recipient cell. DNA polymerase is an enzyme that synthesizes DNA molecules
from deoxyribonucleotides, which are the building blocks of DNA. The enzymes play an essential
role in DNA replication, usually working in pairs to produce two matching DNA strands from a
single DNA molecule.
The relaxosome transferasome is the complex of proteins that facilitates plasmid transfer
during bacterial conjugation

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Synthesis of Complementary Strand.The donor and the recipient strand both contain a single strand
of the F-plasmid. Thus, a complementary strand is synthesized in both the recipient and the donor.
The recipient cell now contains a copy of F plasmid and becomes a donor cell.
Transduction :- This is another process through which genetic material may be exchanged between
bacteria . In this process , bacterial DNA is transferred from a donor cell to a recipient cell inside
a virus that infects bacteria ( bacteriophage).
Transduction is of two types:
Generalized Transduction.
Specialized Transduction.
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Generalized Transduction
In this type, the bacteriophage first infects the donor cells and begins the lytic cycle. The virus then
develops its components using the host cell machinery. The host cell DNA is hydrolyzed into small
fragments by the viral enzymes.
Specialized Transduction
In this, only a few restricted bacteria are transferred from donor to recipient bacteria. This is carried
out by temperate bacteriophage which undergoes the lysogenic cycle. The virus enters the bacteria
and integrates its genome within the host cell DNA. It remains dormant and passes on from
generation to generation. When the lysogenic cell is exposed to some external stimulus, the lytic
cycle begins.
The virus genome is induced in the host cell genome. Due to this, the phage genome
sometimes carries the bacterial genome with it and integrates it into the genome of the
recipient cell. Here, only the restricted genome has the possibility of entering into the recipient
cells.
Previously asked questions.
1. Draw a neat labelled diagram of Bacteria -2marks.

2. Bacterial growth cycle- 5 marks.( repeated).
3. Discussion classification of microorganisms .5 marks.
4. Distribution pattern of Flagella. 2 marks.

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5. Bacteriophage - 2 marks.
6. Conjugation -2 marks ( repeated).
7. Enlist the differences between prokaryotic & Eukaryotic organisms .give egs. for each- 5
marks.
8. Differences between gram + ve and gram – ve organisms.2 marks.
9. Explain the genetic exchange in Bacteria Transformation – 5 marks.
10. Ultra structure of Flagella - 2 marks.
11. Spirochaetes – 2 marks.( repeated).
12. Write the different phases of bacterial growth curve. Give the reasons for each phase. 5
marks.
13. Contribution of Louis Pasteur- 2 marks.
14. Describe in brief the composition & structure of bacterial cell wall – 5 marks.
15. Bacterial cell membrane – 2mark.
16. Differentiate gram+ve & gram- ve cell wall. 5 marks.
17. Discuss transformation & transduction . 5 marks.
18. Formation of bacterial spore & capsule – 5 marks.
19. Transduction 2 marks.
20. Binary fission - 2 marks.
21. Fimbriae – 2 marks.
22. Plasmid – 2 marks.
23. Examples of Gram +ve organisms . 2 marks.
24. Flagella staining – 5 marks.
25. What is Lag time – 5 marks.
26. Write the principle and procedure involved in flagella staining- 5 marks.
27. List four method of genetic transfer in bacteria – 5 marks.
28. Define the following .1) Transduction 2)Conjugation.
29. Describe different phases of growth curve of bacteria based on total count. 5 marks.
30. Describe the factors affecting bacterial growth and survival .5 marks.
31. Explain the different factors affecting the growth of microorganisms- 5 marks.
32.Describe in brief the composition & structure of Gram + ve and Gram – ve bacterial
cell
wall? 5 marks. Repeated .
33.Bacterial cell membrane? 2 marks.
34. Formation of bacterial spore and capsule ? 5 .
35.Fimbriae ? 2 marks.
36. Flagella staining ? 2 marks.
37. write the principle & procedure involved in Flagella staining? 5 marks.
Fungi
Fungi are a group of non motile eukaryotic organisms which exist as saprophytes or parasites .
They posses differentiated nuclei surrounded by a nuclear membrane and reproduce either by
budding or by forming spores. They have rigid chitinous cell walls. Morphologically fungi
may be either simple oval cells or long tubular septate hyphae showing true lateral branching .
All fungi are chemohetrotropes, requiring organic compounds for energy and carbon. Fungi
are aerobic or facultative anaerobic .The majority of fungi are saprophytes in soil and water .
They divide asexually, sexually or by both processes . They may be unicellular or
multicellular.

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The study of fungi is called Mycology.
Media containing high carbohydrate source , nitrogen source are required for the growth of fungi
at pH range of
5 to 6, and a temperature range from 15 to 370 C. Media generally contain a source of carbon ,
nitrogen and vitamins . Glucose ( Dextrose) is the most widely utilizable carbon source , and
hence is the most commonly used in growth media . Fructose and mannose are the next
most commonly utilized sugars by fungi and are found in media from natural sources.
Nitrogen sources include peptone , yeast extract, malt extract , amino acids, ammonium and
nitrate compounds . Fungi have natural deficiencies for vitamins that are satisfied at µgm to
ngm concentrations. The most common naturally occurring vitamin deficiencies are thiamin
and biotin.
Comparison of selected features of fungi and bacteria.
Characteristics Fungi Bacteria

Cell type Eukaryotic Prokaryotic
Optimum pH 4 to 6 6.5 to 7.5
Optimum temperature 25 to 300C (Saprophytes) 32 to 370C
32 to 370C ( parasites) Mesophilic
Cell membrane Sterols present Sterols absent except mycoplasma
Aerobic to Anaerobic
Oxygen requirement Strictly aerobic ( moulds )
Facultative anaerobic ( some yeasts)
Light requirement None Some photosynthetic groups require
Carbon source Organic Inorganic/ organic
Concentration of sugar in 4 to 5 % 0.5 to 1%
laboratory media
Cell wall components Chitin , cellulose or hemicelluloses Peptidoglycan
Susceptibility to antibiotic Sensitive to Griseofulvin Resistant to Griseofulvin

Resistant to Penicillin, Sensitive to Penicillin, Tetracyclines etc.
Chloramphenicol etc.
Classification of Fungi
Fungi are placed in the phylum Thallophyta which contains the irregular plant masses that
lack definite root , stem and leaf structures . Thallophyta are divided into two main groups
the algae and the fungi.
Depending on cell morphology , fungi can be divided into four classes ; moulds , yeasts ,
yeast like fungi and dimorphic fungi.
1. Moulds and Fleshy fungi : Fungi which form mycelia are called moulds or filamentous fungi.
The thallus
(body) of a mould or fleshy fungus consists of long filaments of cells joined together ,
these filaments are called hyphae. They are 2 to 10 µm in diameter. In most moulds the
hyphae contain cross walls called septa (septum),which divide the hyphae into distinct , uni-
nucleate or multi-nucleate cell like units. These hyphae are called septate hyphae .

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In few classes of fungi , the hyphae does not contain septa and appear as long , continuous
cells with many nuclei . These hyphae are called coenocytic or non-septate hyphae.
When environmental conditions are suitable, the hyphae grow and form a mass called
mycelium, which is visible to the naked eye. The portion of the mycelium concerned with
obtaining nutrients is called vegetative mycelium.
The portion concerned with reproduction is called reproductive or aerial mycelium

because it projects above the surface of the medium on which the fungus is growing. On
artificial medium they are seen as a filamentous mould colony which may be dry and
powdery. eg.Aspergillus fumigatus , Penicillium notatum, Trichophyton rubrum, Microsporum
gypseum etc.
2. Yeasts:- Yeast are round , oval or elongated unicellular fungi . Most of them reproduce by
an asexual process called budding in which the cell develops a protuberance ( is something
that sticks out, like a swelling ) which enlarges and eventually separates from the parent cell .
On culture, they form smooth, creamy colonies eg. Saccharomyces cerevisae, Cryptococcus
neoformans. The difference between yeast and mould is given in table .
Yeast cells showing stages of budding.

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Difference between Moulds and Yeast

Mould Yeast
Mould contain multiple identical nuclei. It grows Yeast is round, oval or elongated unicellular
in the from of mycelium hyphae of filaments . fungi .yeast contains only single cell.
It has an indistinct appearance on surface of It forms white, circular, smooth creamy colonies
media forming black , green , brown , orange or on surface of media.
pink colour.
Strictly aerobic Aerobic, some yeasts are facultative anaerobic.
Optimum temperature for growth 22 to 280C Most suitable temperature is 32 to 370C
Reproduce through small spores, which can be Most of them reproduce by an asexual process
either sexual or asexual . called budding.
Most of species are used for production of Yeast species are used in ethanol production and
enzymes and antibiotics . bakery industry.
Eg.Aspergillus niger, Aspergillus fumigates, Eg.Saccharomyces cerevisae, Cryptococcus
Penicillium notatum. neoformans.
Yeast like Fungi :- In some yeasts like Candida albicans , the bud remains attached to the
mother cell and elongates, followed by repeated budding, forming chains of elongated cells
known as pseudohyphae. These can be differentiated from true hyphae because they have a
constriction at the septa are also present at the branching point . On solid media moist creamy
coloured colonies are produced .
Pseudohyphae
Dimorphic fungi:- Some fungi , mainly pathogenic species exhibit dimorphism ie. two forms
of growth . Such fungi can grow either as a mould or as a yeast . The mould like forms
produce vegetative and aerial mycelium
.The yeast like forms reproduce by budding . Frequently dimorphism is temperature as well as
CO2 dependent. At 370C, the fungus grows yeast like and at 250 C it shows mould like growth
eg. Mucor rouxii , histoplasma capsulatum, Blastomyces dermitidis and sporothrix schenckii.
The systematic classification of Fungi:- Based on their sexual spore

formation , fungi are divided into four classes.
a) Phycomycetes : They are fungi having non septate hyphae ( lower fungi). They form
endogenous asexual spores (Sporangiospore ) contained within sac like structures called
sporangia .Phycomycetes also produce sexual spores known as oospores and zygospores eg.
Mucor, Rhizopus etc.
b) Ascomycetes : They form sexual spores within a sac and are called ascopores . The sac is
called ascus . They form septate hyphae. Ascomycetes include both yeasts and filamentous
fungi eg. Histoplasma , Candida etc.

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c) Basidiomycetes : They reproduce sexually and form septate hyphae .
Importance of Fungi :-
1. Fungi are important sources of antibiotics eg. Penicillin ( Penicillium

notatum ), Griseofulvin ( Penicillium griseofulvum), Cephalothin ( Cephalosporium
species) etc.
2. Yeasts and moulds are good sources of different enzymes eg. amylase produced
from Aspergillus species .
3. Moulds ( Aspergillus species ) are used in the production of citric , oxalic and gluconic
acid.
4. Fungi have been used to alter the texture , improve the flavor , increase the
palatibality and digestibility of natural or processed foods eg. Penicillium species is
used for ripening of certain varieties of cheese.
5. Edible wild or domesticated varieties of mushrooms are important as food sources.
6. Yeasts are used for fermentation purposes in production of beverages and juices
as well as brewing and baking eg. Saccharomyces cerevisiae.
7. Moulds are also used for production of industrial alcohol by fermentation eg. Fusarium
species .
8. Fungi have capability to break down complex organic substrates .This is an essential
activity in the recycling of carbon and other elements.
Cultivation of Fungi :- Natural and synthetic types of fungal culture media are commonly used
for growth and cultivation of fungi . Natural media are composed of natural substrates such
as herbaceous or woody stems , seeds , leaves , corn meal , wheat germ , oat meal etc.
Natural media are usually easy to prepare but they have the disadvantage of their unknown
composition eg. Corn meal agar and potato dextrose agar. Synthetic media contain ingredients
of known composition . These types of media contain defined amounts of carbohydrates ,
nitrogen and vitamin sources eg. Czapek –Dox medium , glucose asparagine and
Neurosporacrassa minimal medium. General purpose media which are commonly used for
fungal culture are Sabouraud dextrose agar (SDA). Selective media like inhibitory mould
agar and dermatophyte test media are important in the isolation of fungal pathogens such
as Cryptococcus neoformans and dermatophytes . Agar supplemented by rice , casein and
other nutrients like corn meal agar with Tween 80 have been used to differentiate Candida
species and Trichophytan species . Potato dextrose agar used to enhance conidia and pigment
development of Trichophytan rubrum.
Sabouraud agar is a type of agar growth medium containing peptones . It is used to

cultivate dermatophytes and other types of fungi and can also grow filamentous bacteria such
as Nocardia . It has utility for research and clinical care. It was developed by Raymond
Sabouraud. The acidic pH (5.6) of traditional Sabouraud agar inhibits bacterial growth.
The optimal recovery of fungal pathogen , a modified media should be used such as media
with or without cyclohexamide and media with or without an antibacterial agent
(Chloramphenicol, Gentamicin and Ciprofloxacin ). Antibacterial agents are used to kill the
contaminating bacterial species . If the sample is taken from sterile site, it is not necessary to
use media containing antibacterial agents.
The standard temperature for incubation of fungi is 30 0C and cultures should be incubated in
a humidified environment for 21 days . They should be inspected daily for at least a week and
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at least 3 times weekly thereafter. Some fungi are very slow to grow and may need longer
incubation times. Different types of media used for growth of fungi are as follows .
Brain heart infusion (BHI ) agar : It is a non selective fungal culture medium that permits the
growth of all clinically relevant fungi . It is used for the primary recovery of saprophytic
and dimorphic fungi.
Czapek’s agar :- It is used for the subculture of Aspergillus species for their differential
diagnosis.
Inhibitory mold Agar :- Primary recovery of dimorphic pathogenic fungi. Saprophytic fungi
and dermatophytes will not be recovered .
Mycobiotic agar :- It is generally Sabouraud’s dextrose agar with cycloheximide and

chloramphenicol and used for the primary recovery of dermatophytes.
Potato dextrose agar (PDA) :- It is relatively rich medium for growing a wide range of fungi.
Sabouraud’s heart infusion (SABHI) agar :- Primary recovery of saprophytic and dimorphic
fungi.
Sabouraud’s dextrose agar (SDA) :- It is not recommended as a primary isolation medium

because it is insufficiently rich to recover certain fastidious pathogenic species , particularly
most of the dimorphic fungi . SDA 2% is most useful as a medium for the subculture of
fungi recovered on enriched medium to enhance typical sporulation and provide the more
characteristic colony morphology.
Potato flake agar: - Primary recovery of saprophytic and dimorphic fungi , particularly
fastidious and slow growing strains .
Cornmeal Agar (CMA) :- It is used for growing a wide range of fungi , particularly members
of the fungi imperfecti; provides a good balance of mycelial growth and sporulation .
Malt extract agar (MEA) :- It is a good growth medium for soil fungi , isolated from wood .
Potato dextrose yeast extract agar (PDYA) : This media is good for growing culture derived
from mushrooms.
Yeast:Saccharomyces. Saccharomyces cells are elliptical , measuring about 6 to 8 µm. They

multiply asexually by the budding process . A raised scar remains present on the cell, where a
bud has formed .Shown in figure. During budding , the nucleus divides by constriction and a
portion of it enters the bud along with other organelles. Hyphae are absent. The inability to
utilize nitrate and ability to ferment various carbohydrates are typical characteristics of
saccharomyces. Saccharomyces colonies grow rapidly and mature within 2 to 3 days . They
are flat, smooth , moist and cream coloured.

4
Saccharomyces cerevisiae showing bud and bud scars
There are more than thirty species of Saccharomyces . The best known is Saccharomyces
cerevisiae, strains of which are used in the fermentation of beer and wine and in the
production of bakery products, It is found in nature on ripe fruit . Grape wines are often
made by spontaneous fermentation by yeasts growing on the surface of the fruit . Thus
Saccharomyces cerevisiae is a yeast of great importance in fermentation . Other member of
this genus include Saccharomyces bayanus, used in making wine and Saccharomyces
boulardii used in medicine . Genetically engineered strain is also used to produce different
types of proteins .
Mould : Penicillium.
There are more than 150 known species of the genus Penicillium. They occur as saprophytes
in soil and decomposing organic debris.
On Sabouraud’s dextrose agar at 200C for 1 to 4 days , the colonies become velvety white and
later become blue green. Microscopic examination of these colonies show brush like
arrangement of conidia , sterigmata and conidiophores . Penicillium have septate vegetative
mycelia which penetrate the substrate and then produce aerial hyphae on which develops
conidiophores . Conidiophores may be branched and have brush like heads bearing spores .
Clusters of sterigmata are usually in one plane and form a chain of conidia . The colour of the
mature fungi is useful in helping to identify species .They grow best at temperature ranging
from 15 to 30 0C.
Penicillium species.
Many species of Penicillium are used in the ripening of cheese . Some are used in industrial
fermentations for productions of antibiotics eg. Penicillin is produced by Penicillium notatum
and Penicillium chrysogenum.
Virus
Viruses are so important obligatory intracellular Parasites that their study is treated as a
separate branch known as virology. They share the characteristics of both living and non
living organisms. The first viral disease of plants ie. Tobacco mosaic was discovered in
1892 by a Russian bacteriologist Dmitri Iwanowaski . Just at that time, in 1886 the Dutch
chemist Adolf Mayer had already shown that tobacco mosaic was transmissible from a
diseased plant to a healthy plant. The Tobacco mosaic virus ( TMV) was finally
crystallised & isolated in 1935 by American chemist, Wendell M. Stanley. Bawden &
Pierie ( 1937) studied the chemical nature of TMV particles& found it to be nucleoprotein.

4
In 1900 Walter Reed & his associates discovered the virus of yellow fever, the first known
viral disease of man. Bacteriophages ( Virus infecting bacteria) were discovered by Twort
( 1915)& d. Herelle ( 1917) independently . The word Virus is derived from a Latin root
meaning a slimy, noxious liquid, sort of living snake venom. Viruses cause many diseases
of humans , animals & plants. Newly recognised viruses eg. human Immuno deficiency
Virus (HIV) are referred to as Emerging viruses.
General Characteristics:- Viruses were originally distinguished from other infectious

agents because they are especially small (filterable) & because they are perfect obligatory
intracellular parasites. The truly distinctive features of viruses are related to their simple
organizational structure &their mechanism of replication. The general characteristics of
viruses are :
1. Viruses contain a single type of nucleic acid , either DNA or RNA , never both.
2. They contain a protein coat (sometimes itself enclosed by an envelope of lipids,
proteins& carbohydrates that surround the nucleic acid.
3. They multiply inside living cells using the synthesizing machinery of the cell.
4. They cause the synthesis of specialized structures that can transfer the viral nucleic acid to other
cells.
5. They are easily transmitted from one organism to another, not affected by antibiotics & can be
crystallised.
6. They reproduce solely through their nucleic acid & are unable to grow or undergo binary fission.
Structure & Morphology: Individual particles of virus are known as virions. A virion is a
complete, fully developed viral particle composed of nucleic acid & surrounded by a
protein coat that protects it from the environment& is a vehicle for transmission from one
cell to another. Viruses are not cellular & therefore do not have a nucleus, cytoplasm or cell
membrane.
The genetic information that permits new generations is usually contained in a single
molecule of either DNA or RNA . The nucleic acid of a virus can be single stranded or
double stranded and accordingly viruses can have either single or double stranded DNA or
single or double stranded RNA depending on the virus, the nucleic acid may be linear or
circular . The protein coating surrounding the nucleic acid core is called a Capsid.
It is the second major portion of the virion . Each capsid is composed of identical structural
protein subunits called Capsomers. The arrangement of capsomers is characteristic of a
particular type of virus. The core with it’s capsid is called Nucleocapsid . These virions are
called Icosahedral virions . The icosahedron is a regular polyhedron with 20 triangular faces
and 12 corners . egs are picrova viruses, adeno viruses , papova virus . Many mammalian
viruses have an envelope outside the capsid , which usually consists of lipids, protiens &
carbohydrates . Viruses whose capsids are not covered by an envelope are called non-
enveloped viruses . 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 .
On the basis of their capsid architecture viruses may be classified into several morphological
types .
1. Helical viruses.
2. Polyhedral viruses.
3. Enveloped viruses .
4. Complex viruses .

4
Helical viruses:- In helical viruses the viral nucleic acid is found with in hollow ,
cylindrical capsid , which has a helical ( spiral or forming a continuous curve round a
central point or axis) structure.
Helical viruses of mammals generally have a lipid containing envelope . The virus that
cause Rabis is eg. of helical viruses .
Polyhedral Viruses :- Many bacterial plant & animal viruses are many sided or
polyhedral . The capsids of most polyhedral viruses have the shape of an icosahedron a
regular polyhedron with 20 triangular faces & 12 corners . eg.herpes simplex virus .
Enveloped viruses :- They are roughly spherical, helical and polyhedral viruses enclosed by
envelope are called as enveloped helical or enveloped polyhedral viruses .
Complex viruses :- They posses complicated structures . eg. bacteriophage .Some

bacteriophages have capsids to which additional structures are attached . eg. Pox virus .
Size and shape :- Most animal virus are almost spherical. The pox viruses are brick shaped
with rounded corners . Influenza virus is usually spherical but sometimes may occur in long
filaments . Plant viruses are rod shaped & bacteriophages are generally tadpole shaped.
Classification :- The history of classification & taxonomy of viruses is similar to that of

bacteria. In 1948 Holmes classified viruses on the basis of their host .
1. Animal viruses.
2. Plant viruses.

5
3. Bacterial viruses .
Animal Viruses ( Zoophagineae) :- They usually have DNA but may also have
RNA & infect man, pigeon , parrot , dog , cow etc & arthopods – insects .
Plant Viruses ( Phytophagineae) :- They have RNA & infect angiosperms like potato,
tobacco, sugar cane and any other higher plants.
Bacterial viruses( Phagineae) :- They have DNA & are called bacteriophages or simply
phages .
Identification :- Because of their minute size viruses cannot be seen with out the help
of an electron microscope , but they are easily identified by serological methods .
These tests are based on detection and identification of virus on the basis of their
reaction with antibodies , which are highly specific for the virus that cause their
formation. Antibodies are useful in the laboratory f o r identifying , quantifying &
isolating virions .
Viral Multiplication :-Bacteriophages are obligate intracellular parasites that infect

bacteria . Almost every group of bacteria is subject to infection by one or more
viruses or bacteriophages . Bacteriophages attacking E. coli are called Coliphages and
are designed as T type. These were numbered T1, T2, T3------ T17. T even phages are
T2, T4, T6 & T3, T5 are called T odd phages .
Morphologically 7 types of Bacteriophages .
T4 is among the largest phages , it is approximately 200 nm long & 80- 100 nm wide.
Other phages are smaller . Most phages range in size from 24-200 nm in length .
Structure :- Bacteriophages are of different shapes .
Head or Capsid : -All phages contain a head structure which can vary in size & shape .
Some are icosahedral ( 20 sides) others are filamentous . The head or capsid is
composed of nucleic acid molecule ( usually DNA) coiled within a protein coat or
5
capsid ( made of capsomeres).
Tail:- The tail is a hollow tube through which the nucleic acid passes during infection.
The tail includes a core , a tail sheath, base plate , tail pins & tail fibres all of which are
composed of different proteins . The base plate & tail fibers are involved in the
binding of the phage to the bacterial cell.
Replication of Viruses :-
Five steps are typical in viral reproduction .
1. Absorption ( or attachment) :- The virus attaches to receptors on the host cell wall.
2. Penetration:- The nucleic acid of the virus moves through the plasma
membrane & into the cytoplasm of the host cell.
3. Replication( Biosynthesis of phage components):- The viral genome contains all
the information necessary to produce new viruses . Once inside the virus induces
the host cell to synthesize the necessary components for it’s replication.
4. Assembly(Maturation):- The newly synthesized viral components are assembled
into new viruses .
5. Release :- Assembled viruses are released from the cell.
Reproductive cycle of Bacteriophages:- Bacteriophages can undergo two

alternative life cycles.
A. Virulent or Lytic infections.
B. Lysogenic infections.
Virulent or Lytic infections. Lytic infection ends in lysis ( the virus destroys the
host cell) and death of host cell. In E.coli lytic infections are caused by a group
seven phages known as the T- Phages .
Lysogenic infections :- The phage reproduces with out killing it’s

host.
Lytic cycle of Bacterial virus eg. Bacteriophage T4.
1. Absorption ( or attachment)- The tail fibers of phage or by some analogous
structure on those phages that lack tail fibres attach to specific receptors on the
bacterial cell. The host specificity of the phage ( ie. The bacteria that it is able
to infect) is usually determined by the type of tail fibres that a phage has .
Tail fibres bend to anchor the pins & base plate of the cell surface.
2. Penetration:- Tail sheath contracts & hollow tail fiber is pushed through the
bacterial envelope . Phages that do not have contractile sheath use other
mechanisms to get the phage particle through the bacterial envelope. Some
phages have enzymes that digest various components of the bacterial
envelope.
3. Replication :- The phage nucleic acid takes over the host biosynthetic
machinery & phage specified m-RNA & protein are made. Phage utilizes the
host cell’s nucleotides to make copies of it’s genetic material & the cell’s
amino acids to make proteins. Early m-RNA’s code for early proteins which
are needed for phage DNA synthesis & for shutting off host DNA, RNA &
protein biosynthesis & for shutting off host DNA,RNA & protein biosynthesis.
After phage DNA is made late m-RNA & late proteins are made.
4. Assembly:- Finally the nucleic acid and protein in components are synthesized,

5
the virus particle is assembled with the protein capsid surrounding the phage
DNA. Enzyme lysosyzme is synthesised.
5. Release:- Bacterial cell wall is weakened by lysozyme action leading to lysis of
bacteria & the release of the mature viruses which spread to nearby cells .
Infect them & complete the cell cycle. The lifecycle of T- phages takes about
25- 35 minutes to complete.
Eclipse period:- The time between infection & assembly of new phage is called
Eclipse period.
Latent Period:- The time between infection & release of viral particles by
destruction of host cells is called latent period.
Lysogenic Cycle Bacteriophage:- The first two steps of this processes are exactly
the same as the first two steps of the lytic cycle ( attachment & penetration).
Those steps are then followed by integration & replication. In the case of some
bacterial viruses , the phage DNA becomes incorporated into the host bacterial
DNA & is referred to as a prophage or temperate phage.
When the bacterial DNA replicates prophage DNA also replicates . Bacterial cells
carrying prophages are called lysogenic cells. Certain external conditions such as
UV light & X-rays can cause temperate viruses to reconvert to a lytic cycle & then
destroy their host cell.

5
Virus Assay:-
1. Particle count:- Direct counts can be made with an electron microscope.
Indirect count can be made using methods such as hemagglutination ( virus particles can
cause red blood cells to clump together or agglutinate).
2.Infectious unit counts are based on the observation that many virion particles may not be
infectious . Plaque assays involve plating dilutions of virus particles , on a lawn of host
cells . Clear zones results from viral damage to the cell . Results are expressed as
Plaque forming units (PFU).
Plaque forming unit (PFU) method:- For determination of bacteriophage number , the
sample should be diluted following serial dilution method. There are two methods for
determining the titres of bacteriophages , first broth clearing method ( in which the end
product is the highest dilution of the sample able to lyse the bacterial cells ) and the
second plaque assay method ( where the plaque titre is determined by counting the number
of plaques of which each represents a single infective virus in the suspension that is
represented by counting the number of PFU multiplying by dilution factor.
Requirements:-
Nutrient broth
Phage stock tubes
Nutrient soft agar
Nutrient agar plates
E.coli culture
Pipette

5
1. Suspend the phage in Tryptophan broth by serial dilution method .

2. Add 0.1ml bacterial culture in molten soft agar tubes kept at 450 c in a water
bath.
3. Now take out 0.1ml from each dilute broth sample of virus & transfer to the soft
agar tubes containing E.coli.
4. Mix the mixture & pour them on the Nutrient agar plate.
5. Incubate the plates overnight at 370 c and observe the plaques.
Since 0.1ml of diluted stock is used. Count the number of plaques then multiply by
6. The number of plaques is PFU/ml of stock.
Determination of one step growth curve of Bacteriophage:-

There are several steps occurring during multiplication of bacteriophage . The phage
comes in contact with suitable bacterial host cell wall ( adsorption ), it’s genome ( DNA )
is injected inside the bacterial cell( penetration ) , where the vegetative phages synthesis
it’s own machinery with the support of host cell to organise viral particles ( synthesis &
assembly) leading to lysis of bacterial cell resulting into cell release of several hundred
phages .This process is called lytic cycle of bacteriophage.
Requirements:-
T4 Phage suspension.
Pure culture of E.coli
Nutrient Agar
Soft Agar
Test tube
Pipette
Water bath
Incubator
Procedure:-
1.Transfer 2ml overnight grown culture of E.coli into a test tube & add 0.1ml of T4
phage suspension.
2.After 5-10 minutes, take out 0.1ml suspension & transfer to 9.9 ml of Nutrient
broth in first tube.
3.Take out 0.1ml from first tube & transfer in the second test tube containing 9.9ml of
nutrient broth ( to obtain 102 &104 dilution respectively).
4.Mix the contents well & incubate the second tube at 370C.
5.Mean while prepare double agar plates by making first the nutrient agar plate
& pouring over it the molten soft agar containing well mixed 3-4 drops of E. coli
suspension.
6.Take out 0.1ml sample from the second tube ( step 4) at different duration ( 0, 15,
30,45 and 60 minutes) and mix with 0.1ml soft nutrient agar containing E. coli & pour
the contents on petri dishes & incubate them overnight at 370C .Observe the plates for
plaque formation .
Result:- Many clear plaques of different size and shape are formed . Count the plaque
number & calculate the PFUs by multiplying with dilution factor.

5
Virus Haemagglutination test for the presence of Antigens:-
Antigens may be any chemical , bacterial cell wall or secondary metabolites or even viral
particles which show antigenicity in blood. The antigenic properties of antigens can be
tested following haemagglutination test . More over when a particular antigen is mixed with
it’s specific antibody in the presence of electrolytes at suitable temperature and pH, the
particles are clumped or agglutinated . Agglutination of erythrocytes is called
Haemagglutination. Therefore agglutination serves as a convenient method for the
detection & assay of antigens.
Requiements:-
Permanent ‘U’ or ‘V’
Microtiter dropper ( 0.025 ml)
Microtiter diluters.
Diluent ( Such as complement fixation test diluents is suitable for influenza.
Antigen ( eg. haemagglutinin).
Serum (heated to 560C for 30 minutes) suitably treated to remove nonspecific
inhibitors ( filter sterilized). RBC of optimum concentration of a mammal like
sheep, goat, mouse etc.
Procedure:-
1. Put one drop of dilution in about 11 wells of the micro titre plate. Use the last well
as control.
2. Put one drop of antigen & 1 drop of diluent in the first well so as to
get 1:2 dilution.
3. Further serially dilute the antigen in the subsequent wells and discard the last
drop ( 0.025 ml) from the last well.
4. Add 1 drop of diluent of each well used .
5. Incubate the well at appropriate temperature so as to settle the cells of control
well to the bottom.
Results:- Read the titre of the antigen has the dilution that gives Haemagglutination.
Cultivation of Viruses:- One major unique characteristic of viruses is that they cannot
multiply outside a living host . Because they contain no metabolic enzymes . Viruses
are unable to use environmental nutrients . They therefore must have nutrients &
enzymes provided by living cells ie. living plants , animals , human hosts or bacterial
cells . Viruses that use bacterial cells as a host ( bacteriophages) are more easily
grown on bacterial cultures .
Cultivation of Chick embryos:- Chick embryos are live animals & one procedure for
introducing viruses is to inoculate them into the chorioallantoic membrane. Viruses
can also be cultivated in embryonated eggs . A hole is drilled in the shell of the
embronated egg and a viral suspension is injected into the fluid of the egg. Virus is
injected in any membrane of the egg , which is most appropriate for it’s growth .

5
Viral growth is indicated by the death of the embryo, by embryo cell damage or by the
formation of typical pocks or lesions on the egg membrane .Embryonated eggs once
most popular for isolation & growth of viruses are now used only for the production of
certain vaccines.
Cultivation of cell culture: Embryonated eggs have been virtually replaced for
experimental purposes with mammalian , avian & other vertebrate cell grown in
culture. All cell culture work requires care & skill to exclude microbial
contamination where as some antibiotics are often used to suppress contamination.
Careful design & maintenance of aseptic area are equally important for this type of
work . cell cultures are more convenient to work with than whole animals or
embryonated eggs because these are rather homogenous collections of cells , which can
be handled & propagated much like bacterial cultures . Viruses can be grown in
primary or continuous cell lines.Cultures made directly from live tissues are primary
cell cultures . Primary cell lines are derived from tissue slices & tend to die out after
only a few generations . Subcultures from the primary culture are called Diploid cell
strains . Diploid cell lines developed from human embryos can be maintained for about
100 generations & hence widely used for cultivating viruses that require a human host.
Multiplication of Human Viruses :- Virus depends on the synthetic machinery of a

host cell for replication because it lacks biosynthetic enzymes. Early study on viral
replication employed the bacteriophage as a model. There are some similarities in the
pattern of multiplication of bacterial and animal or human viruses .The steps of virus
infection and replication can be divided into six sequential phases .
1. Adsorption or attachment .
2. Penetration.
3. Uncoating .
4. Biosynthesis of viral components.
5. Maturation .

5
Chapter 3.
Nutritional requirements, growth and cultivation of bacteria and virus.
Study of different important media required for the growth of aerobic and
anaerobic bacteria and Fungi. Differential media, enrichment media and
selective media , maintenance of laboratory cultures.
Culture :-
Bacteria grow well in vitro on artificial media. They differ in their growth requirements,
so that many different kinds of media must be used, the majority of pathogenic bacteria
grow on blood agar.
Culture medium is a mixture of nutrients used in the laboratory to support growth and
multiplication of microorganisms. The survival and continuous growth of microorganisms depend
on an adequate supply of nutrients and favourable growth envoronment. The nutrient on which
microorganisms are grown in the laboratory is known as a culture medium and the growth itself is
called a culture. Media are used for cultivation, isolation and seperation of microorganisms and
sterility testing. The characteristics of an ideal culture media are:
1. It must give a satisfactory and rapid growth from a single inoculum.
2. It should maintain sterility through out the experiment.
3. It should be reasonably cheap and easily reproducible.
4. It should maintain pH during storage and transport.
General types of Media :-

Media used for the growth of microrganisms are classified as follows.
I) On the basis of state:-
a) Liquid media - eg. Nutrient broth.
b) Semi solid media - eg. Nutrient broth + 0.5% agar.
c) Solid media - eg. Nutrient agar.
II) Based on composition of media :-
a) Simple media or basal media - eg. Peptone water, nutrient agar.
b) Complex media - eg. Fluid thioglycollate media.
c) Synthetic media or defined media - eg. Ashby’s medium.
d) Special media.
i) Enriched media - eg. Blood agar, egg media , chocolate agar.
ii) Enrichment media - Selenite – F broth, tetrathionate broth.
iii) Seletive media - eg. Deoxycholate citrate medium which contains nutrient
agar.
iv) Indicator media - eg. Wilson and Blair media for Salmonella typhi.
v) Hiss’s serum - eg.sterile water with 25 % serum.
vi) Transport media - eg. Stuart’s transport media , Amies transport media.
vii) Sugar media - eg. Peptone water+ 1% sugar.
viii) Storage media - eg. Robertson cooked meat media.
III) a) Aerobic culture media.
b) Anaerobic culture media
c) Facultaive culture media.
Peptone, Casein hydrolysate , meat extract, malt extract , blood , serum, agar , yeast extract and

5
water are the common ingredients for different types of culture media. Culture media gives
artificial environment stimulating natural conditions necessary for growth of bacteria. The basic
requirement of culture media contains energy, carbon, and nitrogen sources, salts like sulphates,
phosphates, chloride and carbontes of sodium , potassium, magnesium and Calcium. Culture media
must provide satisfactory temperature and pH , and adequate oxidation- reduction potential.
Basal ingredients of culture media: Various ingredients , some of which are of plant origin and
animal substances are used in natural media. Often some chemical substances are added to the
natural media to fortify nutrition. In a synthetic medium ingredients are mainly chemical
substances of known quantities. In special cases , when required to study metabolism and other
biochemical factors, some growth factors such as vitamins , amino acids , hormones etc, are also
added to the synthetic media.
1) Protein hydrolyzates:- A wide variety of sources are generally used to provide protein
hydrolysis. Plant animal and milk proteins are also used. They are concentrated and
hydrolyzed using heat with acid. Eg. Meat protein – peptone, milk protein – casein
hydrolyzate. These are rich in nitrogen and carbohydrate.
2) Meat extract and inclusions: Meat or beef is extracted in water and concentrated into semi
solid mass or powder. Meat extract and meat infusions for microbiological use should
not contain any toxic substances and the contents of metal should be low. The extract
should be free from fermentable carbohydrates. These substances are rich in minerls ,
organic micronutrients , protein derivatives , carbohydrates and vitamins.
3) Yeast extract: Yeast extract is prepared by autolysis or plasmolysis of cells of
saccharomyces sp. by heating the cell in water at 50 -60 0C for 30 minutes and concentrate
the extract. It is available in powder and paste form.
4) Agar:- Agar is a solidifying agent used for getting solid medium. These are rich in mineral
and organic nutrients. It dissolves in water at 100 0 C and forms a gel at temperature below
450C. agar is a polysaccharide , which is extracted from several red sea weeds. It is a
mixture of two polysaccharides agarose and agaropectin.
5) Gelatin:- Gelatin is prepared from bones, which is defatted and demineralized to yield a
low ash bone matrix. This is also used for solidification.
6) Carbohydrates: various types of carbohydrates are used in media as energy sources for
heterotropic microorganisms . The complex carbohydrates are polymers of the simple
sugars or their derivatives and are present in plant cell in various forms
Growth requirements :- Most pathogenic ( disease causing ) bacteria are heterotrops ,

( they use the organic compounds as an energy source) their nutritional & metabolic requirements
are sophisticated & they need organic materials ( eg. Carbohydrates, amino acids ) and sometimes
specialized growth factors for culture in the laboratory.
Media:- Basically there are two types of culture media for the cultivation of bacteria. A synthetic
or chemically defined nutrients. A complex medium is the one that contains ingredients of
unknown chemical composition . It is a rich, natural, complex media containing digested extracts
from meats , fish and plants providing the nutrients , vitamins and necessary minerals . In the early
days of microbiology natural, empirical media such as milk , urine, diluted blood , vegetable juices
etc. were used. These are known as empirical media.
Constituents of Culture Media:- While making a culture medium for any microorganism , the
primary goal is to provide a balanced mixture of the required nutrients, at concentrations that will
permit good growth. The following constituents are generally desirable in any culture medium.
1. Water.
2. Sodium chloride & other electrolytes.
3. Peptone - a protein digest prepared from animal or vegetable protein by enzymatic action,
contains peptones, proteases , amino acids etc.

5
4. Meat extract, yeast extract, used to enrich media, contain, protein degradation products ,
carbohydrates , inorganic salts & growth factors.
5. Blood usually defibrinated horse blood, sometimes serum from other animals.
6. Agar- A carbohydrate derived from seaweed , it’s unique property is that it melts at 90 0C
but does not solidify until cooled at 400C. Heat sensitive ingredients can therefore be
added just before the medium sets.
Nutrient Broth :- Consists of water, beef extract & peptone, a protein supplement from plant
or animal sources. Now a days media are prepared from dehydrated ingredients supplied by
commercial firms, these are simply reconstituted & sterilized in the laboratory before use. Such
powdered, dry readymade media manufactured by Himedia , Difco, Oxoid are generally
available in India.
Commonly used solid Media :-
Medium Main constituents Use.
Nutrient agar Nutrient broth, agar. General culture.
Blood agar Nutrient agar, 5-10 % horse General culture mainly used in
blood. medical bacteriology.
Chocolate Agar Heated blood agar. Isolation of H. influenza,
N.gonorrhoeae.
Mac Conkey Agar Peptone – water, Agar, bile Culture of Entero bacteria.
salt, lactose , neutral red .
Desoxycholate citrate agar. Nutrient agar, Sodium desoxy Selective medium for shigellae
cholate, sodium citrate, , salmonella .
lactose, neutral red.
CLED Agar (cystine lactose Peptone- L-cystine, lactose, Culture of enterobacteria.
electrolyte deficient medium) bromothymol blue.
Xylose lysine desoxy cholate ( Nutrient agar, xylose, lactose, Selective medium for
XLD) agar. sucrose, lysine, sodium shigellae, salmonella.
desoxycholate, ferric
ammonium citrate, phenol red.
Lowenstein–Jensen A mineral salt solution, Culture of M.tuberculosis.
(Contains no agar but glycerol, malachite green,
solidified by egg) whole egg.
Antibiotic sensitivity Peptone in a semisynthetic Antibiotic sensitivity test.
medium to avoid antibiotic
inhibitors .
Solid media :- solid media are solidified fluid media , dispensed in plastic petri plates.
Agar- The settling agent, added in a concentration of 1.5% to give the medium the consistency of
a firm jelly. When agar is added to solidify, the nutrient broth, the product is called Nutrient agar.

6
Nutrient agar Nutrient agar
Chocolate Agar Blood agar
Fluid Thioglycolate medium : For sterility testing in Pharmaceutical formulations.

L- Cystine 0.5gm
Dextrose 2.5gm
Granular agar 0.75gm
Yeast extract 5.0 gm
Pancreatic digest of casein 15.0 gm
Sodium thioglycolate 0.5gm
Resazurin(0.10% fresh solution) 1.0ml
Distilled water to 1000ml
Alternative Thioglycolate medium :

L-Cystine 0.5gm
Sodium Chloride 2.5gm
Dextrose 5.5gm
Yeast extract 5.0gm
All the media are adjusted to pH 7.1± 0.2 and sterilized by autoclaving at 121 0 C for 20 minutes.
Sterility of these media should also be tested .
Method of Inoculation :- The specimens or cultures of bacteria are plated or streaked out on the
medium with a wire loop in such a way as to ensure a reducing inoculam ,this means that after
incubation , separated colonies will develop where individual bacteria have been deposited.

6
Advantages & uses:-

1. Solid medium is of great value because it allows separate colony formation.
2. Colonial morphology enables many bacterial species to be identified . A film made from
the colony & stained by Gram’s method establishes the microscopic features of the
bacterium; Gram+ ve or Gram-ve coccus or bacillus.
3. Quantitation:- The numbers & relative proportions of different bacterial species originally
present in the specimen can be estimated.
4. Pure cultures:- Can be obtained by picking isolated bacterial colonies on the fresh solid
medium necessary for full investigation .
Enrichment media :- Enrichment media are fluids , which encourage the preferential growth of a
particular bacterium they contain inhibitors for contaminants that might other wise overgrow the
pathogen . for eg. Whole blood is added to nutrient medium to encourage the growth of
Neisseria species, the media is heated before solidified, the process disrupts the red cells & release
the haemoglobin. As the media finally has charred brown appearance , it is also known as
chocolate agar.
Disadvantages:-
1. Identification of bacteria by morphology is not possible.
2. No estimate can be made of the numbers or the relative proportion of different species
of bacteria originally present in the specimen.

6
1.Identification of bacteria by morphology is not possible.

2.No estimate can be made of the numbers or the relative proportion of different species
of bacteria originally present in the specimen.
Selective media:- are solid media containing ingredients , which inhibit unwanted contaminants
but allow certain pathogens to grow. Mannitol salt agar encourages the growth of staphylo cocci
but inhibits other gram (+ve) bacteria. Another eg. is Eosin methylene blue ( EMB) agar that
contains carbohydrates fermented by E.coli & other Gram (-ve) bacteria , but it also contains eosin
& methylene blue, which inhibit gram+ve bacteria.
Mannitol salt Agar
High salt ( Nacl) concentration in medium favours organisms that tolerate high salt concentration
eg. Staphylococcus .
MacConkey agar was the first formulated solid differential media but now known as a selective and
differential culture media commonly used for the isolation of enteric Gram-negative bacteria.Based on
the bile salt-neutral red-lactose agar of MacConkey.
The presence of Crystal violet and bile salts incorporated in MacConkey agar help to inhibit the
growth of Gram-positive bacteria making it a selective media and fastidious Gram-negative bacteria,
such as Neisseria and Pasteurella. Gram-negative enteric bacteria can tolerate bile salts because of
their bile-resistant outer membrane.

6
MacConkey agar is selective for Gram negative organisms, and differentiate lactose fermenting gram
negative rods from non lactose fermenting gram negative rods. It is primarily used for detection and
isolation of bacteries members of family enterobacteriaceae and Pseudomonas spp.
MacConkey agar is used for the selective isolation and identification of Enterobacteriaceae from
samples such as feces, urine, wastewater and foods.
Principle of selective and differential ability of MacConkey agar
Those Gram-negative enteric bacteria that grow on MacConkey agar are differentiated by their ability
to ferment lactose (Sugar). Fermentation of lactose by bacteria leads to the production of the acid that
drops the pH of the media. The drop in pH is indicated by the change of neutral red indictor to pink
(Neutral red appears pink at pH’s below 6.8).
Strongly lactose fermenting bacteria produce sufficient acid which causes precipitation of the bile
salts around the growth. It appears as a pink halo surrounding individual colonies or areas of
confluent growth. Pink halo in not seen around the colonies of weaker lactose fermenting bacteria.
As for the selective ability of MacConkey agr,Gram-negative bacteria that grow on MacConkey agar
but do not ferment lactose appear colorless on the medium and the agar surrounding the bacteria
remains relatively transparent.
Composition of MacConeky Agar and the functions:
Enzymatic digest of Gelatin, Casein and Animal tissue: provides nitrogen, vitamins, minerals and
amino acids essential for growth.
Lactose: fermentable carbohydrate providing carbon and energy.
Bile Salts: selective agents and inhibit Gram positive organisms.
Crystal Violet: Gram positive bacteria are generally inhibited by crystal violet.
Sodium Chloride: supplies essential electrolytes for transport and osmotic balance.
Neutral Red: pH indicator. which is red in color at pH’s below 6.8.
As a resulut of lactose fermentation, the pH of the medium decreases, changing the color of neutral
red to pink
Agar : Solidifying agent
Composition of Mac Conkey Agar

Proteose peptone or polypeptone – 3g
Peptone or gelysate -17 g
Lactose -10 g
Bile salt No.3 - 1.5 g
Nacl -5g
Neutral red - 0.03g
Crystal violet - 0.001g
Agar -13.5g
Distilled water - 1 liter
According to manufacturer instruction,Suspend the measured amount of powder (See in the agar
bottle and generally 50 gram) in 1 L of distilled water and mix thoroughly. Heat while constantly
agitating and boil for 1 minute to completely dissolve the powder. Autoclave at 121°C for 15 minutes.
Colonies appearance on MacConkey Agar

Lactose-fermenting organisms appears pink to brick red colonies with or without a zone of
precipitated bile.
Non-lactose fermenting organisms appears colorless or clear colonies

6
Identification of lactose fermenters and non lactose fermenters MacConkey Agar
Lactose Fermenter Organisms as seen on MacConkey Agar:

 Citrobacter spp.: colonies are light pink after 48 hours.
 Klebsiella spp.: Mucoid lactose fermenter colonies
 Escherichia coli: Lactose fermenter; flat, dry, pink colonies with a surrounding darker pink area of
precipitated bile salts.
 Serratia spp.: Late lactose fermenter; S. marcescens may be red pigmented, especially if the plate is
left at 25°C
Non Lactose Fermenter Organisms as seen on MacConkey Agar
 Proteus spp.: may swarm depending on the amount of agar in the medium; characterize by a foul
smell
 Shigella spp.: Shigella sonnei produces flat colonies with jagged edges.
 Yersinia spp.: may be colorless to peach.
 Salmonella spp.: Not a lactose fermenting
 Other organisms showing colorless colonies on MacConkey agar are; Edwardsiella spp, Hafnia spp.,
Morganella spp., Providencia spp.
As for gram positive bacteria like Staphylococcus aureus,no growth is observed
Liquid media :- Dispensed in tubes with compressed paper cotton wool stoppers or in screw
capped bottles. Growth is recognised by turbidity in the fluid.
Medium Main constituents Use

Nutrient Broth. Peptone water, General culture.
Meat extract.
Peptone water. Peptone, Nacl, H2O General culture, Basal
medium for sugar
fermentation tests.
Glucose Broth. Nutrient broth, glucose. Culture of delicate
organisms.

6
Robertson’s Meat Nutrient broth, minced meat. Culture of anaerobic &

medium aerobic bacteria.
Tetra thionate Broth Nutrient broth. Sodium Enrichment culture for
thiosulphate, Iodine salmonella.
Selenite ‘F’ Broth Peptone water sodium selenite. Enrichment culture for
shigellae & salmonella.
Nutrient broth in screw cap tubes
Nutrient broth in conical flask
Robertson’s Meat Medium
Tetra thionate broth

Selenite – F Broth is used as an enrichment medium for the isolation of Salmonella from feces,
urine, water , foods and other materials.
Sodium selenite inhibits the growth of gram positive and many gram negative bacteria including
Enterococci and Coliforms , where as the Salmonella are not affected .
Sodium selenite is highly toxic at near neutral pH.

6
Uses:- Some bacteria particularly if present in small numbers , will grow only in fluid media,
occasionally a specimen contains inhibitory substances eg. Antibiotics, which are diluted by
inoculation into a fluid medium, thus allowing bacterial growth.
Fluid media with special constituents ( eg. Sugars) are widely used to test the biochemical activities
of bacteria for identification.
Media for blood cultures :-
It is packed in two bottles with rubber seals & a perforated metal cap ; inoculated by injection of
aseptically collected blood through the hole in the cap with a syringe, they contain.
1. For aerobic culture : nutrient broth sometimes with an agar slope set on the narrow
side of the bottle so that colonies can be observed.
2. For anaerobic culture : nutrient broth with sodium thioglycolate (a reducing agent),
alternatively. Robertson’s meat medium.
Transport medium:- It is used for the preservation of delicate pathogens during transit to the
laboratory. For eg.Stuart’s transport medium is a semi solid non nutrient agar with thioglycolic
6
acid & electrolytes . Originally devised for the general transport Neisseria gonorrhoeae it is used as
a general transport medium.
Incubation:-
1.Atmosphere:- a) Most human pathogens grow in air but the addition of 10 % CO 2 is essential for
the isolation of some species & enhances the growth of many others. b) Anaerobic bacteria require
incubation without oxygen . Plates of media are inoculated & are placed in a sealed jar from
which the air is removed . Hydrogen & CO 2 are liberated inside the jar by means of a
commercial gas generating system, the remaining oxygen combines with hydrogen in the presence
of a catalyst to form water.
2. Temperature :- The optimal temperature for the growth of most pathogens is body
temperature ie. 370c, a few bacteria require a higher & some a lower temperature.
Pure culture Technique:- For various reasons a pure culture of microorganisms may often be
required but in nature microorganisms usually occur in mixed cultures. Thus preparation of a pure
culture involves the isolation of a given microorganism from a mixed natural microbial
population & the maintenance of the isolated individual & it’s progeny in an artificial
environment is easily created for example within the confines of a test tube, a flask or petri dish
to exclude the external environment , which is the primary source of contamination. The whole
operation of transferring the inoculum into a sterilized container having sterilized medium can be
carried in an aseptic hood or before a laminar flow bench, to minimise the chances of
contamination . The inoculum is commonly introduced on a metal wire or loop that is rapidly
sterilized immediately before it’s use by heating in a flame where as liquid culture can be
transferred by pre-sterilized pipettes. Commonly used methods are briefly described below.
Plating method :- Pure cultures of some microorganisms eg. Yeast, most bacteria , many fungi &
unicellular algae form discrete colonies on solid media. Isolation of such microorganisms involves
the separation & immobilization of individual organisms on or in a nutrient medium generally
solidified with agar . Under favourable growth conditions each viable organism gives rise to a
colony from which transfers can be readily made. In plating method Petri plates containing

6
solidified nutrient medium are prepared. A dilute suspension of microbial material ( mixed culture)
is also prepared. A sterilised bent wire is dipped into a suitable diluted suspension of organisms &
is then used to make a series of parallel , non-overlapping streaks on the surface of already
solidified agar plate. This mode of streaking causes gradual dilution of the inoculum & hence
well developed isolated colonies develop along the lines of later streaks. Alternatively , successive
dilutions of the inoculum can be poured on about to solidify media poured plates & mixed ,
subsequent dilutions give rise to well developed isolated colonies.
Plating methods are generally satisfactory for the isolation of bacteria & fungi because most of
these microorganisms can grow well on solid media. However , some of the larger celled
bacteria , many protozoa & algae are cultivable only in a liquid medium. The simplest procedure of
isolation in liquid media is the dilution method. The inoculum is subjected to serial dilution in a
sterile medium & a large number of tubes of medium are inoculated with aliquots of each
successive dilution.
There may be situations in which a pure culture is difficult to obtain & in such cases the
alternative is to obtain the next best degree of purification in the shape of a two membered
culture, which contains only two kinds of microorganisms. Two membered culture is the only
possible way to maintain viruses, since these organisms are all obligate intracellular parasites of
cellular organisms. Many of the protozoa , which feed in nature on smaller microorganisms , are
also most easily maintained in the laboratory as two membered cultures in association with their
smaller microbial prey.
Methods of obtaining pure cultures of organism.
Microorganisms are found in almost every environment ( soil, water, air) as a mixed population. To
study the specific role played by a specific microorganism in it’s environment, it is imperative to
isolate them in pure culture. A pure culture contains only one kind of microorganism & involves not
only the isolation of individual microorganisms from a mixed population but also the maintenance
of such individuals and their progenies in artificial media, where no other microorganisms find way
to grow. Pure cultures are essential in order to study, colony characteristics, biochemical
characteristics, morphology, staining reactions & immunological reactions or the susceptibility to
antimicrobial agents of a particular strain of bacterium or fungus. However it is not easy to isolate the
individual microorganisms from natural habitat & grow them under imposed laboratory conditions .
A great deal of laboratory manipulations is required if inoculum from natural habitat is taken &
allowed to grow in a culture medium, a large number of diverse colonies may develop due to
crowdedness may run together & there by lose individuality. There fore it is necessary to make the
colonies well isolated from each other so that each appears distinct , larger & shows characteristic
growth forms, such colonies may be picked up easily & grown separately for detailed study.
Several methods of obtaining pure cultures are in use.
The most commonly used methods are.
1. Streak plate method.
2. Pour plate method.
3. Spread plate method.
4. Serial dilution method.
Streak plate method:- The method was developed by Loeffler & Gaffkey & is most commonly used
method to isolate pure cultures of bacteria. A small amount of mixed culture is placed on the tip of
an inoculum loop/ needle which is streaked across the surface of the medium. The successive streaks ‘
thin out’ the inoculum sufficiently & microorganisms are separated from each other in the form of
discrete colonies that grow from a single cell spore. Generally the second plate is streaked out by the
same loop needle with out re-inoculation.

6
Continuous streaking method
Discontinuous streaking method
Principle :-
The method is based on the principle that by streaking a dilution gradient gets established across
the surface of the petriplate as bacterial cells are deposited on the agar surface. Because of this
dilution gradient , confluent(together) growth takes place on the part of the medium where few
bacteria are deposited .Each colony is the progeny of a single microbial , cell thus representing a
clone of pure culture. Such colonies may be picked aseptically & re- streaked on to fresh media to
ensure purity of a particular strain.
2.Pour plate method:- In this method , successive dilutions of the inoculum ( original specimen
serially diluted) are added into sterile petri plates to which is poured melted & cooled agar medium &
thoroughly mixed by rotating the plates which is then allowed to solidify ( or the inoculum is diluted
in tubes containing liquefied agar medium). After incubation, the plates are examined for the
presence of individual colonies growing through out the medium. The pure colonies are isolated ,
transferred into broth cultures & thus pure culture is obtained.
Principle :- The main principle is to dilute the inoculum in successive tubes containing liquefied
agar medium so as to permit a through distribution of bacterial cells with the medium. The mixed
culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the
liquid state at the temperature of 42 0C -450c. The bacteria & the melted medium are mixed well. The
contents of each tube are poured into separate petri plates, allowed to solidify & then incubated .
When bacterial colonies develop, the isolated colonies develop both within the agar medium ( sub-
surface colonies) & on the medium ( surface colonies). These isolated colonies can be picked up &
streaked on to another agar plate to ensure purity of the strain.
Procedure 1:-
1.Prepare liquefied agar tubes & allow to cool to 450 C. Also numbering the test tubes 1-5.
2.Transfer 1ml of a suspension of a mixed culture of organisms to test tube 1. Shake well.
3.Transfer 1ml of the suspension from tube 1 to tube 2and repeat the same procedure up to tube 5 to
get the appropreiate dilutions of 10-1 ,10-2,10-3,10-4,10-5.
4.Transfer 1ml of the bacterial suspension each from tubes 1-5 to sterile petri plate using sterile
pipettes.

7
5.Pour the liquefied agar medium in tubes into each petri plate containing 1ml of diluted
suspension.
6.Rotate the plates gently to ensure uniform distribution of bacterial cells in the medium & allow the
medium to solidify.
7.Incubate the plates for 24-48 hours at 370C in an inverted position.
Observation:-The progressively poured plates will contain lesser number of colonies which will be
distributed more or less sparsely in the plates. These isolated colonies may be sub-cultured on to
other fresh media to ensure purity of strain.
Procedure 2
1. Prepare liquefied agar tubes by placing the solidified agar tubes in a water bath at desired
temperature.
2. Inoculate tube 1 with loopful of bacteria . Mix thoroughly.
3. Transfer a loop ful of suspension from tube 1 to tube 2 shake well.
4. Transfer a loopful of culture from tube 2 to tube 3. Shake vigorously to allow thorough
distribution of inoculum with in the tube.
5. Pour the contents of each tube separately on three sterile petri plates. After the medium gets
solidified incubate the plates at 370C for 24- 48 hrs in inverted position.
6. Evaluate the plates for pure culture of a particular strain.

7
Pour plate method for pure culture
3.Spread plate method:-

In this method, microorganisms are spread over the solidified agar medium with a sterile L- shaped
glass rod called spreader when the petri plate is spinning on a turn table.
Principle :- The method relies on the fact that petri plate spuns , a stage reaches when single cells will
be deposited with the bent glass rod on to the agar surface. Some of these cells will be separated from
each other by a distance sufficient to allow the colonies that develop to be free from each other.
Spread plate method
Procedure:-
1. Take three Nutrient Agar plates& label them with the name of the organisms to be
inoculated.
2. Aseptically inoculate the plates with a loopful of the given organisms.
3. Place plate 1 on the turn table ( revolving).
4. Sterilize the spreader by putting it first in ethanol ( 95%) in a beaker, then on the flame of
Bunsen burner & cool the rod for 30 seconds.
5. Remove the lid of plate & spin the turn table.
6. Touch the spreader gently on the surface of agar & move it forth and back to spread bacterial
cells on the agar surface when the turn table is spinning.
7. When turn table stops spinning , put the lid over the lower half of petri dish.
8. Sterilize the spreader again & repeat the same process for the other two plates.
9. Incubate all the plates at 370C for 24 hrs.
Result:- Some colonies will be seen that grow individually with out over lapping with other
colonies. It may be picked up & purified further by sub-culturing.
4.Serial dilution method ( viable plate count method).

This method is commonly used to obtain pure culture of those microorganisms that have yet not been

7
successfully cultivated on solid media & grow only in liquid media. Here the inoculum is subjected
to serial dilution in a sterile liquid medium & a large number of sterile liquid medium& a large
number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution. The
aim of the 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 instance , we have a culture
containing 10 ml of liquid medium & 1000 microorganisms meaning 100 microorganisms /ml of
liquid medium. If we take 1ml of this medium& mix it with 9 ml of sterile liquid medium, we have
then microorganisms in 10 ml or 10 microorganisms /ml. If we add 1ml of this suspension to another
9 ml of a sterile liquid medium, each ml would contain a single microorganism. If this tube shows any
subsequent growth of microbial colonies in it, there is probability that this growth resulted from the
introduction of a single microorganism in the medium& represents the pure culture of that
microorganism.
Procedure :-
1. Take 9 ml of sterile physiological saline or sterile distilled water in a sterile test tubes
numbering from 1-5( the number of dilutions is not fixed for any particular bacterial
suspension/specimen).
2. Transfer 1ml of suspension of a given mixed culture to test tube 1. Shake the tube well.
3. Transfer 1ml of suspension from test tube 1 to test tube 2. Shake well.
4. Repeat the same process for the other test tubes so as to get the desired dilutions
(10-1,10-2,10-3,10-4,10-5 ).
5.Transfer 1ml of the suspension from each dilution to sterile petri plates separately to which
melted & cooled agar medium is poured.

6 The plates are rotated gently to allow distribution of microorganisms in the medium.
7.The plates are allowed to solidify & incubated at 37 0c for 24-48 hrs in inverted positions.
Serial Dilution Method
Incubate at 370C for 24 hours
Observations:- The individual colonies will appear incubation , which may be picked up & sub-
cultured on fresh media to ensure purity of a particular strain.
Types of cultures:-
The different types of cultures obtained after their isolation from different specimen in their pure

7
form ( pure cultures ) are classified as

1.Streak culture – Such cultures are obtained by drawing an inoculating needle/loop containing
specimen or suspension in straight lines of different patterns over the surface of agar solidified
media.
2.Stab culture:- These cultures are also called stick cultures & are prepared by inserting an
inoculation needle for some distance straight into the agar deep tubes ( half way). Growth of such
media occur along the lines of insertion that results in change in the characteristic of media.
3.Stock cultures:- These are pure cultures of known species of microorganisms maintained in ideal
laboratory condition for longer periods of time in viable state. Such cultures act as a stock of a
particular microbial species of their strains.
4.Starter Culture:-These are some microorganisms which act up on specific substances in the
medium and result in the production of specific end products. These end products serve as boasting
dose for the activation of some other microorganisms ( utilized by some other microorganisms )
which carry further the desirable reactions in the medium to serve the purpose of their cultivation.
These cultures are called starter cultures & are most frequently used in fermentation processes. eg.
Lacto bacillus , are used as starter culture in dairy industry in manufacturing fermented milk
products.
5.Enrichment culture:- In this culture , growth of a particular organism is favoured against a
mixed population by adjusting nutritional requirements ( like carbon source, oxygen source, nitrogen
source , source of light etc. & environment requirements ( like temperature , pH etc.).
6.Batch culture:- Such cultures are used to study the growth characteristic of microorganisms.
Growth of microorganisms in a limited volume of culture medium represents a batch culture. For
instance , a flask with constant volume of medium inoculated with a given microorganism &
metabolic end products get accumulated in the medium which slows metabolism & hence growth rate
of the microorganism.
7.Continuous culture:- In such cultures , fresh nutrients are continuously supplied & end products
used continuously , maintaining exponential growth of bacterial population at constant rate for
longer periods of time.
8.Synchronous culture:- A culture in which all microbial cells growing are physiologically identical
& in the same stage of cell division.
9.Feb- batch culture :- Feb batch culture is a hydride of batch culture & continuous culture &
involves ordinary batch growth of microorganisms until the medium present in the fermentation
products.
For isolation of anaerobic bacteria by plating method the inoculated plates must be incubated in
closed containers , from which the oxygen is removed either by chemical absorption or evacuation.
A modification of this method is known as dilution shake culture. A tube of melted & cooled agar
medium is inoculated & mixed , approximately 1/10 of it’s contents is transferred to a second
tube , which is then mixed & used to inoculate a third tube in a similar fashion. After 6 to 10
successive dilutions have been made , the tubes are rapidly cooled & sealed, by pouring a layer of
sterile petroleum jelly & paraffin on the surface. Such sealing prevents access of air to the agar
column. In this method the colonies develop deep in the agar column & are thus not easily
accessible for transfer. Successful cultivation of strictly anaerobic microorganisms would
obviously require extraordinary measures to exclude even traces of oxygen at all times.
In roll tube method , the tube contains a few millilitres of molten agar medium that has been
reduced chemically to remove dissolved oxygen & is tightly stoppered with a butyl rubber bung.
The molten agar is inoculated with appropriate dilutions of the bacterial sample by inserting them
through the rubber stoppers with a sterile syringe. The tubes are allowed to solidify with rolling so
as to deposit a thin layer on the inner wall. After incubation the colonies are picked from the agar

7
with a needle or capillary tube. A redox dye, resazurine , is often used to ensure that some entry of
air has not inadvertently occurred. Strict anaerobes can also be isolated using conventional streak
plate techniques.
Maintenance & Preservation of Pure Cultures :-
After the isolation of any microorganism in pure culture, it is imperative to maintain the viability &
purity of such microorganism by keeping the pure culture free from any contamination. To preserve
microorganisms , it is necessary to reduce their metabolism up to it’s minimum due to which the
processes that lead to ageing & death are slowed down & the microorganisms can be maintained
in its inactive state for several years .
There are several methods of preservation of pure cultures but some commonly used methods are
1) Refrigeration.
2) Paraffin method ( store in mineral oil).
3) Lyophilization (freeze drying).
4) Cryopreservation ( storage in liquid Nitrogen).
5) Storage at – 700C.
6) Storage on glass beads at -600C to – 700C.
7) Storage in gelatin discs.
8) Storage in soil.
1.Refrigeration :- Pure cultures can be stored successfully in refrigerators or cold rooms at

0- 40C. This method is applied for short duration ( 2-3 weeks for bacteria & 3-4 months for fungi) ,
because the metabolic activities of the microorganisms are greatly slowed down but not stopped.
Thus their growth continuous slowly , nutrients being utilized & waste products released in the
medium, which finally results in death of the microbes after some time.
2.Paraffin method ( Storage in mineral oil):-

This is a simple & most economical method of maintaining pure cultures of bacteria & in fungi. In
this method , sterile liquid paraffin or pre sterilized mineral oil or medical paraffin ( specific gravity
0.83- 0.89) is poured over the slant ( slope) of culture & stored up right at room temperature. The
layer of paraffin ensures anaerobic condition ( reduced oxygen tension ) & prevents dehydration of
the medium. This condition helps microorganisms of pure cultures to remain in a dormant state &
therefore , the culture is preserved for several years.
Procedure:-
1.Grow the culture in suitable medium in a culture tube till full growth is achieved.
2.Sterile the liquid paraffin in an oven for 1 hr at 1800C.
3.Add sterile paraffin to a level of 1 cm above the highest point of agar slope.
4.Plug the mouth of the tube with cotton plug or metal cap & store in refrigerator .
Revival of the culture :- The culture is revived by putting a loopful of the culture on to a fresh
agar( plates) or broth (tubes) which are incubated till growth resumes.
3.Lyophilization ( Freeze drying ):- In this method the culture is rapidly frozen at a very low
temperature -700C and then dehydrated by vaccum. Under these conditions the cells are dehydrated &
the metabolic activities are stopped, the microbes undergo dormant state & retain viability for years.
Those lyophilized pure cultures are then sealed & stored in the dark at 4 0C in a refrigerator.
Procedure :-
1. Sterilize the ampoules in an oven for 1 hr at 1800C.
2. Pour 0.5 ml of a broth culture into a sterilized ampoule.
3. Push a cotton plug completely into ampoule.
4. Connect the ampoule to a hole in the centrifuge head of freeze drying apparatus .

7
5. Centrifuge is continued for 5-10 minutes which results in rapid dehydration & freezing of
the contents of the ampoules .
6. Allow these for primary dehydration for 5 hrs.
7. The necks of the ampoules are drawn out & the ampoules are given secondary dehydration for
12 hrs.
8. Seal the ampoules under vacuum & store these at 4 0C on the desk.
9. Examine the ampoules for sealing using vacuum tester . Production of a blue glow is
indicative of sufficiently low pressure. ie. The proper sealing of the ampoules.
Revival of the culture:- During opening of an ampoule , the ampoule neck is flamed, a
stretch is made with a file around the ampoule, corresponding to the equator of the cotton –
wool plug & open the ampoule by applying pressure at the stretch mark.
10. Remove the cotton wool plug, flame the ampoule neck.
11. Add 0.2ml of broth into the ampoule with a sterile pipette & keep it for
rehydration for a few minutes.
12. Using a sterile pipette, transfer the rehydrated culture to a broth tube.
13. Incubate the tube at 370 c for 24 hrs.
14. From the culture , streak the agar plate aseptically.
15. Incubate the plate at 370 c for 24 hrs & study morphology of a given culture.
4.Cryopreservation ( Store in liquid Nitrogen):- This method is suitable for long term preservation
of microorganisms that do not survive freeze drying. Some microorganisms are sensitive to the
effects of cooling & warming. Such loss is reduced by the use of cryoprotectants such as glycerol &
dimethyl sulphoxide ( DMSO). In Cryopreservation the microorganisms of a pure culture are rapidly
frozen in liquid nitrogen at -196 0 c in presence of stabilizing agents ( glycerol/ DMSO) that prevent
the formation of ice crystals which may kill frozen cells. By this method , some cultures retain
viability for 10- 30 years with out any alteration in their physiology or morphology.
Procedure :-
1. Grow the culture on a suitable medium in sterile petri dish.
2. Add 4ml of 10% glycerol ( v/v) or 5% DMSO on the growing culture plate.
3. Scrap the culture with sterile pipette .
4. Four 0.5 ml of the cell suspension in cryo tube ( meant for storage ).
5. Transfer & store the culture in liquid nitrogen.
Revival culture :- The culture can be revived by keeping it at room temperature for 2- 3 hrs
and there after transferred on to suitable media aseptically .
6. Incubate the plates at 350 c for 24 hrs until growth resumes.
5.Storage at -700 c :-
The low temperature deep freezers are recommended for storage of certain microorganisms.
Procedure :-
1. Grow cell under cryopreservation conditions.
2. Transfer 0.5 ml suspension in small glass screw capped tube.

7
3. Store the tubes at -700 c.
6.Storage of glass beads at -600 c to -700 c.

Some microorganisms fail to show viable colonies due to repeated freezing & thawing method. For
such frozen bacterial cells with a cryoprotectant in glass beads is a reliable method. In this method ,
the individual glass bead is removed when needed with out thawing the entire sample. It is
mention worthy that various groups of microorganisms can be maintained by using beads of
different colours. The animal pathogens are stored on red beads , plant pathogens on green
& microbes require special growth factors on blue . The alkalinity should be neutralized by using
dil.Hcl.
Procedure :-
1. Grow the culture on a suitable media.
2. Prepare the broth medium recommended containing 15 % glycerol & auto clave it.
3. Suspend the culture in 10ml of the above medium & remove the culture by scrapping to
form a thick suspension.
4. Pour 0.5ml of suspension in each bead containing screw capped vial & freeze vial at -60 0
c to -700 c.
Revival of culture:- The culture can be revived by removing the beads by rubbing over
the surface of suitable solid medium with the support of a wire loop.
5.Incubate the plates at desired temperature till growth resumes.

The glass beads are neutralised by dil.Hcl, washing with tap water followed by through rinsing with
deionized water is recommended . After drying , 20-30 beads , in screw cap glass vial are added &
used for autoclaving.
7.Storage in gelatin discs.

In this method , bacterial strain after growth on a suitable medium is suspended in molten nutrient
gelatin . The drops of the molten gelatin are allowed to solidify on petridish & allowed to dry over a
dessicant. The method is recommended for preservation of various species of Entero bacteriaceae,
Staphylococcus etc.
Procedure :-
1. Grow the culture on a suitable medium , add 3ml of gelatin containing pre molten
medium consisting of
Gelatin powder – 10 gm
Meso – inositol- 5gm
Nutrient powder – 2. 5 gm.
2. Pipette out 0. 5ml of bacterial suspension .
3. Place 0.02ml suspension on a petri plate , cover it & keep at -20 0C until frozen. The
drop will become opaque.
4. Transfer the plate to freezer dryer for dryness.
5. The silica gel containing screw capped glass tube or vial & transfer thesolid drop or gel
into it , plug it tightly.
6. Store at 50C to be revived & used a pure culture.
8.Store in soil. Various fungi like Rhizopus, Aspergillus, Fusarium, Alternaria, Penicillium,
Melanospora etc, can be stored in sterile soil for longer periods of time.

7
Procedure:-
1. Grow the culture on a suitable medium until spore are formed.
2. Suspend the spores in 1ml of sterile water.
3. Place the garden soil in a bottle, auto clave twice or thrice & pour sterile water
containing spore suspension in to it.
4. The bottle is left at room temperature to allow the culture to grow for 10 days.
5. Store the bottle with cap loose in refrigerator.
Revival of culture:- The culture can be revived by spraying few soil particles on of a given
culture.
Cultivation of anaerobic microorganisms.

The cultivation of aerobes and facultative anaerobes is comparatively easy .Growing strict anaerobes ,
on the other hand , requires special equipment and nutritional media. Some anaerobic
microorganisms grow in complete absence of oxygen, while few can tolerate small amount, if
exposed to air. A number of methods namely Wright’s tube method, brewer’s Petri dish method,
vacuum and gas displacement method are known. The candle method and the gas Pak anaerobic jar
method are more popular. Each method has it’s particular application.
Experiment 1: Candle method for cultivation of Anaerobic Bacteria.
This method is based on a lighted candle placed in a jar tightly sealed with Vaseline. When candle
stops burning , it shows that the oxygen has been used and anaerobic condition created.
Requirement
Candle
Match box
Bacterial culture
Bell jar
Nutrient agar plates.
Procedure:
1. Streak the culture on the surface of solid nutrient agar in two Petri plates.
2. Keep one plate out side and the second inside the jar.
3. Place a candle inside the jar and wait for some time . The candle will stop burning due to
absence of oxygen and production of CO2. This method is recommended for the growth of
Neisseria . For most of the organisms, however this method is considered too toxic due to
production of CO2 as a result of burning the candle.
4. Observe the growth of bacteria and compare the results with the plate kept outside.

7
Result: If the bacteria is aerobic , induced growth in the plate kept out side will be observed as
compared to bacterial growth in plate kept inside the jar. On the other hand, if the bacterium is
anaerobic, induced growth in culture kept inside the jar will occur as compared with that of out side
bacterial culture.
Exp. 2 Candle Jar method for cultivation and differentiation of anaerobic Bacteria.
Requirement
Bell jar
Vaseline
Candle
Bacterial culture
Nutrient agar plate
Procedure:
1. Place the petri dish streaked with all the three cultures namely, bacillus subtilis, cl.
sporogenes and cl. rubrum in the centre of the glass jar.
2. Fix a lighted candle over it.
3. Apply Vaseline on the bottom of bell jar to ascertain that the jar to glass plate seal is air
tight.
4. Once the candle flame quits, place the entire assembly at 37 0C in the incubator for 24 – 48
hours .
5. Record the growth.
Results:
The anaerobic bacteria such as cl. sporogenes and cl. rubrum will show profuse growth while bacillus
subtilis will be inhibited due to lack of oxygen.
Exp. 3 GAS PAK anaerobic jar for cultivation of Anaerobic bacteria.
This method is based on hydrogen generation in the jar. It can react with available oxygen to form
water. Palladium pellets catalyse the reaction at room temperature. To ensure whether anaerobic
atmosphere exists in the jar , an indicator strip of methylene blue is used that becomes colourless
in the total absence of oxygen. If the strip is not decolourised within two hours, the gas contains
oxygen and chemical reaction failed to occur. The jar is not sealed properly. The entire assembly is
placed at 370 C in an incubator for 24- 48 hours and the growth is recorded.
Requirements:
Gas Pak anaerobic jar
Palladium pellets
Methylene blue
Bacterial culture
Nutrient agar plates.

7
Procedure:
1. Keep the streaked plates in the jar containing gas pak indicator strip of methylene blue and
gas pak generator envelope.
2. Pull the strip half the way so as that the change in colour is visible.
3. Cut the envelope and add 10 ml of the tap water into the open envelope.
4. Close the whole assembly as per instruction and keep at 37 0C.
5. Check the jar after 2-3 hrs to note if the indicator strip has lost it’s blue colour.
6. After 24-48hrs of incubation , remove the lid. If a vacuum holds the inner lid firmly to the
jar, break the vacuum by sliding the lid to the edge.
7. Examine the plate after staining and evaluate the plate and observe the spores.

8
Chapter 4. Different method used in isolation and identification of

bacteria with emphasis to different staining techniques and
biochemical reactions. Counting of bacteria- Total and viable
counting techniques.
Microbiological Methods :-
The very small size of the microorganisms poses certain problems in their study & this
makes microbiology a different science. One of the basic microbiological methods is
making preparations for microscopic examination.
1.Hanging drop preparation:- Microorganisms in a natural state are best viewed when
suspended in a clear fluid. A clean glass cover slip is taken & a drop of the fresh culture
( 6 hrs old ) is placed in the centre. Soft paraffin is applied at the margins of the cover
slip to protect the drop from the external environment, a clean , grease free ,
grooved glass slide is inverted over the cover slip & quickly reversed ,taking care that
the drop does not touch the slide, but remains in the centre . The hanging drop
preparation is then examined under microscope.
and reveals the size, shape , arrangement & one most important characteristic , the motility of
the organisms.
2.Wet ( unstained )films:- Drops of liquid specimens are placed on a slide & covered with
a cover slip. The microorganisms in the wet film are examined with high power dry
objective (40X), final magnification with 10X eye piece is 400X. A wet film is used to
observe bacteria & other cells, specially bacterial motility.
3.Stained Films:- A drop let of sample ( eg. Suspension of bacteria in water is smeared
on the glass slide & dried in air. It is passed through a flame. This treatment kills any
live bacteria & fixes them over the slide. The process is called heat fixing. Flooding with
appropriate dye solutions ( basic dyes such as crystal violet or methylene blue ) stains
heat fixed smears of specimens of bacterial cultures. Basic dye have +ve charge & are
attracted to cytoplasm that is -vely charged. Thus simple staining stains the cytoplasm
of bacterial cell. Stained bacteria are examined with the oil immersion objective (100X)
using 10X eye piece, the final magnification being 1000X.
4.In negative staining :- The back ground is coloured as against simple staining in
which bacteria is coloured. The sample of bacteria is mixed with an acidic dye
(nigrosin,Eosin, Indian Ink). This mixture is then smeared on a slide & air dried.
Because of its -ve charge the acidic dye is repelled by the cytoplasm & hence gathers
around the –vely charged cells (when an acidic dye is added, the bacteria form a
complex with the cations of the dye which are colorless & the anion of the acidic dye
which is coloured gives a coloured back ground).
The bacterial cells appear as clear or white objects on a coloured back ground. An
important advantage is that cells are observed in near natural condition as the technique
avoids chemical reaction& heat fixing.

8
Microscopy :-
Light microscopy :- The basic microscopic system used in microbiology is the

compound microscope ( light microscope or bright field microscope), a two lens system
with the objective lens nearer the objective & the ocular lens nearer the eye. Light
passes through the objective lens, forming an inverted real image. This image serves
as an object for the ocular lens which re-magnifies the image & forms the virtual
image. The lens system of the eye perceives (understand) this image & captures it on
the retina.
Usually there are three objective lenses in a light microscope , the low power lens (10
X), the high power lens ( 40X) & the oil immersion (100X) . The ocular lens further
magnifies the real image . Thus a 10X ocular lens will show (10X10 =100X) or (40X10
=400X) or (100X10= 1000X) ie. 100 times, 400 times or 1000 times magnification.
When 10x, 40X or 100 X objective lenses are employed . The oil ( eg.Caedar wood oil)
used in oil immersion microscopy provides a homogenous pathway for light from the
slide to the objective & resolution of the object increases.
The resolving power (RP) or resolution of a lens system is

calculated as RP= λ/ 2xNA.
Where λ represents the wave length of light ( usually set at 550 nm), and NA is the
numerical aperture of the lens. NA refers to the size of the cone of light that will
enter the objective & the medium in which the lens is suspended ( usually air).
RP = 550nm/ 2x0.25 = 1100 nm= 1.1μm.
The RP of an oil immersion lens with a NA of 1.25 can be calculated as

RP=550nm/2x1.25 = 220 nm=0.22 μm.
Thus in the first case an object larger than 1.1 μm. & in the second case an object
larger than 0.22 μm would be visualised.
Q. Write the functions of different components of compound microscope. 2 marks.
Parts of a microscope.

8
1)Oculars : A series of lenses (5X, 6X, 10X,15X) that magnify the object and correct s some of the
defects of the objective , Huygenian, , Ramsden and compensating oculars are commonly
used in microscopy.
2)Objectives : The objective is the most important lens on a microscope because its properties
make the final image . The objective lenses generally equipped with microscope are low power ,
high power and oil immersion lens having magnification of 10 X , 40 X or 45X and 100 X
respectively . Functions of the objective lens are to gather the light rays coming from any point of the
object and to unite the light at a point of the image and magnify the image . There are three types
of objectives like achromatic, fluorite and apochromatic.
3.Condenser : This component is found directly under the stage and contains two sets of lenses
that collect and concentrate light passing upward from the light source into the lens system. There
are several different types of condensers , depending upon the type of microscopy eg. Abbe
condenser, variable focus condenser and achromatic condenser.
4.Iris Diaphragm : It is equipped with a condenser . It controls the intensity of light entering the
condenser . A lever is equipped with it to adjust the light intensity .
5.Illumination ( Light source ) : The light source is positioned in the base of the instrument . Some
microscopes are equipped with a built in light source to provide direct illumination . Others are
provided with a mirror , with one side flat and the other concave . An external light source , such as
a lamp , is placed in front of the mirror to direct the light upward into the lens system. The flat side
of the mirror is used for artificial light and the concave side for sunlight.
6.Body tube : Above the stage and attached to the arm of the microscope is the body tube . The upper
end of the tube contains the ocular or eye piece lens . The lower portion consists of a movable
nosepiece containing the objective lenses . It also provides sufficient space for image formation .
7) Revolving nose piece : A base in which the objectives are fixed and it holds 2 to 4 objectives
and which can be revolved to align the required objective .
8.Focus adjustment knobs : There are two focus adjustment knobs , a coarse adjustment and a fine
adjustment . Coarse adjustment knob is used to bring the object into focus and fine adjustment
knob is used for fine and clear focus of specimen.
9.Mechanical stage : It is a platform on which the specimen to be viewed is placed . Some stages
have clips to hold the glass slide . Others have a mechanical stage , which makes it possible to
move the slide across the stage .
2.Dark ground microscopy :-
Use of a special condenser results in oblique illumination of the specimen. Light

does not enter the objective directly but only when scattered by bacteria , which
appear brightly illuminated against a dark background. The technique is helpful in the
diagnosis of diseases caused by spiral bacteria. Eg. Treponema pallidum that causes
syphilis . The unstained organism ( dia≡ 1.5 μm) are seen moving about with
characteristic rotary motion.
3.Phase – contrast microscopy:- A special condenser & objective are used, direct
light from the source & light scattered by structures in the field are so that when
direct & diffracted beam unite they are not in phase. Details of the object appear as
differences in intensity & the contrast produced shows details of the fine structure
of unstained, living micro organisms. Internal details not visible with the light
microscope & fine structures of yeasts, molds & protozoa are studied by this
technique.
4.Fluorescence microscopy :- This uses ultra violet light. Bacteria or cells coated
with fluorescein, auramine or other suitable fluorescent dye alter the wavelength &
become visible as bright objects against a dark ground. Fluorescent antibody
technique is based on fluorescence microscopy & used to identify the unknown

8
organisms.
5.Immunofluorescence :- It combines serology (diagnostic identification of
antibodies in serum) with fluorescence microscopy by using antibody labelled
with a fluorescence dye ( eg. Fluorescein isothiocynate, lissamine, rhodamine) to
detect specific antigens (is the substance that binds specifically to the respective
antibody) & to identify bacteria.
6.Electron microscopy:- Electron microscopy has immensely added to our
understanding of the structure & functions of microorganisms by permitting us
access to their inner most secrets. A beam of electrons allows resolution
(separation) of extremely small objects .eg.0.001 mm, the electrons are focused by
electromagnetic fields & the image is visualised & can be photographed on a
fluorescent screen, used for the study of the ultrastructure of bacteria.
Electron microscope is of two types . Transmission Electron microscopy ( TEM) is used
to photograph detailed structures with cells. A total magnification of about 20 million
times can be achieved & objects as small as 2.0nm can be seen. Scanning Electron
microscopy (SEM) is used to visualise the surfaces of objects in the natural state ,
without sectioning . SEM offers even greater effective resolution than TEM.
Staining :- The cytoplasm of bacteria lacks colour & therefore staining techniques
greatly facilitate the study of microorganisms by imparting them colour.
Staining is further divided into two categories.
a) Simple staining
b) Negative staining.
c) Differential staining. This is further divided into several methods.
1) Gram’s staining . 2. Ziehl –Nelsen staining (acid fast staining).
Fixed stained preparations are used for observations of the morphology of various types
of bacteria and other microorganisms. This method has various advantages.
The cells are clearly visible after staining.

The difference between the cells of different species and also within the same species
can be demonstrated by using appropriate staining technique.
Stains are classified on the basis of nature of staining component into acidic, basic and
neutral dyes.
An acidic dye (anionic) is one in which the dye ion is negatively charged. Eg.
Nigrosin. A basic dye (cationic) is one in which the dye ion is +vely charged.
Eg. Crystal violet.
Hence positive staining can, easily stain the bacteria using any basic dye. In
positive staining basic stains are used because the bacteria have lipids,
phosphate , nucleic acids etc. making them acidic in nature.
Hence a complex is formed between the positive part (or basic part) of the dye
that is coloured and the negative part of the bacteria to stain the bacteria. Eg.
Methylene blue is represented as MB+ or Cl- . ie. Methylene blue chloride. The
ion exchange occurring may be represented as :
Na+ (Bacterial cell) + MB+ Cl- --- ( Bacterial cell component) MB+ + Nacl.

8
Composition of staining reagents:-
1. Loefflers methylene blue: Methylene blue - 0.2 gm.
Absolute alcohol -10ml.
Distilled water - 90ml.
Procedure- Dissolve methylene blue in alcohol, add water and filter.
2. Crystal violet Solution A- Crystal violet -2gm.
Ethyl alcohol (90%) -20ml.
Solution B –
Ammonium oxalate -0.8gm.
Distilled water -18ml.
Procedure – Mix solutions A and B store for 24 hours.
3.Safranine : Safranine -2.5 gm.

Alcohol - 10ml.
Water- 90 ml
Procedure- Dissolve Safranine in alcohol and
add water.
Procedure for staining:-
1. Clean the plane slide thoroughly with cleaning powder and wash with water. Heat
the slide gently to destroy organisms adhering to it. Again wipe it with clean cotton
piece.
2. Sterilize the inoculation loop; transfer a loop full of distilled water on to the slide.
3. Transfer a small quantity of colony on to the drop aseptically with the help of a
loop and make a suspension of organism in water.
4. Spread the suspension with inoculation loop to make a smear.
5. Fix the smear by gentle warming by passing the slide over a burner.
6. Flood the smear with stain and allow it to stand for 2 minutes.
7. Wash the excess stain by exposing it to a thin stream of distilled water. Dry the
slide in air after wiping with blotting paper.
8. observe the slide under oil immersion lens using cedar wood oil ( 100 X).
Negative staining:
The principle involved here is that when an acidic dye is added, the bacteria form a
complex with the cations of the dye which are colourless and the anion of the acidic
dye, which is coloured, gives a coloured back ground. Hence the bacteria remain
colourless over a coloured background and can be observed under a microscope.

8
Bacteria + ( Cations)+ (anions) → ( bacteria) ( Cations) + anions
Colourless complex coloured
Negative staining has certain advantages over positive
staining.
1. In positive staining , fixation of the bacteria is done to the slide by slightly

warming. This may change the shape of the bacteria, which is prevented in
case of negative staining, as the smear is not fixed.
2. Slightly acidic bacteria, with basic dye when stained , do not get properly
coloured or stained, as they are less acidic. Therefore such organism, negative
staining can be done. Eg. for lower class of bacteria like spirochetes.
3. Negative staining is also done for capsulate bacteria to study the capsules.
Some of the acidic dyes are Nigrosin, eosin, Indian ink etc.
The disadvantage of the negative staining is that when the smear is kept for a
long period of time, the cells may shrink which is not seen in case of positive
staining.
Procedure.
1. Take a clean slide and dry it.

2. Place a drop of the acidic dye at the end of the slide.
3. With the help of an inoculation loop following aseptic technique add a loop
full of organisms and mix it with the dye.
4. With the help of another clean slide spread the suspension by dragging it
along the first.
5. Dry the smear in air, add a drop of cedar wood oil and observe under the
microscope using 100 X objective.
Q. Simple staining ? 2 marks.
Simple staining:- In simple staining only one dye is used. A smear of the sample is

8
fixed on a clear glass slide & any simple aniline dye solution is applied by flooding the
smear with it for about a minute. Then it is washed off with gentle stream of cool water
dried by pressing between two pieces of filter paper& finally examined under a
microscope . Bacteria generally take up basic dye such as methylene blue, crystal violet,
safrarin, Eosin Y & basic fuchsin. This is because most bacteria react towards stains
as though they are composed of acidic components; nucleic acid, acidic
polysaccharides , proteins etc.
Q. Explain the principle and procedure of Gram’s staining ? 5 marks.
Gram’s staining
Developed by Christian Gram in 1884 in Denmark, Gram staining is one of the most
fundamental & most widely used technique for the differentiation & identification of
bacteria . It is not only reveals the shape & size of the bacteria but also enables them
to be differentiated immediately into two categories . Gram +ve & gram-ve hence it is
also known as differential staining.
In this procedure the cells are stained with a basic dye (crystal violet), treated with an
iodine – potassium iodide mixture to fix the stain, washed with alcohol or
acetone ( decolorizer) , & counter stained with a polar dye of a different colour ( eg.
Safranin).All bacteria take up the initial blue- purple stain but only gram+ve ones
retain it during the subsequent steps. Gram –ve, bacteria takes up the counter stain &
appear as red objects in contrast with violet cells of gram-ve bacteria.
The mechanism of gram staining is related to the properties of cell wall. The gram +ve
organisms have a much thicker cell wall, which retains the dye iodine complex by
preventing it’s elution. In ageing cultures the cell wall deteriorates & hence gram+
ve cells often become gram –ve. It is believed that crystal violet & iodine form a
chemical complex in the bacterial cytoplasm. Since gram-ve bacteria have a high lipid
content in their cell walls, alcohol dissolves the lipid & permits crystal violet iodine
complex to leak out the cytoplasm . Gram +ve bacteria trap less of the complex
because of their considerably less peptidoglycan content.
Gram’s Staining
Aim :- To study the morphology of a given organism by Gram’s staining method.
Requirements:- Microscope, plane slide, Bunsen burner , Inoculation loop, Staining

material , culture tube containg organisms.
Discussion :- Gram’s staining is an emperical method of differentiating bacterial species

into two large groups ( Graam – positive and Gram negative based on the chemical and
physical properties of their cell walls. The gram staining is almost always the first step in
the identification of bacterial organism. While Gram staining is a valuable diagnostic tool
in both clinical and research settings , not all bacteria can be definitively classified by this
technique , thus forming Gram variable and Gram indeterminate groups as well.
Mechanism:-
Gram positive have a thick mesh like cell wall made of peptidoglycan ( 50 – 90% of cell
wall), which stains purple while Gram negative bacteria have a thinner layer ( 10 % of
cell wall), which stains pink. Gram negative bacteria also have an aadditional outer
membrane which contains lipids , and is seperated from the cell wall by the periplasmic
space. There are four basic steps of the gram stain , which include applying a primary stain
( crystal violet) to a heat fixed smear of a bacterial culture, followed by the addition of a
trapping agent or mordant ( Gram’s iodine) rapid decolorization with alcohol or acetone,
and counter staining with safranin.

8
Crystal violet ( CV) disssociates in aqueous solution into CV + and chloride ( Cl-) ions.
These ions penerate through the cell wall and cell membrane of both Gram+ ve and Gram
– ve cells. The CV+ ion interacts with negetively charged components of bacterial cells
-
and stains the cells purple. Iodine (I- or I3 ) interacts with CV+ and forms large
complexes of crystal violet and iodine ( CV-1) within the inner and outer layers of the
cell. Iodine is often referred to as a mordent, but is a trapping agent that prevents the
removal of the CV-I complex and therefore colour the cell.
When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the
cell membrane. A Gram –ve cell will lose its outer lipopolysaccharide membrane and the
inner peptidoglycan layer is left exposed. The CV-I complexes are washed from the Gram
–ve cell along with the outer membrane . In contrast , a Gram +ve cell becomes
dehydrated from an ethanol treatment. The large CV-I complexes become trapped with in
the Gram +ve cell due to the multilayered nature of it’s peptidoglycan. The decolorization
step is critical and must be timed correctly; the crystal violet stain will be removed from
both Gram +ve and Gram -ve cells if the decolorizing agent is left on too long.
After decolorization , the Gram +ve cell remains purple and the Gram –ve cell loses it’s
purple colour. Counter stain , which is usually +vely charged safranin or basic fuchsin, is
applied at last to give decolorized Gram –ve bacteria a pink or red colur.Gram positives
sometimes get converted to Gram –ve which may be due to
a) Removal of magnesium ribonucleic acid.

b) Rupture of the cell wall.
c) Treatment with lysozymes or antibiotics.
d) In old cultures.
2. Special stains :- These are used for demonstration of volution granules

(Albert’s or Neisser’s stain), capsules ( Muir’s method ), spores ( Schaffer &
Fulton’s ), flagella , spirochaetes ( Fontana’s or Levaditi’s method ) , rickettsias
( Giemsa’s or Machiavelli’s stain ) etc. for identifying particular chemical
constituents, for identifying antigens (fluorescent antibody technique), & for staining
bacteria in tissue sections etc.
Staining for acid- and alcohol fast bacilli (Usually tubercle bacilli).
.Ziehl- Neelsen method OR Acid fast staining method:– The heat fixed smear of the
material to be examined is flooded with concentrated carbol fuchsin, heated to 900 C
over a steam bath for 4 minutes, washed decolourised with acid & alcohol, and finally
counterstained with methylene blue or malachite green. Red bacilli are observed
against a blue back ground. The technique is used to identify members of the genus .
Mycobacterium eg. M. tuberculosis causing tuberculosis. The Mycobacterium species
is said to be acid fast or acid resisted because of a very high content of mycolic
acids, a waxy material in the cell. As it differentiates acid- fast bacteria from non-acid
fast ones this method of staining is also called as differentiated.
Requirements:-
Cultures:- A) Broth culture ( 72 hrs ) of Mycobacterium tuberculosis and B)
Broth culture ( 24 hours) of Staphylo coccus aureus.
Stain:- Carbol fuchsin, methylene blue, malachite green.
Reagents: Acid- Alcohol( 3% HCl+ 95% ethanol), 20 %
H2SO4.
Principle:- Acid – fast staining is another widely used differential staining
procedure in bacteriology. This stain was developed by Paul Ehrlich in 1882. Ziehl
and Neelsen independently proposed acid fast stain in 1882-1883, which is
commonly used today. Some bacteria resist decolourisation by both acid and
alcohol and hence they are referred as acid fast organisms. Acid- alcohol( 3% Hcl+
95% ethanol) is a very intensive decolouriser . This staining technique divides

8
bacteria into two groups 1. Acid fast and 2. Non acid fast .This procedure is
extensively used in the diagnosis of M. Tuberculosis and M. Leprae.
Acid fastness property in certain mycobacteria and some species of Nocardia is
correlated with their high lipid content ( 60% w/w). Due to high lipid content of
cell wall, acid fast cells have relatively low permeability to dye and hence it is
difficult to stain. For the staining of these bacteria, penetration of primary dye is
facilitated with the use of 5% aqueous phenol which acts as a chemical intensifier.
Heat is also used which acts as a physical intensifier. Once these cells are stained, it
is difficult to decolourise with acid and alcohol.
The acid fast staining uses three different reagents.
i) Primary stain ( carbol fuchsin):- Carbol fuchsin , a phenolic stain that is
soluble in the lipoidal material which constitute the major portion of the
mycobacterial cell wall allows penetration and retention of red stain.
Penetration is increased by the application of heat, which drives the carbol
fuchsin through the lipoidal wall and into the cytoplasm. All cells appear red
in colour after application of primary stain.
ii) Decolourising agent ( acid- alcohol or 20% H 2SO4): Smear is cooled before
decolourisation which allows the waxy cell substances to harden . On
application of decolourising agent, acid- fast cells shows resistance to
decolourisation. Primary stain is more soluble in the cellular waxes than
decolourising agent. Hence primary stain is retained by mycobacterium
species but this is not the case of non acid fast bacteria that lack cellular waxes.
Primary stain is easily removed during decolourisation and non acid fast cells
observed as colourless.
iii) Counter stain ( Methylene blue or malachite green):- Non acid fast bacteria
absorb the counter stain and appear as blue or green colour while acid- fast cells
retain the red colour of primary stain.
Procedure:- Prepare a smear of given bacterial suspension . Allow smears to air
dry and then heat fix it. Flood the smear with carbol fuchsin stain and heat the
slide from below till steam rises for 5 minutes. Do not boil the stain and ensure
that stain does not dry out. Allow the slide to cool for 5 minutes to prevent the
breakage of slide in the subsequent step. Wash with tap water. Decolourise the
slide by using acid- alcohol or 20 % sulphuric acid until carbol fuchsin fails to
wash from smear. Wash with water and counter stain with 1% aqueous solution of
malachite green or methylene blue for 1 to 2 minutes. Wash smear with tap water,
dry and examine under oil- immersion objective.
Observations:- A) Smear A shows thin bright red, slightly curved bacilli with
beaded appearance showing palisade arrangement. Thin bright red, slightly curved
, beaded appearance are the features of M. Tuberculosis ie. Acid- fast bacteria.
B) Smear B shows blue coloured cocci arranged in clusters which
means that the organisms may be staphylococci.
Auramine method :- It is a modification of Ziehl –Neelsen method in which carbol

fuchsin is substituted by auramine . Auramine is fluorescent dye. The culture is treated
with auramine – phenol decolourised with acid & alcohol & finally KMno 4 is applied.
Fluorescent yellow bacilli are observed in the dark field under UV light.
Bacterial identification.
Identification of bacteria isolated by culture from a specimen proceeds in the
following stages.Colonial & microscopic morphology – The appearance of bacterial
colonies is often characteristic & colonies of many pathogenic bacteria can be
recognised on the primary cultures. Colonies or growth formed on different solid
8
media gives important information about the unknown organisms. Colonies are
observed in respect to size, margin, elevation, type of growth & type of liquefaction. A
stained film of the colony may be required for identification & in any event most
bacterial species require confirmatory test.
1.Conditions required for growth:- These also help to identify bacteria. Eg. Whether
aerobic or anaerobic growth conditions are required ability to grow on simple or only
on enriched or selective media.
2.Biochemical Tests:- The organisms having same morphology & cultural

characteristics may have marked differences in their biochemical tests listed in Table
are principally used for Enterobacteria.
Common Biochemical Tests for Identification of Bacteria:-
Substrate Test Observation
Carbohydrate Metabolism
Various Carbohydrates. a) Aerobic (Oxidative) Acid, sometimes with gas.
b)Anaerobic( fermentative) Acid, sometimes with gas.
Glucose Voges Proskauer Acetyl methyl carbinol.

Sodium citrate Citrate utilization Growth.
Protein Metabolism
Gelatin Gelatin liquefaction Liquefaction.
Lysine, ornithine, Amino acid Carbon dioxide.
Arginine Decarboxylases.
S containing amino acids. H2S production. H2S
Phenyl alanine Phenyl alanine deaminase Phenyl pyruvic acid.
Tryptophan Indole Indole
Other Tests.
Urea Urease Ammonia
Hydrogen peroxide Catalase Oxygen
Redox dye Oxidase Oxidation to purple colour.
Potassium nitrate Nitrate reduction Nitrite.
Method:- After inoculation & incubation , results are scored to give a numerical profile.
This is compared to a profile complied statistically from type cultures & enables
identification to be made with a known degree of certainty.
Recognition of Enzymes:- Although enzyme production is the basis of most of the
reactions included in the biochemical tests listed in the table, some bacteria can be
identified primarily by production of a characteristic enzyme for
eg. a) coagulase : - clots plasma ( characteristic to Staphylo coccus aureus.
b) Lecithinase:- produces opacity in egg or serum medium ( if inhibited by

Clostridium perfringens antitoxin, identifies this organism- the Nagler reaction.
Antigenic structure:- Serology is the definitive method of identification. In bacteriology,
it depends mainly on recognition of antigens in flagella , cell wall, capsule or
liberated from the bacteria as toxins. It is particularly useful for the large numbers of
biochemically similar enterobacteria ( eg. Salmonella). Various immunological
techniques are used , most often agglutination, but also precipitation, sometimes

9
neutralisation of toxin.
Typing of bacterial strain:-
It is often necessary to identify individual strains or types within a bacteria species to

trace epidemic spread of an organism.
1. Antigenic typing:- To distinguish different strains of bacteria by their

antigenic structures.
2. Bacteriophage typing:- Strains of an organism differ in their susceptibility to a
series of bacterial viruses or bacteriophages; patterns of lysis with more than
one phage are usually detected.
3. Bacteriocin Typing:- Bacteriocins are proteins released by bacteria, which
inhibit the growth of other members of the same species, strains may be
differentiated on the basis of bacteriocines they produce by observing the
patterns of inhibition of a series of test strains.

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Enumeration of Microorganisms:- In order to standardise an inoculum or to

determine the extent of microbial contamination or to illustrate the phenomenon of
growth & multiplication or other wise, we may have to ascertain the number of
microorganisms in any population. Numerical counts of bacteria are determined by
various methods of enumeration.
1. Direct methods:- In direct methods the number of bacteria present in a
specimen is counted after making suitable dilutions so that the numbers are
easily manageable & counting is simplified . Total count includes the
numbers of both live & dead bacteria where as viable count excludes the
dead or non- viable organisms.
a) Cell counts :- The method is similar to that used in counting of RBCs in a
haemocytometer. The total count per cubic millilitre from a suitably diluted
specimen is first obtained & multiplied by the dilution factor. Numbers of the
order of at least 1x107 per ml of suspension are sufficient for statistical validity
of the count.
b) Smear counts:- In this method an exact volume of the culture is smeared over
an exact area of a slide , stained with any appropriate dye & organisms are
counted in a known portion of the total area. Such smear counts give total
count.
c) Comparative :- We know that 1ml of blood contains above 5 million
erythrocytes per cubic mm. If 1ml of male human blood is mixed with 1ml of
culture& a smear of the mixture is prepared , an estimate of the number of
bacteria may be obtained by counting both blood and bacterial cells in a
certain number of fields and noting
their proportions. Finally we can estimate the total number of bacterial cells

9
by comparing their proportions associated with erythrocytes in the mixture.

This method also gives the total count.
d) Membrane filter counts:- The method consists of passing appropriate
dilution of the specimen suspension through a sterile membrane filter and
directly counting the retained bacterial cells after staining. The filter can be
made transparent by saturating it with immersion oil to facilitate the
counting. This gives a total count of live and dead bacteria.
e) Electronic counters:- A dilute suspension of specimen is passed between
electrodes that causes an interference with the electron beam due to different
conductivities of the cells and the suspending fluid. The extent of interference
is recorded electronically and is proportional to microbial cell size. This is
very rapid and accurate method of making total count.
2. Indirect Methods :- Unlike the direct methods we count the number of cells
indirectly based on certain constant parameters of bacterial cells.
a) Determination of total volume:- A standard volume of the culture is placed in
a graduated centrifuge tube, subjected to centrifugation at a standard speed
for an exact time, and the packed average volume of the individual cells and
the reading of their packed volume, an estimate of total numbers can be made.
This is a total estimate.
b) Turbidometric methods:- A measured volume of the culture is placed in a
special , clear glass tube of known diameter and interposed between a
unit source of light and a photoelectric unit, attached to a galvanometer. Of
the total light from the unit source, the percentage transmitted through the
tube will be diminished in proportion to the turbidity. This is the quickest ,
simplest and reasonably accurate method and is widely employed in
microbiology. Using numerically standardised suspensions of bacteria,
turbidity readings can be standardised in terms of number of cells by
haemocytometer counts or electronically . Turbidometric measurements are
also made in microbiological assays.
c) Chemical methods :- Such methods are more commonly useful industrially in
measuring heavy growths of filaments microorganisms but seldom for bacteria
. These methods are based on quantitative determination of substances. ( eg.
amino acids, nitrogen, nucleic acids or phosphorous ) that are always present
in fairly constant amounts in living cells & calculating the numbers from the
knowledge of content of a substance per cell. Proteins usually contain
about 18% nitrogen & can be determined by Kjeldahl method or indirectly
by means of Folin reagent , which gives a colour reaction with tyrosine &
tryptophan, two amino acids always present in protein in fixed amounts.
d) Dry weight measurements:- These measurements are also more useful in
measuring the growth of molds in certain phases of industrial work. The
specimen is effectively washed , dehydrated completely or to a constant
degree and accurately weighed. The increase in weight represents
biological synthesis & with the data on cell volume available can be used
to calculated cell numbers.
3) Dilution methods :- These methods involve the dilution of the specimen to
a degree when the last dilution contains easily countable microorganisms .
Serial dilutions :- As the name implies, the serial dilutions ( usually decimal or ten
fold) of the specimen are prepared & placed in tubes of nutrient broth, incubated
& the presence or absence of growth is recorded. By using the dilution factor & the
numbers present in a particular dilution , the total count , usually a range , present in
the specimen per ml can be calculated mathematically . This number of organisms
per ml is known as the indicated number, which is the reciprocal of the highest
dilution. But if the determinations are simultaneously made in duplicate or triplicate
then the most probable numbers can be calculated from the tables, given in standard
books. However the most probable number is also not the exact number . Any
organism that will grow as colonies on solid laboratory media can be enumerated by

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serial dilution method, if the bacterial suspension from highest dilution , is inoculated
, incubated & developed colonies are counted . Samples or dilutions of fluid
specimens may be mixed with melted nutrient agar at a temperature of about 420C
in cylindrical vials called roller tubes. While agar is still fluid these vials are rotated
rapidly until the agar solidifies in a thin film evenly distributed over the inside surface
of the tube. After incubation , colonies are easily counted in the film of agar . The
method is especially valuable in field trips. Methods based on cultivation of cells
give only viable count & not the total count because in viable count only viable
organisms grow to form colonies. The total count of a sample may be equal to or
usually greater , but never less, than the viable count.
The pharmaceutical microbiologist must be able to enumerate microorganisms
present in a pharmaceutical product and production environments. Direct observation
using a microscope and microbial replication to production environments. Direct
observation using a microscope and microbial replication to produce visible colonies
or turbidity are common techniques used for counting the microorganisms. Direct
microscopy methods are laborious in nature and difficult in determining cell viability.
Hence it is less encountered than plating techniques.The basic unit of microbial
enumeration is the colony forming unit or c.f.u. The underlying assumption is that
one viable microorganism produces one c.f.u
Direct microscopy method :
Aim :- to determine the total count of microorganisms in a culture ( liquid ) by

using direct microscopy method ( haemocytometer method).
Requirements:- Escherichia coli or Bacillus subtilis liquid culture.

Chemicals : acetone, alcohol. Saline solution.
Apparatus: R.B.C. pipette, coverslip, Bunsen burner, test
tube.
Equipments:- microscope, counting chamber
( Haemocytometer)
Principle:- In the haemocytometer or counting chamber method , a minute drop of the
culture is placed in a tiny , shallow , rectangular glass slide called Neubauer’s slide.
This is a special slide accurately ruled into squares that are 1/400 mm2 in area. A
suspension of unstained bacteria can be counted in the chamber , using a phase
contrast microscope.
Total number of bacterial cells/mm3 = No. of cells counted x dilution
Area counted x depth of fluid

Since cells are counted in 5 bigger squares and such a square is further divided
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into 16 small squares.

Each small square is equal to 1/400 sq.mm.
Hence , area of 5x16 = 80 squares = 80/400 mm2 = 1/5 mm2
It is a special microscope slide with a counting chamber 1/10 mm deep so that
volume of liquid over a one square = 1/10 mm .( The depth between the coverslip
& the counting chamber is 1/10mm).
Dilution = 1:200 =200. As the dilution is
200. Hence number of bacterial cells
counted = = N x200
--------- = Nx200x50= Nx10000 cells/mm3
1/5x1/10
Advantages:-
1. Direct microscope counts can be made rapidly.

2. Minimum equipments required for this technique.
3. Morphology of cells are also observed.
4. Concentrated suspension of cells can be counted if they are diluted appropriately.
Disdvantages:-
1. Live as well as dead microorganisms are also counted.

2. Due to motility of cells , counting errors are possible.
3. Accuracy might decline, if the suspension is very dense or concentrated.
Procedure:-
Keep the slide under the microscope and adjust the squares at 10x. Then keep

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coverslip and adjust at 45x. Take bacterial culture up to 0.5 mark of R.B.C. pipette
and dilute the suspension by using diluting fluid up to 101 mark. Pipette is rotated
rapidly by keeping it horizontal for mixing the culture. Discard first few drops from
the pipette and a small volume of fluid is introduced on counting chamber under
coverslip.
Allow the cells to settle for 2 to 3 minutes. Place the counting chamber on the edge of
stage of microscope. Adjust the light and count total number of microorganisms by
using large
R.B.C. squares in the centre with 25 small squares.
Observations and Results:- Observe the morphology of microorganisms in form of

size , shape and arrangements. Check the motility of microorganisms. Total number of
microorganisms found in given culture by direct microscopic method is total number
of cells counted in 80 squares x 10000 /mm3.
Plate count method ( Serial dilution method)
Aim :- To determine total viable cells in a bacterial culture by plate count method or
serial dilution method.
Requirements:-
Culture:- Liquid culture of Bacillus subtilis or Escherichia coli , water, milk or

food samples.
Media:- 20 ml nutrient agar deep

tubes (3). Apparatus:- Test tubes,
pipette, petri plates,
Equipments: Autoclave, hot air oven, incubator, Quebec colony counter, water
bath, incubator.
Principle:-
The plate count method is most commonly used for enumeration of viable cells in
water, milk, food and many other pharmaceutical substances. This method is used for
determination of the number of the cells that multiply under defined conditions. A
measured amount of sample or bacterial suspension is introduced into an agar
medium ( liquid form at 450C) and after mixing, add into petri plate. All organisms
grow, reproducing a visible mass of microorganism called colony. The development
of one colony from one microorganism can occur when the bacterial suspension is
homogenous. If microorganisms have a tendency to aggregate ( eg. Staphylo cocci,
strepto cocci, diplo cocci) that resulting counts will be lower than the actual number of
individual cells.Hence counts of microorganisms are often reported as colony
forming units/ ml rather than number of bacteria/ml. The original sample is usually
diluted so that the number of colonies developing on the plate will be in the range of
30 -300. Within this range the count can be accurate and the possibility of mixing of
the growth of one organism with other is minimized.
The total count of microbial suspension is obtained by multiplying the number of cells
per plate by the dilution factor.
Procedure:
Mix the bacterial suspension by rolling the test tube between the palms of hands to
ensure even dispersion of cells in the culture. By using sterile pipette , aseptically

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transfer 1ml from the bacterial suspension to first flask containing 99ml saline
solution. Discard the pipette in the beaker of disinfectant. The bacterial suspension has
been diluted 100 times (10-2). Mix the contents of the first flask and transfer 1ml
suspension to the second flask ( containing 99ml of saline solution) with a sterile
pipette. Thus original culture is diluted (10-4). Mix the contents of the second flask and
transfer 1ml suspension to the third flask containing 99 ml of saline solution with a
sterile pipette. Finally in the third flak bacterial suspension is diluted to 10-6.
Add approximately 15 to 20 ml nutrient agar medium into three large size test tubes,
sterilize by autoclave at 121 0C for 15 minutes and cool to 45 0C. Mix all the dilutions
and transfer 1ml from each dilution to large size test tubes . Mix the bacterial
suspension by rolling the test tubes between the palms of hands to ensure even
dispersion of culture in the medium. Immediately pour the medium of three test
tubes into three sterile petri plates s shown in figure . Allow the plates to solidify ,
incubate these plates in an inverted position for 24- 48 hrs at 370C.
Observations: - Observe all petri plates & count total number of colonies by using
Qubec colony counter.
Number of cells/ml= number of colonies x dilution factor.
Spread plate Method:-

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Aim : to determine total viable count of bacterial suspension by spread plate method.
Requirements:
Cultures: Liquid cultures of Staphylococcus aureus , water food or any pharmaceutical
liquid sample.
Reagents-
95% alcohol
Meida-Nutrient agar
Apparatus – test tube, pipette, petri plate, spreader .
Equipments- Auto clave, Quebec colony counter, hot air oven, incubator.
Principle: The method in which bacterial suspension is uniformly distributed on solid
agar by glass spreader is called spread plate method. The spread plate technique is
used for the separation of mixed population of microorganisms so that individual
colonies can be isolated and counted . In this method samples are spread over the
solidified agar medium with the help of glass spreader after proper dilution. All
microbial cells are separated from each other to develop isolated , free colonies on the
media. The total microbial count of sample is obtained by multiplying the number of
cells per plate by the dilution factor. The count of microorganisms are reported as
colony forming units/ml.
Procedure:- Prepare the dilution of given sample with the help of sterile pipettes. Add
0.1ml of any prepared dilution on sterile nutrient agar plate. Spread the dilution
with the help of glass spreader, sterilized by 95% alcohol. Glass spreaders are
sterilized by flaming after dipping in alcohol and are allowed to become cool between
two burners. Incubate the petri plates at 35 to 370 for 24 hrs.
Observations and results:- Observe all petri plates and count total number of
colonies by using Quebec colony counter or mechanical hand counter. The number of
microorganisms per ml of sample is calculated by multiplying the number of colonies
by dilution factor. Eg. Number of cells/ml = number of colonies x dilution factor.
( suppose , number of colonies present in 10-6 dilution = 180)
Number of cells/ml = 180x 106 = 18x107 colony forming units ( c.f.u)/ml of sample.

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Dry weight and wet weight measurement:
In determining microbial growth some time it is necessary to measure the mass of

cells present in culture rather than actual number. However it is obvious that cell
mass depends on cell number. With increasing cell number, cell mass also
increases. The cell mass can be measured on dry weight basis.
Requirements:
Bacterial cell culture ( eg. E.Coli, Salmonella sp).
Growth medium centrifuge and it’s accessories.
Electronic balance.
Millipore filter paper (45 mm) with assembly .
Oven.
Procedure:
1. Prepare growth medium for the given bacteria and transfer into a fermentor .
2. Autoclave the medium at 121 0C for 30 minutes.
3. Run the fermenter for desired period at temperature 25 0C.
4. After proper growth take out 5-6 ml of suspension containing 2-5 mg dry bacteria/ml,
transfer into cuvettes and centrifuge at 5000 rpm for 30 minutes to get the pellets of
bacterial cells.
5. Decant the supernatant , wash with sterile distilled water to remove the nutrients ( if
pathogenic bacteria is used , wash with 0.15 M Nacl containing neutralized commercial
formaline(10 %) which kills most of bacteria ) and finally filter the pellets through pre
weighed Millipore filter paper ( 45mm). Care should be taken to filter the pellets of all the
cuvettes through a single filter paper.
6. Repeat steps 4 & 5 to get large quantity of pellets from 100 ml of suspension.
7. Measure wet weight of pellets by using a sensitive balance ( weight of wet Millipore filter
paper containing bacterial pellets - weight of empty filter paper same size).
8. Dry the filter paper containing Pellets and filter alone separately in an oven at 90 0C for 16-

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20 hours.
9. Measure dry weight of bacteria ( dry weight of filter paper containing pellets – dry weight of
empty dried filter paper).
Sample No Wet weight Dry weight % of dry mass

1
2
3
Average dry mass
% of dry mass = Dry weight of pellets x100

-------------------------
Wet weight of pellets
Turbidimetry Method:
Turbidity of a culture can be measured with a photocolorimeter and it can be

transformed into the number of organisms. Further it is advisable to perform the plate
count for one culture of known turbidity.
The instrument calibration is necessary for determination of turbidity of a sterile

nutrient broth to establish 100 % transmittance. After it is calibrated , turbidity in
terms of transmittance , is read by inserting a cuvette of the culture into the sample
holder of the instrument. The principle is based on when the spectrum of light
falls on a dark screen with a slit only that portion of spectrum fallen on the slit
passes through the sample and activates a phototube which in turn shows
percentage of transmittance on a galvanometer. The higher is the % transmittance
, the lesser will be the cells in suspension.
For illustrating the direct proportional relationship between the concentration of
bacterial cells and the absorbance of light ie. Optical density, values are converted
to optical density and plotted on a graph.
1
Requirements:-
Broth culture of E.Coli.
Photo colorimeter cuvette
Pipette – 5ml
Capacity test tube
Bottle of sterile Nutrient broth
Procedure:
1. Calibrate the instrument , rinse the cuvette several times with distilled water
to get it clean before use.
2. Thereafter , dispense 4ml of sterile nutrient broth into test tubes 1:2, 1:4,1:8
, and 1:16.
3. Shake vigorously the culture tubes containing E.coli.
4. Dispense 4ml of the culture into test tube 1:2.
5. Mix well and take out 4ml from the tube and dispense in 1:4, again mix it thoroughly.
6. Transfer 4ml of the culture in next tube 1:8 and lastly transfer 4ml into 1:16
which contains 6ml of diluted organisms.
7. Record the % transmittance of the 5 tubes.
8. Convert this value into optical density (OD) for eg. The trnsmittance of the
sample is 53.5%, hence the optical density can be calculated by Optical
density= 2- log53.5
Direct total cell counting:
Use fluorescence microscopy to count bacteria retaining filters after appropriate staining
of the cells on a portion of the membrane filter or by use of phase contrast microscopy
on a clarified portion of membrane. Counting of bacteria is carried out by the total fields
of the objectives or by using a eyepiece graticule ruled in square. The most popular
method used for total count is the acridine orange direct count (AODC) by fluorescence
microscopy.
Requirements:

1
Fluorescence microscope
Acridine orange (0.01%).
Fluorescence isothiocynate.
Sterile distilled water

Procedure:
1. Stain the bacteria by adding acridine orange (0.1%) before or after

filteration for 1 minute or 0.01% fluorescein isothiocynate.
2. Rinse the sample with sterilized distilled water.
3. Prepare a smear on the slide and observe under fluorescence microscope.
Direct Viable cell counting:
Natural samples contain innumerable amount of bacteria. It is advisable to dilute

the sample when bacterial population is high before placing on filter. Keep the filter
on a suitable nutrient agar for counting the colonies formed after incubation. Viable
count is made by following methods.
a) Nalidixic acid method: The main objective of the method is to allow the cells
to grow bigger but not to divide. The procedure is as follows.
1. Add the sample with 0.25 % yeast extract as a growth substrate , 0.002 %
nalidixic acid as an inhibitor of DNA synthesis.
Incubate it at 300 C for hours.
2. Filter the sample and stain with 0.01% acridine orange or fluorescence
isothiocynate for fluorescene microscopy.
3. Observe under fluorescence microscope.
b) Vital fluorogenic dye: In this method 0.02% to 0.05 % fluorescein diacetate
is added to the sample. The dye enters in the cell in non fluorescent form but
hydrolysed by esterase’s in the cell . The released fluorescein is easily
observed by fluorescence microscope.
Biochemical Tests:
Bacterial species differ in their capacity to attack different carbohydrates, proteins and
fats. Bacterial species that cannot be differentiated by morphology and cultural
characters may show metabolic differences which can be exploited. Most of the
biochemical tests are based on
a)Presence of specific enzymes in bacterial cultures such as coagulase , oxidase ,

urease gelatinase , lecithinase , catalase and others.
b)Production of metabolic end products of some compounds like sugar present in the
culture media which are the out come of enzymatic action of bacteria. Some of the
widely used biochemical tests are listed below.
1.Sugar fermentation :- Break down of sugar is tested in various sugar media –
glucose, sucrose, lactose etc. Acid production is indicated by development of pink
colour of the medium and gas produced accumulates in inverted Durham’s tube.

1
Glucose broth:
Peptone - 5gm.
Beef extract - 5gm
Water -1ltr
Glucose -5gm
Bromocresol purple -qs.
Lactose Broth:-
Lactose - 10 gm
Peptone - 5gm.
Beef extract - 5gm
Water -1ltr
Sucrose Broth:-
Sucrose -5gm
Peptone - 5gm.
Nacl -5gm
Water -1ltr
2.Voges Proskauer ( VP) test ( Barrit’s method):
Some bacteria produce acetyl methyl carbinol ( CH3.CHOH.CO. CH3) or it’s reduction
product 2,3 butylene glycol (CH3.CHOH. CHOH. CH3) from pyuric acid in the media.
It can be detected by colorimetric method . In the presence of alkali and atmospheric
oxygen, the small amount of acetyl methyl carbinol present in the medium is oxidised
to diacetyl (CH3.CO. CO. CH3) which reacts with the peptone of the broth and produces
red colour.
In a 48 hr growth in 2.5ml glucose phosphate broth, 0.5ml of 40% KOH and 1.5ml of
5% alpha naphthal in absolute alcohol are added. In a positive test, deep pink colour
appears in 2 to 5 minutes which deepens into magenta or crimson colour in 30 minutes.
The media remains colourless for 30 minutes in –ve test. For maximum aeration the
tube is to be shaken at intervals . Traces of pink colouration should be ignored.
Glucose phosphate broth:-

Peptone : 10 gm
Dipotassium hydrogen Phosphate :5gm
Glucose : 5gm
Distilled water qs to : 1000ml.

1
3.Methyl red test (MR) : the test is employed to detect production of sufficient acid
during fermentation of glucose by bacteria and sustained maintenance of pH below 4.5.
In a 3 to 5 day old culture in glucose phosphate broth( buffered glucose broth) , 4 to
5 drops of 0.04% methyl red solution is added, mixed well and read immediately.
Positive reactions are bright red ( indicating persistent activity) and –ve reactions are
yellow( signifying low or transient acidity).
4.Citrate utilization : This test the ability of an organism to use citrate ( sodium citrate
in the presence of Citrase) as the sole source of carbon and energy, and ammonium
salt ( ammonium dihydrogen phosphate as the sole source of nitrogen. Koser’s citrate is
a liquid medium and Simmon’s citrate contains agar and an indicator in addition.
Positive rection in koser’s citrate medium is denoted by turbidity ie. Bacterial growth;
whereas in Simmon’s citrate medium bacterial growth occurs along the streak and
development of blue colur ( due to change of pH) denote + ve reactions.
Koser’s Citrate Broth:
Ammonium dihydrogen phosphate -1.5 gm
Dipotassium phosphate - 1.0 gm
Magnesium sulphate -0.2gm
Sodium citrate -3gm
Distilled water qs to - 1 ltr
Simmon cirate Agar:
Sodium chloride - 5 gm
Ammonium dihydrogen phosphate -1 gm
Dipotassium phosphate - 1.0 gm
Magnesium sulphate -0.2gm
Sodium citrate -2gm
Agar -20gm
Distilled water qs to - 1 ltr
Bromothymol Blue -qs.
Citrase Acetic acid Citrase
Citrate → OAA+ AA → Pyruvic acid + CO2.
Oxalo acetic acid
5.Indole production : Certain bacteria decompose amino acid tryptophan present in

peptone into indole. Bacteria are grown in tryptophan rich peptone water for 48 to 96
hours.
Tryptophanase
Tryptophan→ Indole + Pyruvic acid+ NH3.
a)Kovac’s method: To the bacterial growth in peptone water, 0.5ml kovac’s reagent is
added and gently shaken. A red colour near the surface indicates + ve reaction.
Kovac’s reagent:
Paradimethyl aminobenzaldehyde - 10 gm
Amyl or iso amyl aalcohol - 150ml
Con. Hcl - 50 ml
The reagent is prepared in small amount and stored in refrigerator.
b)Ehrlich’s method: Ehrlich’s reagent contains
paradimethyl amino benzaldehyde - 1 g,
absolute alcohol- 95 ml and concentrated Hcl -20 ml
In 5-6 cc bacterial growth of peptone water, about 0.5ml of xylene is added and shaken
thoroughly . Then few drops of Ehrlich’s reagent is added, pink colour develops at the
junction of two fluids.

1
Peptone water 1% Peptone - 10gm

Distilled water - qs to 1000ml.
Four metabolic test ( Indole, methyl red, Voges- Proskuer and Citrate utilization)
collectively known as IMViC tests are used to distinguish different enteric bacteria.
6) Hydrogen sulphide production: Sulphur containing amino acids are decomposed by
certain bacteria with production of hydrogen sulphide as one of the end products. When
media containing lead acetate, ferric ammonium citrate or ferrous acetate is inoculated
by these organisms and incubated for 24 hrs , the media turn black or brown.
Alternatively , the test can also be performed by lead acetate strips ( filter paper
soaked in 10% lead acetate solution and dried) . The strip is inserted between the cotton
plug and culture tube suspending it above the culture. The lead acetate strip turns black
which indicates H2S production.
7. Phenyl alanine deaminase test: Certain bacteria deaminate ( removal of amine
group ie.NH2 and release it as free NH3) phenyl alanine with production of phenyl
pyruvic acid ( PPA) which on reaction with ferric salts produce green colour.
Nutrient agar slope containing DL- Phenyl alanine is inoculated with a fairly heavy
inoculum of culture and incubated at 37 0 C for 4- 24 hours. A few drops( 4-5
drops) of 10 % ferric chloride solution is added, + ve reaction is indicated by
development of green colour in the medium and in the fluid.
8. Urease : A 4- 24 hours growth of an organism in Christensen’s urea medium

( peptone water, urea, phenol red) acquires pink colour by urease producing
organisms. Urease producing bacteria reduce urea to ammonia (NH2CO NH2+ H2O →
2NH3+ CO2) which changes colour of the medium. Urease production is not to be
considered -ve till a 4- day old culture is tested.
9. Oxidase reaction: This reaction is due to presence of cytochrome oxidase that

catalyses the oxidation of reduced cytochrome by molecular oxygen. The organism is
cultured on a solid medium like nutrient agar and then a freshly prepared 1 - 1.5%
solution of tetramethyl p- phenylene diamine dihydrochloride is poured on the surface
of colonies and the excess amount is thrown out. In positive reaction , the colonies
become maroon , purple and black in 10- 60 seconds. Alternatively filter paper strip
soaked in the reagent is smeared by the organisms. A + ve reaction is indicated by
an intense deep purple colour appearing with in 5- 10 seconds, where as the colour
change occurs in 10- 60 seconds in a delayed + ve reaction. Important oxidase +ve
organisms are Neisseria, Vibrios, Pseudomons etc.

1

1
9.Catalase production : The test demonstrates the presence of catalase in bacteria which
releases oxygen from hydrogen peroxide. Most aerobic organisms during respiration
produce H2O2 which may be toxic to their enzyme system & hence lethal for such
organisms. The enzyme catalase present in these organisms breaks down this H2O2 into
water & oxygen and thus helps them in their survival . When 1ml of 3% H2O2 ( 10
vol) is added on colonies on nutrient agar , prompt effervescence indicates production
of catalse.
2H2O2 → 2H2O + O2
1. Cultivation of anaerobic organisms . 5 marks.

1
2. Different methods used in isolation of pure culture-. 5 marks.

3. Bacteriophage - 2 marks
4. What are enriched , selective & differential media give examples for each. 5 marks.
5. Selective media – 2 marks.
6. Acid fast staining -2 marks.
7. Discuss any two methods of bacterial cultivation -5 marks.
8. Classify culture media & explain the types with examples . Why Agar is
called an ideal solidifying agent. 10 marks. ( repeated).
9. Enumerate various methods of isolation of pure culture . explain any
one of them in detail – 5 marks.
10. Roller tube method – 2 marks.
11. Describe the method & precautions for culturing anaerobic bacteria. 5 marks.
12. What are pure cultures. Explain how you preserve pure cultures. 5 marks.
13. Nutrient broth- 2 marks.
14. Explain a) Isolation of pure cultures b) Maintenance of a pure culture. 10 marks. Or
15. Describe the methods involved in isolation of pure cultures. 10 marks.
16. Simple staining – 2 marks.
17. Explain the principles and procedure of Gram staining method. 5 ma
18. What are cultures. Explain how pure cultures can be preserved. 5 marks.
19. Nutritional requirements of animal cell culture – 5 marks.
20. Write the cultivation of anaerobic bacteria – 5 marks.
21. Write the different methods used in isolation of Bacteria – 5 marks
22. Write the functions of different components of compound microscope. 5 marks.
23. Describe different phases of growth curve of bacteria based on total count. 5 marks.
24. Cup plate method- 2 marks.
25. Complex synthetic media – 2 marks.
26.Write briefly about anaerobic culture technique -5 marks.
Chapter 5.Detailed study of different methods of sterilization

including their merits and demerits. Sterilization methods for all
pharmaceutical products. Detailed study of sterility testing of
different pharmaceutical preparations. Brief information on
1
validation .
Sterilization .Laboratory work and patient care service require the use of apparatus , culture
media, instruments and dressings and parenteral drugs that are free from all living microorganisms
and spores. It is there fore , essential to understand the principles of sterilization , disinfection and
antiseptics for control of infection .
Definition - Sterilization is a process by means of which an article , surface or medium is

made free from all living microorganisms including spores .
Disinfection is a process of destruction of vegetative forms of pathogenic organisms which are

capable of producing infection but not necessarily resistant spores .
Antispetics : Substances which either kill microorganisms or inhibit their growth .
Disinfectants : Compounds that kill microorganisms ( except bacterial endospores ), Since these
may be harmful to human tissue , disinfectants are usually reversed for inanimate (non living)
objects.
Sterilant-Chemical agents that destroy all microorganisms , including spores .
Sporocide- Generally a chemical agent that destroys both bacterial and fungal spores.
Fungicide – Chemical substances that destroys fungi.
Virucide- Chemical agent that destroys viruses.
Bacteriostat- Chemical agent that inhibit bacterial growth.
Sanitizer-An agent , usually a detergent , that reduces the number of bacteria to a safe level.
Sterilization is the complete inactivation of all forms of microbial life , this can be accomplished
using physical , gas or chemical steriliants.
Methods of Sterilization .
Method Concentration ( Level of Activity )
A.Physical Sterilants
1. Steam under pressure 1210C for 15 -20 minutes or 1340C for 3 minutes .
2. Dry Heat 1600C for 1hr 30 minutes , 1710C for 1 hr.
3. Filtration 0.22 to 0.45 µm pore size; HEPA filters.
4. UV radiation Exposure at 254 nm wavelength for variable time.
5. Ionizing radiation Exposure to microwave or gamma radiation for variable
time.
B. Gas vapour sterilants .

1. Ethylene oxide 450- 1200 mg/L at 29 0 to 650 C for 2 to 5 hrs.
2. Formaldehyde vapours 2- 5 % at 60 0 to 800C.
3. Hydrogen peroxide vapour 30 % at 550 to 600C
4. Plasma gas Highly ionized H 2O2
C. Chemical steriliant.
Glutaraldehyde - 2%, Paraldehyde -0.2%
Sterilization by heat .
Physical sterilants such as moist and dry heat are effective means of sterilization and are most

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common sterilizing methods.
Principle .
Moist heat is a much more effective sterilant than dry heat because of the following:
i)Moist heat kills the microorganisms by coagulating and denaturing their enzymes and structural
proteins a process that requires participation of water . Hence all parts of the load / material to be
sterilized have to be kept in direct contact with water molecule or steam . Sterilization by
saturated steam under pressure ( 1210C) takes 15 minutes .
ii) Dry heat kills the microorganisms by destructive oxidation of intracellular contents.
Procedure of sterilization by dry heat .
1.Red heat . Metallic objects such as inoculating wires, tips of forceps and needles are held in the
flame of a bunsen burner for instant sterilization .
2. Flaming : Certain articles like glass slides , needles , cotton wool plugs are passed over flame ,
These are not allowed to get red heat . This method is of uncertain efficiency.
3. Incineration : This method is employed for destruction of infective materials . The soiled
dressings , bedding, pathological materials like sputum and stool are reduced to ashes by burning.
4. Hot air oven – It is the most widely used sterilizer of dry heat particularly for glass ware .
Sterilization of articles by exposure to hot air is the accepted method.
Dry heat sterilization By Hot Air Oven:-
The design of an oven for the sterilization must try to satisfy the following requirements.
Every article inside must receive the correct exposure where ever it is placed
The sterilizing temperature must be reached quickly and maintained with little variation.
These aims are most neatly fulfilled by electrically heated thermostatically controlled ovens with
far circulation.
Ovens of this type consists of an Aluminium or stainless steel chamber separated from the outer
case by a thick layer of glass fibre insulation.
The hollow flanged ( external or internal ridge or rim for support) door is also filled with insulation
and carries as asbestos gasket that provides a tight seal .
The reflecting inner surfaces, the lagging ( material providing heat insulation) and the gasket all help
to prevent heat losses.
In the best types the heaters are fixed to the outside of the chamber .
Heat is transferred from the source to the articles in a hot air oven by conduction, convection and
radiation.
Conduction , along the shelves from the hot lining cannot play a major part because of the limited
pathways from the heat source and frequently small area of contact .
Convection is more important especially in ovens with heaters restricted to beneath the floor , but
air is a very poor heating agent because of it’s low specific heat.
Even in a carefully loaded oven articles near the walls tend to screen those in the centre from
radiation, there fore , it is important to make maximum use of the heating capacity of the hot air
oven.
By circulating the air with a fan, more of the air molecules are made to collide with the load &
with hot chamber surfaces.

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A common arrangement is to project a fan from the back of the chamber & put a baffle ( a device use
to regulate the flow) in front , the air is sucked through holes in the centre of the baffle .In a loaded
oven the temperature variation usually taken as the difference between the temperature at the
centre and any other point should not exceed 50C, once the sterility temperature has been reached.
Method :
For the process to be reliable , every article no matter where it is placed in the oven , must be at the
correct temperature through out it’s mass, for the whole of the sterilization period .
Consequently extra time must be allowed for the heat to penetrate the material & raise every part
of it to the correct temperature .
In some cases such as large containers of poor conducting substances( eg. powders) this is
considerable.
The most satisfactory procedure is to set up a preliminary experiment in an oven loaded as for a
normal process with thermocouples in the centre of containers in different parts of the chamber and
one in the oven. Then the difference between the times at which the air & most slowly heated
container reach the sterilizing temperature is taken as the lag and added to the sterilization time in
subsequent processes.
To prevent over heating of more rapidly penetrated articles, only one type of material( powder , oil
etc) in one size & type of container should be sterilized at one time & lag should be determined
previously for each.
After the articles have been suitably packed they are carefully arranged on the oven shelves, taking
into account the following point.
1. They must be well spaced to interfere as little as possible with the air flow. In an over
loaded oven heating up is very considerably delayed & heat distribution is very uneven.
2. Contact with walls & floor must be prevented because these are hotter than the oven air &
may damage certain materials & char cotton wool plugs & paper wrapping.
3. It is much better to wrap the pipettes individually & the petri-dishes in 2 or 3.
4. The screw caps of containers should be loosened half a turn to prevent bursting of the
container from the expansion of the contents & the entrapped air.
Next the following are checked .

1. The thermo-regulator – This is adjusted if necessary.
2. The thermometer if it is not built into the oven it should be fitted & must project as far as
possible into the oven ( eg. 15 cm) to prevent significant effect on the reading from the
cold exposed stem.
3. The vent (on the top of the oven) to obtain the most accurate control of the temperature
these should be closed .
4. The doors can then be shut & the heaters & fan switched on. When the thermometer shows
that the oven air has reached sterilization temperature , heating is continued for the lag
exposure time.
5. After switching off the door is left closed until the temperature has fallen considerably
( ideally to about 40 0C). This prevents breakages. The bottle caps are tightened as soon as
possible.
Procedure of sterilization by moist heat:-

a)At a temperature below 100 0C – Following techniques are employed.
1.Vaccine bath- Serum or body fluids are sterilized in water bath at 56 0C for 1 hr and bacterial
vaccines are sterilized by a special water bath ( Vaccine bath ) at 60 0C for 1 hr as most
vegetative bacteria are killed at this temperature and time.
2.Pasteurisation of milk :- This is done by maintaining a temperature of 63 0 C for 30 minutes

( holder method ) or 72 0C for 20 seconds ( Flash method ). Method vegetative bacteria like
Mycobacterium , Salmonella and Brucella are rapidly killed in the temperature range of 60 – 65 0
C except Coxiella burnetii , a relatively heat resistant organism which may not be killed in holder
method.

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3.Fractional sterilization :- Inspissator is a water jacketed copper box fitted with a heater and
thermostat for automatic regulation of temperature . Serum or egg medium is tubed and placed in
special racks and slow solidification of serum or egg is carried at 80 0C to 850C for one 1 hr
( inspissation) and for sterilization of the medium , the process is repeated for three consecutive
days ( fractional sterilization ).
Serum inspissator :
It is a chamber made of copper plates fitted with a water jacket around it and closed with a
glass lid. The temperature is automatically regulated by a heating element and thermostat fitted
in it. There are holes in the water jacket located at level higher than that of water , so that water
vapour may keep the medium moist. The serum medium tubes with plugs are kept in slanting
position in the chamber in such a way that the media solidifies in slopes . The chamber is closed
and the media are further sterilized at 75- 80 0 C for 20 minutes for two successive days.
Uses:- Sterilization of Loeffler’s serum medium, Dorset’s egg medium, L.J. Medium.
b)At a temperature of 1000 C. The following methods are in use .
1.Boiling at 1000C: Although boiling for 10 -30 minutes kills most of the vegetative forms of
bacteria , many spores withstand boiling for a considerable time. So boiling is inadequate for
sterilization purpose . When better methods are not available or absolute sterility is not essential ,
glass syringes , tubes , rubber stoppers and small surgical instruments are sterilized by boiling.
2.Steam at atmospheric pressure at 100 0C for 90 minutes . Gas or electrically operated steamer
like a Koch or Arnold steam sterilizer is employed to sterilize bacteriological media such as
sugar media which decompose if subjected to be higher temperature of autoclave. The articles to
be sterilized are kept on perforated tray through which free steam passes which then escapes
through an opening at the top. . The 90 minutes sterilization time includes loading and heating
time from 0 to 100 0C. Most of the vegetative organisms are destroyed by this process except
thermophiles .
3.Streaming at 1000C for 3 successive days ( Tyndailisation) .The culture medium is steamed for
30 minutes each on three successive days. The vegetative cells destroyed at first exposure , in the
intervals between the heating, the remaining spores germinate in the favourable nutrient media
which are killed on subsequent heating.
C) At a temperature above 100 0 C: When water boils in a closed and airtight container under
increased pressure , the temperature of steam inside rises above 100 0C which can effectively
destroy all thermal resistant bacterial spores. This is the principle of sterilization by an autoclave.
Autoclave:
Autoclaving is the process of sterilization by saturated steam under high pressure above 100 0C.
Essentially it is a modified pressure cooker or boiler which may be horizontal or vertical. It is a
double walled or jacketed chamber made of stainless steel or gun metal with a supporting
frame . The steam circulates within the jacket and is supplied under high pressure to the closed
inner chamber where goods are kept for sterilization . One fifth part of the cylinder is filled with
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water and the materials to be sterilized are placed inside. The lid is closed securely with discharge
tap on it open. The heater is put on . Safety valve is adjusted to required pressure . For some time
after boiling of water inside the chamber the steam and air mixture is allowed to escape till the
cylinder becomes air free. Then the discharge tap is closed. When the desired pressure in the
chamber rises to the one chosen for autoclaving. ie. 15 lbs per square inch (121 0C), the sterilizing
period is maintained for fixed time ( 15- 20 minutes).
Types of autoclaves .
1. Simple iron jacketed.
2. Low pressure low temperature type.
3. High pressure high vacuum type where as much as 98% of the air present is rapidly
expelled by an electric pump and sterilization is done without delay.
Sterilization time :-
Autoclaving is usually done at a temperature of 121 0 C and chamber pressure of 15 lbs pressure
/inch2 for 15-20 minutes . Sterilization can also be done at higher temperature , at 126 0 C ( 20 lbs
pressure /inch2) for 10 minutes or at 133 0C ( 30 lbs pressure / inch2) for 3 minutes . A comparative
study of the four principal methods of sterilization are shown below.
Sterilization time, Temperature and indicators .
Method Temperature Holding time Chemical indicator Biological
indicators.
(0C) ( minutes)
Moist heat 121 15 Colour change
B.stearothermophilus
(autoclaving) 126 10 indicators
134 3
Dry heat 160 60 Colour change B.subtilis var niger

(hot air oven) 170 40 indicators
180 20
Ionising -- -- Radiochromic B. pumilus

Radiation chemicals.
Principles of autoclaving:- Steam saturated at a high pressure and temperature is a better

sterilizing agent than dry heat. Bacteria are intrinsically more susceptible to moist heat as
bacterial protein coagulates rapidly .
Saturated steam heats the articles to be sterilized rapidly by releasing latent heat which
participates in bacterial killing.
Moist heat sterilization :- Moist heat in the form of saturated steam under pressure
( autoclaving) appears to be most reliable method of sterilization . Microorganisms are destroyed
by moist heat due to coagulation of proteins in the cell. Upon heating , wet proteins release the -
SH groups and form smaller peptide chains , which in turn form complexes different from the
original protein molecules .
An important advantage of moist heat is that it is quicker in heating the exposed articles and in
penetrating porous materials such as cotton wool , stoppers , papers , cloth wrappers , bundles of
surgical linen etc.
A house hold pressure cooker is the simplest form of an autoclave , which can be used in a
laboratory . The operation is simple. A pressure regulator regulates the pressure. It is also provided
with the interlocking lid with an automatic safety valve.
Autoclaving- Steam under pressure provides more heat than boiling water or free flowing steam.
The higher the steam pressures, the higher will be the temperature . Steam under pressure is used as
a means of obtaining temperatures high enough to destroy microorganisms.
Disadvantages – Autoclaving – Diluting of unprotected cutting edges , destroys heat

sensitive materials , may rust or corrode steel instruments.

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Dry heat – Destroys heat labile items.
Ethylene oxide- Long time need for liquid and rubber material.
Unsaturated chemical vapour- Destroys plastics , chemical odour in disinfected areas .
A portable or bench autoclave consists of an upright aluminium or stainless steel cylindrical vessel
of about 15 litres capacity . Pressure within the autoclave is indicated by a pressure gauge. Such an
autoclave is shown in figure.
Operation :- Two liters of water is placed in the vessel and the containers to be sterilized are
sealed and loaded into it. The lid is fitted , air vent is opened and the heat is turned on. After some
time the steam starts issuing freely from the vent, which is allowed to continue for about 5 minutes
and then the vent is closed . The temperature and pressure now start rising till the controlled
conditions are attained . Heating time is computed from this stage , heating continued for the desired
period and then it is allowed to cool. The vent is opened only after the autoclave has attained the
atmospheric conditions. The lid is opened and the autoclave is unloaded.
Bench autoclaves are suitable only for small loads of vacuum bottles or for one or two transfusion
bottles.
The mode of operation may depend upon the material to be sterilized . The sterilization cycle
consists of the following stages :-
i) Loading and packing the autoclave.
ii) Raising the temperature and pressure.
iii) Holding the load at this level for a specified time.
iv) Cooling and uploading.
The larger autoclave measure upto 20 ft. in length and 6 ft. each in breadth and height . The larger
models are mostly horizontal and the general design is shown in Figure. In pharmaceutical
industry, double door autoclaves are commonly used in which the load is introduced from one
door and withdrawn from the other.
Design and operation of Steam sterilizer.(Large sterilizers)
The apparatus for sterilization by steam under pressure is called an autoclave or steam sterilizer
Usually the portable types used for small scale production of injections solutions are cylindrical

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upright , they may be less than 0.3 m in internal diameter & depth. The types previously referred
to as large sterilizers are almost always fixed to a steam supply & are generally horizontal &
either cylindrical or rectangular .
The internal dimensions of the cylindrical type may be up to 0.75 m in diameter & 1.2 m in length,
while rectangular forms can be as large as 1.3 m high 1.0 m wide and 1.3 m long internally.
A ) Portable sterilizers :
The 2 types of portable sterilizers are most simply described as pressure controlled & Temperature
controlled.
In a Pressure Controlled Type the pressure gauge is the sole indicator of the internal conditions &
there fore all the air must be removed before the sterilizing exposure begins .
In the temperature controlled type a thermometer or thermostat is used to indicate or ensure

respectively that the exposure temperature has been reached & it is not essential to expel the air.
Portable Autoclave Design:

This autoclave has a strong cylindrical body made of an Aluminium alloy and provided with a
bucket type handle .
Inside , on the bottom are several radial ribs on which rests a light removable inner chamber.
Around the rim are eight bolts that swing up into slots on the lid where they are held in position by
wing nuts.
The curved lid and a rubber gasket that gives a steam tight seal with the body . On the lid are 3
controls , a vent through which air is expelled , a pressure gauge & a safety valve.
At the top of this valve is a knurled nut that can be raised or lowered to decrease or increase the
tension in a spring holding a ball bearing over a hole in lid.
The standard model is heated on a gas ring but an electrically heated model is also available.
Working:-
Water is put into the level of the bottom of the inner chamber & the articles for sterilization are
loosely arranged in the latter.
Tight packing is avoided to leave room for expansion & prevent breakages, also air cannot escape
easily from the narrow pockets between closely adjacent containers.
Caps of bottled fluids should be screwed down tightly , there is no danger of explosion because the
internal pressures are approximately balanced by the steam pressure outside.
The lid is put in position taking care that all the hinged bolts will slip easily into the slots. Then the
wing nuts are lightly turned & afterwards tightened in opposite pairs.
The vent is opened & the safety valve is set at the required pressure , which for the BP injections is
1.7 bars( 1210C or 15 lbs).
Heating is started over a full gas . Steam is allowed to issue freely from the vent for 5 minutes,
which in a portable autoclave is long enough to expel the air.
Timing of this must not start when the first whistle of steam appears , vigorous emission for the
whole time is necessary.
The vent is closed & when the pointer of the gauge reaches 15 lbs timings of the exposure begins .
It is advisable to record immediately the beginning & end of the period .

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This will prevent accidental under or over exposure.
At the required pressure the safety valve opens & noisily releases excess steam.
The flame is now turned down to a height that maintains the pressure but minimizes steam
release.
If the valve is allowed to blow vigorously through out the exposure period the noise & the steamy
atmosphere are objectionable & more important , the sterilizer may boil dry.
If the latter occurs the steam become superheated, solutions in plugged or loosely capped containers
are concentrated & sealed bottles & ampoules may burst.
The BP requires exposure of the whole of the contents to 115- 116 0 C for 30 minutes & there fore
allowance must be made for the lag time.
This should be predetermined by using thermocouples, but if in an emergency the information is

not available a useful general rule is to add 10 minutes for containers of 101 to 250ml, 15 minutes
for 251- 500ml & 20 minutes for 501 – 1000ml.
Small & large volumes should not be sterilized in the same load because the correct time for the
latter might cause overheating of medicaments in the former.
On completion of the exposure the gas is turned off & the pressure allowed to fall to atmospheric .
Then the vent can be opened . This should not be done while the internal pressure is high because
it’s sudden release would cause vigorous boiling & frothing over the liquids in unsealed
containers might lead to bursting of sealed bottles & ampoules.
Opening the vent should not be delayed.

When the atmospheric pressure has been reached because the vaccum produced by further cooling
causes partial evaporation of solutions in loosely closed containers.
If the sterilizer contains the large sealed bottles of fluids , the lid should not be removed as soon as
the vent has been opened.
Large volumes cools slowly & their internal pressure will still be well above atmospheric .
In some hospitals the staff wear fencing masks when removing infusion fluids from large sterilizer
, if unloading is essential while the contents are hot. The danger is slight with a portable
sterilizer if about 10 minutes cooling is allowed after opening the vent.
The instruction to load the sterilizer before the water is heated should be modified if solutions
such as culture media , in plugged or loosely capped containers are being sterilized.
If these are put into a cold autoclave their temperature follows closely that of the heating water
& may be at boiling point during venting consequently steam is produced from them &
concentration results. They should be put in when the water is boiling & allowance made for the
increased lag that will result.

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When the load in the autoclave has been raised to the sterilizing temperature , it is held so for the
required time.
The holding times needed for sterilization are indicated below in terms of the temperatures and
pressure .
However the choice of the combination used depends mostly on the ability of the material to
withstand the imposed conditions.
Autoclaving Conditions
Temperature Corresponding Normal Recommended Minimum
0
C Pressure in Excess of Holding Time
Atmospheric ( lb/sq. inch) ( minutes).
115- 116 10 30
121-123 15 15
126-129 20 10
134-138 32 3
Obviously the total time required for completing the sterilizing cycle is much more than the holding
times mentioned above.
It is always safer to use a holding time longer than recommended , for various reasons. Eg. whether
the autoclave load is light and consists of small items or whether it contains dressings drums or
larger items. Thus the holding time may range between 10- 45 minutes at 121 0C , or 15 to 60 minutes
at 1150C.
When the sterilization period is over , the steam supply to the chamber is discontinued and the
autoclave is allowed to cool .
This is done by venting the steam steadily and slowly through a drain or by pass , taking between
7-30 minutes to reach the atmospheric pressure, according to the load.
Finally the autoclave is opened and unloaded . The load should be removed as aseptically as
possible.
Autoclaving is one of the methods of sterilization , recommended in Indian Pharmacopoeia for

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many official injections .
Advantages of autoclaving :-
Because of the greater penetrating power of the steam under pressure , microorganisms are destroyed
more efficiently than the dry heat , and there fore , a shorter exposure at a lower temperature
suffices.
The method is suitable for a large load.
For sterilizing porous materials , dry saturated steam can be supplied.
The method is applicable for a wide variety of materials.
It is used to sterilize culture media , rubber chamber as temperature of air steam mixture at a given
pressure is lower than that of pure steam.
Contents should be arranged loosely to ensure free circulation of steam inside the chamber.
Sterilization control:-
1. Bacterial spores : Dried bacterial spores in a tube is placed inside the autoclave during
sterilization . Bacillus stearothermophilus is an organism of choice which has to be grown
in an incubator at a temperature of 55- 600C. Its spores get killed in 10-12 minutes at 1210C.
2. Chemical indicators – Such as pellets of sulphur in test tube or Browne’s sterilizer control
tubes may be used . AT 1200C. sulphur pellets gets melted with change in shape and red
solution of Browne’s tube turns red to green , when adequate temperature is maintained
for adequate time.
Browne’s tubes. These are the sealed glass tubes which contains a red fluid that changes
through amber to green on heating .
Hour glass devices.- This makes use of the melting point of a solid .eg. Acetanilide for
1150C, Succinic anhydride for 1210C and para acetotoluidide for 1500C ( for dry heat
sterilization ).
Filter paper strip. Another device involving the melting of a solid & incorporating a time factor ,
consists of a filter paper strip , laminated to aluminium foil to improve heat conduction &
distribution & impregnated at one end with a 2,4 – dinitro phenyl hydrazone (DNP) derivative of
appropriate melting point . DNP compounds are highly coloured . The melted substances creeps
along the paper for a distance corresponding to the time at exposure temperature .
The chief advantages of chemical over culture methods is that they can be read immediately.
Thermocouple :- It is placed in a test article
Filtration :- The fluid to be filtered is sucked through the filter into a receiving flask by
negative pressure with the help of an exhaust pump . This method is useful for making bacteria
free preparations of substances which get damaged by heat process eg. bacteria free filters of
toxin and bacteriophages , sterilization of serum , sugar and antibiotic solutions. The pore size of the
filter is not less than 0.75 micron in diameter and it retains bacteria but allows viruses to pass
through into the filtrate . .It may be noted that filtered preparations are not safe for clinical use
because viruses and Mycoplasma may pass through the filter . Sand filters are employed in
purification of drinking water.
Uses :- The early rather absorptive filters of asbestos or diatomaceous earth were replaced by
unglazed porcelain or sintered glass , and these in turn have been superseded by nitrocellulose
membrane filters of grade porosity . These are used to separation of soluble products of bacterial

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growth ( eg. toxins , sterilization of pharmaceutical preparations ( antibiotic solutions ) and blood
products . These are used in purification of water . High efficiency particulate air ( HEPA ) filters are
used to deliver clean bacteria free air to a cubicle or a room eg. Laminar air flow system.
Principle : This method is mainly involves sterilization by filtration process. Filtration is a general
method employed for the sterility testing of liquids . This is useful for large volumes. Filtration
sterilization method is used to reduce the microbial population in solutions of thermolabile material
. For this purpose , many types of filters are used . In this method , filters remove the contaminating
microorganisms from solutions , rather destroying them directly . This method is also used for
sterility testing of liquids , and for large volume solutions , antibiotic solutions , sera , carbohydrate
solutions and eye drops .
The efficiency of filters depends on their pore size , wall thickness, filtration rate , positive or
negative pressures and nature of liquid to be filtered . Filters do not kill microorganisms but remove
them.
Filters used for sterilization are of the following types :
I.Depth Filters : These are made up of granular or fibrous materials that have been bonded into a
thick layer filled with twisting channels of small diameter . By the application of vaccum, this
layer absorbs microorganism present in solution ; thus the microbial cells are removed by physical
screening or entrapment and also by adsorption to the surface of the filter material .
Depth filters are further divided into the following :
i)Asbestose Filter (Seitz Filter): These are disposable and single use discs that are available in
various grades of pores ( from 0.01 -5 microns). These filters are made up of asbestos such as
magnesium silicate or magnesium trisilicate . The filter is supported on a perforated metal disc
within a metal funnel and then fitted on sterile flask through a silicone rubber bung. Fluid for
sterilization is placed on the funnel and the flask is connected to the exhaust pump.
Seitz Filter Sintered Glass Filter
Seitz Filter Sintered Glass Filter
Asbestos filter have very high adsorbing capacity and tend to alkalinise the filtered fluid. However ,

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the use of these filters is restricted due to the carcinogenic potential of asbestos.
ii) Sintered Glass Filters – These are made up of finely powdered borosilicate glass particles in a
ball mill, and packed into disc moulds and heated until proper adhesion takes place between the
granules. The sintered discs are finally fused in funnels of suitable size and shape . Their
adsorbing properties are low and they are available in different pore sizes, but for filtration
sterilization , a number or grade 5 or 3 must be used . The adsorptive property of sintered glass
filters is low and therefore they can be cleaned easily .
iii)Candle Filters : These are made up of unglazed porcelain ( eg. Chamerlain filters ) or
diatomaceous earth or kieselguhr ( Berkefeld filters). These filters are available in different
grades of porosity and have been used extensively for the purification of water for both drinking
and industrial purpose. These are depth filters with thick cellular walls and are available in the
shape of cylindrical candles . They are available in various pore sizes.
Candle Filters
II.Membrane Filter ( Millipore/ Ultra Filter ) : Now a days membrane filters are widely used , and
have replaced depth filters . These filters are made up of cellulose acetate , cellulose nitrate ,
polycarbonate , polyvinylidine fluoride , or other synthetic materials . They are 150µm thick and
contain millions of microscopic pores ranging from 0.01- 10 µm in diameter.
These are circular in shape , having 0.1 mm thickness and are available in wide variety of pore
sizes ( 0.015 – 12 µm). Membranes with pores of about 0.2 µm are used commonly, because the
pore size is smaller than the size of bacteria . These filters remove most of the vegetative cells of
microbes , but are unable to filter viruses .
Membrane Filter
During the filtration process, the filter membrane is held in special holders and often preceded
by depth filters ( made of glass fibres ) to remove larger particles that might clog the membrane
filter . Membrane filters are supported on a rigid base of perforated metal, plastic or coarse
sintered glass .
After that the solution is forced or pushed through the filter using vacuum or pressure from a

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syringe , peristaltic pump or nitrogen gas bottle , and collected in previously sterilized containers .
Radiation :-
Two types of radiation are in use for sterilization purposes : Ionizing and Non- ionizing.
Ionizing radiation :- Ionizing radiation include gamma ray, X- ray and accelerated electrons .
These are more effective than ultra violet rays and act by formation of radiation tracks in the
DNA of bacteria leading to it’s death . X-rays and gamma rays have only practical use and two
types of machines are available in the market . eg. linear accelerator for X- rays and cobalt -60 for
gamma rays . A dose of 2.5 M rad is adequate to kill both vegetative and spore form of bacteria .
As there does not occur any appreciable increases in temperature , this techniques is also referred to
as cold sterilization.
Uses – Rubber or plastic disposable goods , disposable syringes , surgical catgut (Catgut sutures have
been used to make absorbable stitches ), bone and tissue grafts , adhesive dressings are sterilized by
ionizing radiations.
Non-ionizing radiations :-
Electromagnetic rays with wave lengths longer than those of visible light are used in non ionizing
radiations and these rays are adsorbed to a great extent.
a)Infrared radiation : Infrared radiation is considered as a form of hot air sterilization and is
employed for rapid mass sterilization of syringes.
b)Ultraviolet radiation:- The presence of ultraviolet rays in sunlight has a low energy , non ionizing
radiation ( 290 nm) with poor penetrating power and is the cause of it’s bactericidal action which
occurs in natural conditions . Ultraviolet rays with wavelengths from 240- 280 nm is even more
effective radiation produced by mercury lamps have marked bactericidal activity which act by
Denaturation of bacterial protein.
Damage of DNA.
Induction of colicin production in colicinogenic bacteria.
Inhibition of DNA replication , formation of hydrogen peroxide and other toxic products
in the culture medium.
Commonly used U.V.Lamps : It is of low pressure mercury vapour type whose 95% of emitted
radiation is of wave length of 253.7 nm.
Application – Gram positive bacteria show a slightly greater resistance than gram negative
bacteria and spores are highly resistant to UV radiation . It is mainly used in disinfection of
surfaces and air in bacteriological laboratories and operation theatre .
Gas vapour sterilization:-

1.Ethylene oxide – Sterilant gases like ethylene oxide is an alkylating agent and exert lethal
effect on proteins of bacteria .It is a gas at ordinary room temperature ( boiling point 10.8 0C) and
active against all types of bacteria and spores. Although it is highly inflammable , it forms non-
inflammable mixtures with cabon dioxide .
Uses- It has got a good degree of penetration power , even through plastics . Plastic goods ,
polythene tube , artery and bone grafts , vaccines and culture media can be sterilized by ethylene
oxide . These objects are kept in a cabinet from which air is removed by a vacuum pump and then
a mixture of ethylene oxide and carbon dioxide is introduced in the cabinet.
2.Formaldehyde gas :- Formaldehyde in aqueous solution or as gas has been in use as a
disinfectant for decades . But it’s pungent smell even at very low levels ( 1 ppm) , allergy to
formaldehyde and it’s carcinogenic effect has limited it’s use . Paraformaldehyde vaporized by
heat is used for decontamination of biological safety cabinets.
3.Hydrogen peroxide gas:- Hydrogen peroxide vapours are effective sterilants because of the
oxidizing nature of the gas. It is used for sterilization of instruments. There are several variations
of hydrogen peroxide vapours or gas sterilization . eg. Plasma gas sterilization . Hydrogen
peroxide is vapourised in the instruments and then reactive free radicals are produced either with
microwave frequency or radiofrequency energy .

1
Detailed procedure of Ethylene Oxide sterilization.

The EO sterilization process has been employed by the health care industry to sterilize medical
devices since the early 1940s .In its pure form (100 percent), ethylene oxide vapor is flammable
and explosive when allowed to mix with as little as a 3 percent air by volume.
To safely utilize the gas as an industrial sterilant, the process is designed
using cycle phases which are delivered in such a manner that will never allow the process to enter
into an unsafe condition.
The process may be described in a very simple form as follows:
Using a vacuum-tight chamber, an initial vacuum is drawn to remove air

and prevent an unsafe mixture when EO is injected.
After the vacuum is complete, moisture, usually in the form of steam, is
added to the chamber to replace that moisture which is lost during the initial vacuum phase.
Next in the sequence is the introduction of the ethylene oxide gas to a
predetermined concentration. The concentration has been selected to assure an adequate
sterilization process is delivered.
Finally, after the product is allowed to soak in the EO for a controlled and
predetermined amount of time, a series of washes is performed to rid the chamber of the EO.
A wash consists of pulling a vacuum followed by a pressurization with an inert gas, which is
usually nitrogen. The vacuum and pressurization processes are repeated for a predetermined
number of repetitions until the chamber atmosphere is below the flammability limit of 3% for EO.
A Simple EO Process In Detail
1. Environmental Preconditioning
2. Initial Evacuation.
3. Humidification.
4. Gas Injections and Gas Dwell.
5. Postexposure Gas Purge and Air Inbleed.
6. Heated Aeration.

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1.Environmental Preconditioning.
Most of the EO sterilization processes of today start with conditioning of the products to be
sterilized outside of the sterilization chamber. Preconditioning is usually performed in a room
which has been specially designed to heat and humidify the products to a stable internal
temperature and moisture content prior to entering the chamber. This will assure that the
sterilization process is reproducible regardless of external influences such as varying climatic
conditions.
Items for consideration:
Be aware of the need to heat and humidify the product for 12 to 72 hours. Packaging integrity and
its ability to withstand preconditioning conditions of a nominal 118°F (47°C) and 65 percent
relative humidity must be taken into consideration.
Once preconditioning is complete, the products are placed in a heated chamber which has been
designed to withstand the extreme pressures realized when delivering the sterilization process.
2.Initial Evacuation.
To safely deliver the 100% ethylene oxide process, at least 97 percent of the air must be removed
from the chamber.
Today, the two most common methods of accomplishing this requirement are
(1) pulling a deep vacuum, or (2) performing a series of partial vacuums followed by a
series of nitrogen injections.
This combination, when performed using an adequate number of repetitions, will purge (remove)
the air, thus allowing the process to be performed safely.
The initial vacuum rate is designed and controlled for the product and its ability to withstand
pressure changes.
The evacuation rate is selected to assure package integrity is maintained by allowing the air trapped
inside the package to vent without destroying the seal integrity.
This will assure that the package’s sterile barrier properties are maintained, thus ultimately
protecting the sterility of the product once the process is complete.
The amount of negative pressure (vacuum) designed into the process is dictated by the pressure
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sensitivity of the product.
Some devices and/or components are not designed to withstand deep vacuums and/or high
pressures.
Subjecting them to the extreme pressures required to deliver a deep vacuum or 100% EO process
will result in burst packaging and damaged products.
For those pressure sensitive products, the shallow vacuum or nitrogen soft cycle is utilized for
processing.
During a nitrogen soft cycle, an initial shallow vacuum is drawn followed by a nitrogen injection.
The combination of the vacuum and nitrogen injection is called a nitrogen wash.
This process is repeated several times (several nitrogen washes) to assure an adequate removal of
air from the vessel.
Although less demanding on packaging seal integrity, the nitrogen soft cycle is the less desirable
when compared to the 100% EO process.
By performing the additional washes, which are required for safety purposes, additional time is
added to the total process.
3. Humidification
The total inactivation of microorganisms using ethylene oxide is attained when sterilizing
conditions are met within the chamber.
The four active ingredients required to deliver a successful process are:

 Heat
 Moisture
 Gas concentration
 Time
During the previous preconditioning step, heat and moisture were added to the product to a
predetermined or stable condition. When the initial evacuation phase of the process is performed,
the product can lose a significant amount of moisture.
This moisture must be replaced prior to introducing the ethylene oxide. This is accomplished by
adding humidity in the form of steam injections.
The amount of steam required is calculated to yield a predetermined relative humidity.
After the addition of steam, the product is allowed to dwell or soak for the amount of time
required to replace the moisture lost from the evacuation phase.
The humidification phase of the process can subject the product to elevated levels of moisture and
heat. Care should be taken when selecting packaging to assure that the corrugate strength is
adequate to withstand the process. The levels of moisture are determined by the process design
scientist to assure adequate moisture content within the product for sterilization purposes. Care is
exercised to prevent overheating when injecting steam. It is extremely important to identify any
temperature limitations prior to initial process design.
If necessary, the process design scientist can adjust cycle parameters of an existing processes to
compensate for product sensitivity to heat. The cost of the adjustment may be added time to the
entire process.
4.Gas Injections and Gas Dwell
1
After the humidification phase, liquid ethylene oxide is first heated into a gaseous phase, then
injected into the chamber. The amount of gas or gas concentration3 is dependent on two primary
factors which are addressed during cycle design. The most important factor is to assure that the
minimum gas concentration required to achieve sterility within the product is attained. This
minimum concentration must be balanced against the second factor, which is the maximum
amount of gas that can be injected before difficulties arise due to high levels of post-sterilization
EO residuals. After the gas has been injected, the exposure phase of the process is performed. This
is the phase in which the product is exposed to heat, relative humidity, and gas for a predetermined
amount of time. The amount of exposure time is determined by the process design scientist, after
careful analysis of the product, load configuration, and desired level of sterility. Preliminary
laboratory experiments may be needed prior to validation execution.
5.Postexposure Gas Purge and Air Inbleed.

After the exposure phase of the process, all gas must be removed from the chamber until the levels
of EO fall below the flammable limit for the gas (3 percent or 30,000 ppm). This is accomplished
by performing a series of post-vacuums, each followed with a nitrogen backfill (wash). A maximum
working pressure for the washes is selected by the process design scientist to assure that the
products, which may have been softened during the exposure phase of the process, will not be
damaged. An ample number of washes is performed to reduce product residues and facilitate safe
handling of the product after processing.

Postexposure washes are very similar to the washes performed during initial evacuation for
shallow vacuum nitrogen soft cycles. However, in the case of the 100% EO processes, the
procedure is similar but deeper vacuums are utilized.
6.Heated Aeration.
To reduce the amount of residence time in the vessel, products after sterilization are usually
placed in
a heated room for additional removal of the residual gases. The rooms are maintained at elevated
temperatures and the outgassed residues are continuously removed from the room and scrubbed.
The aeration rooms help contain any airborne EO and continually reduce the in-product residues.
After aeration, the load is moved to the warehouse for storage until release.

The additional aeration time and heat accelerates the outgassing process. The amount of time required
to clean the products is a factor of the product material composition and the intended use of the device
(blood contact, mucus membrane contact, topical, etc.).
Validation of sterilization processes.

The efficiency of any sterilization process can only be assured , it can not be guaranteed because a
process of sterilization cannot be verified by subsequent inspection and testing of the finished
product .
Therefore it is essential that the effect of chosen sterilization process on the product should be
investigated and the procedure validated before it is applied in practice .
Validation is defined as a documented procedure for obtaining , recording and interpreting data
required to show that a process will consistently comply with predetermined specifications.
The sterilization equipment should be installed in accordance with its specifications , should be
safe to use and function with in predetermined limits as per operating instructions .
The data will include certificate of calibration for instruments used to control , monitor or record
the parameters of the process.
Performance qualification data provide evidence that the process equipment will produce a

1
product with the acceptable assurance of sterility when operated as per the operating
instructions .
Physical performance qualification provide that the specified sterilization conditions are attained
within every part of the product in it’s final packaging and are maintained through out the
sterilization cycle.
It also demonstrates that the process has no deleterious effect on the product or it’s packaging .
The measurements performed will depend upon the process being validated and may include
temperature , pressure, relative humidity , chemical concentration , irradiation dose, and filter
integrity.
Biological performance qualification provides evidence that the specified sterilization conditions
deliver the required microbial lethality to the product. This is usually done by measuring the
inactivation of reference microorganisms with known resistance to the steriliant either presented
on carriers or inoculated directly on or in the product.
Sterility assurance also requires that the performance of a sterilization process be monitored
routinely. This is done by monitoring and suitably recording the physical , and when relevant ,
chemical conditions achieved within the load in the chamber through out each sterilization cycle.
Physical methods –
Heat distribution within the sterilizer chamber or load is usually measured using
thermocouples( is a sensor that measures temperature . It consists of two different types of metals,
joined together at one end . When the junction of the two metals is heated or cooled, a voltage is
created that can be correlated back to the temperature) . Pressure measurement is by means of
pressure gauge or through pressure transducers ( transmitter that converts pressure into an
electrical signal) connected to electronic recording instruments. Physical measurement of relative
humidity in sterilizers is difficult . Microprocessor controlled sterilizers utilize multichannel
recording systems, which are able to handle large amounts of data from physical instruments and
give a more detailed control and monitoring of the sterilization cycle.
Chemical methods-
Chemical monitoring is based on the ability of the sterilization process to produce sufficient
change in the physical and or chemical characteristics of chemical substances to be detected
visually or by physical instrumentation .
generally undergo melting and or colour changes . Chemical indicators – Such as pellets of sulphur
in test tube or Browne’s sterilizer control tubes may be used . AT 120 0C. sulphur pellets gets
melted with change in shape and red solution of Browne’s tube turns red to green , when
adequate temperature is maintained for adequate time.
Browne’s tubes. These are the sealed glass tubes which contains a red fluid that changes through
amber to green on heating .
Hour glass devices.- This makes use of the melting point of a solid .eg. Acetanilide for 115 0C,
Succinic anhydride for 1210C and para acetotoluidide for 1500C ( for dry heat sterilization ).
Filter paper strip. Another device involving the melting of a solid & incorporating a time factor ,
consists of a filter paper strip , laminated to aluminium foil to improve heat conduction &
distribution & impregnated at one end with a 2,4 – dinitro phenyl hydrazone (DNP) derivative of
appropriate melting point . DNP compounds are highly coloured . The melted substances creeps
along the paper for a distance corresponding to the time at exposure temperature .
Biological indicators .
A biological indicator ( BI) is a preparation of microorganisms , usually bacterial spores , that is
carried either directly by some of the items to be sterilized or by adventitious carriers such as
filter papers , threads , and porcelain cylinders , and that serves as a challenge to the
effectiveness of a given sterilization process or cycle .

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In order to confirm the efficiency of the sterilization process , the I.P. 1996 recommends the
following biological indicators .
Moist heat sterilization - Spores of Bacillus stearothermophilus or
( autoclaving ) Clostridium sporogenes.
Dry heat sterilization Spores of Bacillus subtilis var niger

Ethylene oxide sterilization Spores of Bacillus subtilis var niger
Radiation sterilization Spores of Bacillus pumilis.
Test for Sterility (IP 1996)
The test for sterility are intended for detecting the presence of viable forms of microorganisms in
phramacopoeial preparations . The tests must be carried under conditions designed to avoid
accidental contamination of the product during the test .
A. Culture media : The following media are recommended in the Pharmacopoeia .

Fluid Thioglycolate medium : It is for use with clear fluid products.
L- Cystine 0.5gm
Dextrose 2.5gm
Granular agar 0.75gm
Yeast extract 5.0 gm
Resazurin(0.10% fresh solution) 1.0ml
Alternative Thioglycolate medium : It is for use with turbid and viscid products and for devices
having tubes with small lumina.
L-Cystine 0.5gm
Sodium Chloride 2.5gm
Dextrose 5.5gm
Yeast extract 5.0gm
Soyabean- casein digest medium

Pancreatic digest of casein 17.0gm
Papaic digest of soya bean meal 3.0gm
Sodium chloride 5.0gm
Dibasic potassium phosphate 2.5gm
Dextrose 2.5gm
All the media are adjusted to pH 7.1± 0.2 and sterilized by autoclaving at 121 0 C for 20
minutes. Sterility of these media should also be tested .
B. Test Organisms : The test microorganisms for different media and their incubation
conditions are given below .
Medium Test Microorganisms Incubation

(Strains specified in IP)
Temperature Condition
(0C)
1.Fluid Thioglycolate. 1.Bacillus subtilis 30 to 35 Aerobic
2.Candida albicans
1
3.Bacteroides vulgates 30 to 35 Aerobic

30 to 35 Aerobic
2.Alternative Thioglycolate. 1. Bacteroides vulgates 30 to 35 Anaerobic
3.Soyabean Casein digest 1.Bacillus subtilis 20 to 25 Aerobic

2.Candida albicans 20 to 25 Aerobic
C. Test Procedure : IP recommends two methods ie. Method A, Membrane Filtration

or Method B, Direct Inoculation.
Method A – Membrane Filtration : This method is to be preferred where the substance being
examined is (a) an oil , (b) an ointment that can be put into solution, c) a non- bacteriostatic
solid not readily soluble in the culture medium and d) a soluble powder or a liquid that possesses
inherent bacteriostatic and fungistatic properties.
The membrane should have a pore size not greater than 0.45 µm and diameter of approximately
47 mm. The apparatus is assembled and whole unit is sterilized.
Diluting fluids : Fluid A : It is prepared by dissolving 1 gm of Peptone in water to make 1 liter,

clarified by filtration or centrifugation , adjusted to pH 7.1±0.2, and sterilized at 121 0C for 20
minutes in 100ml quantities in flasks.
Fluid B : It is fluid ‘A’ to which 1ml of Tween 80/L has been added.
Method of Test :
a)For aqueous solutions : For each medium to be used , the prescribed quantity is transferred
aseptically into two separate membrane filter funnels , the liquid is drawn rapidly through the
filter with the aid of vacuum. The membrane is removed aseptically , cut into two , immersed in
100ml of Soya bean – casein digest medium / Fluid thioglycolate medium and incubated at 20 to
250C for seven days .
b) For liquids immiscible with aqueous vehicles and suspensions : Add a sufficient quantity of fluid
‘A’ to the sample and proceed as above.
c) For oils and oily solutions : Materials of sufficiently low viscosity are filtered with out
dilution .
d)For ointments and creams : Ointments containing a fatty base and o/w emulsions are diluted by
heating to give a concentration of 1% w/v.
e) For soluble solids : For each medium , dissolve the prescribed quantity in fluid ‘A’ .
f) For sterile devices : Aseptically pass a sufficient volume of fluid ‘B’ through the 20 devices to
recover not less than 100ml from each device , the collected fluid is filtered and proceeded with test.
Method B – Direct Inoculation:
a)For aqueous solutions and suspensions : The liquid from the test containers is removed and the
specified volume is transferred aseptically to a vessel of the culture medium and mixed . The
inoculated medium is incubated for 14 days at 30 to 35 0C ( Fluid Thioglycolate medium) and at 25
to 300C ( soya bean - casein digest medium).
b)For oils and oily solutions : Use media to which have been added 0.1% w/v of (4-tert-octyl
phenoxy) polyethoxyethanol or 1% w/v of Tween 80 and proceed with the test .
c)For ointments : Prepare by diluting ten fold in a sterile diluents such as fluid ‘B’.
d)For solids : transfer the required quantity of the material to the medium .
e)For sterile devices : Immerse the device in 1000ml of culture medium .If immersion is
impracticable , flush the lumen of 20 units with fluid thioglycolate medium and soya bean casein
digest medium separately , and recover 15ml of each medium.
D) Observation and interpretation of results:

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At the end and during the incubation period if no growth is observed, the preparation is deemed to
have passed the test.
If growth is observed , the containers showing the growth are reserved and a re -test is performed as
in the original test .
If no growth is observed , the preparation being examined passes the test .
If growth is observed again , the organisms are isolated and identified .
If they are not readily distinguishable from those growing in the containers reserved in the first
test, the preparation fails the test . If they are not readily distinguishable from those growing in the
containers reserved in the first test , a second re-test is performed using twice the number of
samples .
If no growth is observed in the second re-test, the preparation passes the test . If microbial growth is
observed in the second re- test the preparation fails the test.
Chapter 6. Disinfectants – Study of Disinfectants , antiseptics , fungicidal and

virucidal agents factors affecting their activation and mechanism of action .
Evaluation of bactericidal, bacteriostatic , virucidal activities , evaluation of
preservatives in pharmaceutical preparations.
Introduction: Disinfection is a process of destruction or elimination of microbes, and decreasing their

numbers in such a low level, that they become unable to impart any harmful effect. The process of
disinfection is more effective in killing or removing vegetative cells , than endospores, because
vegetative cells are less resistant as compared to endospores ( as they are heat resistant). The
antimicrobial agents that are applied on the surface of nonliving or inanimate objects. Eg. working
area, dishes, bench etc. are termed as disinfectants , where as term antiseptics is used for those
chemical agents that are used on animates or living bodies eg. human body tissues, plant tissues etc.
Generally , disinfectants are bactericidal , through they may sometimes be bacteriostatic.
Antiseptics are specific type of disinfectants that are used in the reduction of number of viable
organisms on the skin or other exposed parts of the living body.
An ideal antiseptic or disinfectant should have the following properties:
1) It should have a broad spectrum of activity.
2) It should be capable of eradicating microbes in a specified time period.
3) It should be active in the presence of organic matter also .
4) It should be effective enough to make contact and show proper wettability.
5) It should be active at all pH.
6) It should be stable.
7) It should have long shelf life.
8) It should have a high penetration power and rapid action.
9) It should be non toxic , non allergenic , non irritant and non corrosive.
10) It should not leave non volatile residue or stain.
11) It should be inexpensive and easily available.
12) It should have a pleasant odour.
Classification:- Disinfectants are classified on the basis of the following factors.

1
1) Based on consistency: On the basis of consistency or state of matter , disinfectants are of

two types
i)Liquid – eg. alcohols , phenols
ii) Gaseous-eg. Formaldehyde vapour.
2) Based on spectrum of activity: On the basis of spectrum of activity , disinfectants are of
three types.
i)High level disinfectants:- these disinfectants are used against certain types of endoscopes ,
cystoscopes and surgical instruments with plastic components which cannot withstand sterilization
procedures such as autoclaving . eg. of high level disinfectants include glutaraldehyde, hydrogen
peroxide, peracetic acid and chlorine compounds.
ii )Intermediate Level Disinfectants:- These disinfectants may not be effective against bacterial
spores , so are used for instruments (eg. laryngoscopes, fiberoptic endoscopes) where contamination
with spores and other highly resistant organisms is unlikely . eg. of these disinfectants include
alcohols, iodophores and phenolic compounds.
iii) Low level Disinfectants:- These disinfectants are used against items which come in contact with
the patients , but they do not penetrate the tissues. Stethoscopes, electrocardiogram, electrodes etc. are
examples of such items. Egs .of these disinfectants include quaternary ammonium compounds and
phenolic disinfectants.
3)Based on mechanism of action:- On the basis of mechanism of action , disinfectants are classified
into:
i) Disinfectants acting on plasma membrane( eg. alcohol, detergent).
ii) Disinfectants denaturing cellular proteins ( eg. alcohol, phenol).
iii) Disinfectants responsible for oxidation of essential sulphydryl groups of enzymes.
Eg.H2O2 ,halogens.
iv) Disinfectants responsible for alkylation of amino, carboxyl and hydroxyl group eg. formaldehyde.
v) Disinfectants damaging nucleic acid eg. formaldehyde.
Mode of Action:-Many substances act as disinfectants like alcohols,bleaches, hydrogen peroxide and
iodine. Different disinfectants have different mechanism of action. An ideal disinfectant would offer
complete sterilization, without harmimg other life forms. Along with these essential properties , an
ideal disinfectant should be inexpensive and non corrosive. However such ideal disinfectants do not
exist and many disinfectants generally show partial sterilization. The bacterial spores, some viruses
and bacteria are highly resistant to many disinfectants.
Mode of action and application of the following disinfectants are discussed below:
1. Alcohols.
2. Phenols and phenolic compounds.
3. Aldehydes.
4. Halogens
5. Heavy metals.
6. Oxidising agents.
7. Surface active agents and
8. Dyes.
Alcohols:- Alcohols show broad spectrum antimicrobial activity. Ethyl alcohol, methyl alcoholand
isopropyl alcohol are most frequently used.
Mode of action:- Alcohols act by dehydrating the cells , by disrupting bacterial cell membrane and by
coagulating the protein content of microorganisms. The ethyl alcohol or ethanol solutions of
concentration between 70- 90 % are very effective against the vegetative forms of microorganisms .
but ethyl alcohol does not completley sterilise an object since it does not affect bacterial endospores .
for eg. it is reported that the endospores of Bacillus anthracis can survive in alcohol for 20 years. In
alcohols the bactericidal property increases in the length of carbon chain. Also alcohols with carbon
chains longer than propyl alcohol and isopropyl alcohol are slightly water soluble 9 even less than
ethyl alcolh) there fore they are not used as disinfectants.

1
Applications:
1.Antiseptic action:- A solution of ethyl alcohol( 70%) and isopropyl alcohol (90%) work as a skin
antiseptic. This solution is used as a disinfectant for clinical orl thermometers and other surgical
instruments.
2. Cleaning agent :- Wipes of alcohol are used to clean skin before tking blood samples.
3. Isopropyl alcohol :- It is prefered over ethanol for disinfecting surfaces.
4. Methyl alcohol:- It kills fungl spores , there fore is best suited for disinfecting inoculation hoods.
Phenols and Phenolic Componds :-

Phenols or carbolic acids are derived from coal tar and are used as disinfectants. It was first time
used by Lister . It has a corrosive property and may produce harm ful effects in sensitive people . egs
of phenolic compounds used as disinfectants include 5% phenol, 1-5% cresol, 5% lysol ( cresol with
soap solution ), hexachlorophene, chlorhexidine and chloroxylenol (dettol).
Mode of action :- Phenols act by disrupting the cell membranes, precipitating the proteins and by
inactivating the microbial enzymes. They alter the selective permeability of cytoplasmic membrane
of microbial cell , there by the vital intracellular substances leak out. Depending on their
concentration to be used , they may be either bacteriostatic or bactericidal.
Application :-
1) Phenol functions as bactericidal, fungicidal and mycobactericidal , but is ineffective against
spores and viruses.
2) The corrosive phenolic is used to disinfect the ward floors, discarded jars in laboratories and
bed pans.
3) Cresol ( Lysol) is a soap solution of phenol like compounds ( 0- phenyl phenol, 0- benzyl- p-
chlorophenol, xylenols). It is useful in disinfecting the inanimate objects like floors, walls ,
table surfaces and contaminated hospital items ( like rectal thermometers , excreta ,
secretions from infected patients etc.).
4) Chlorhexidine is isopropanol solution is used for skin disinfection, and its aqueous solution is
used for wound irrigation . It is also used as an antiseptic hand wash.
5) Chlorhexidne gluconate solution (20%) is used as a general skin disinfectant and also as a
preoperavite hand and skin disinfectant. Chlorhexidne gluconate solution with quaternary
ammonium compounds ( eg. cetrimide ) show stronger and broader antimicrobial effects
( eg. Savlon).
6) Chloroxylenols are topically suitable due to less irritant property. These compounds are more
effective against gram positive bacteria than against gram negative bacteria.
7) Hexachlorophene is a chlorinated diphenyl preparation and is relatively less irritant . It is
highly effective against gram positive bacteria and is less effective against gram negative
bacteria , mycobacteria , fungi and viruses.
8) Triclosan is organic phenyl ether and is very effective against gram positive bacteria and to
some extent against gram negative bacteria.It is also effective against fungi and
viruses.
9) Hexylresorcinol is a complex chlorinated phenolic compound. It is incorporated into
mouthwashes, topical antiseptics and throat lozenges. It is also used as dusting powders and
creams.
Aldehydes:-
Formaldehyde works as a bactericidal , sporicidal and virucidal agent. It is used in aqueous as well as
gaseous form. For general purposes a 10% aqueous formalin solution is used.
Glutaraldehyde is highly effective against bacteria eg. M. tuberculosis, fugi and viruses (like HIV).
Its toxicity and irritancy to skin and eyes is comparatively less than formaldehyde. Generally it is used
as a 2% buffered solution.
Mode of action:- Aldehydes cause alkylation of amino, carboxyl or hydroxyl groups and damage

1
nucleic acids. They are effective against all microorganims and spores.
Applications:-
1) A 40 % formaldehyde solution ( formalin ) is used to disinfect surface. It is used to fumigate
rooms, chambers, operation theaters , biological safety cabinets, wards ,sick rooms etc.
2) It is used as a preservative for histological examination of tissues.
3) It is used for sterilizing bacterial vaccines.
4) It is used for preparing toxoid from toxin.
5) Fumigation process is performed by boiling formalin, heating paraformaldehyde or treating
formalin with potassium permanganate.
6) It is also used for sterilizing bedding , furniture and books.
7) 10 % Formalin solution with 0.5% tetra borate is used for sterilizing clean metal
instruments .
8) 2% Gluteraldehyde solution is used for sterilizing thermometers, cystoscopes, bronchoscopes,
centrifuges, anasethetic equipments etc. Gluteraldehyde action requires an exposure of at
least 3 hours at alkaline pH.
9) It is also used for sterilizing plastic endotracheal tube, face masks, corrugated rubber
anaesthetic tubes and metal instruments.
10) 2% formaldehyde solution is used at 40 C for 20 minutes for disinfecting wool and 0.25%
0
formaldehyde solution is used at 60 C for 6 hours for disinfecting animal hair.

0
Halogens:-Halogens being strong oxidizing agents are highly reactive and destructive towards vital
cellular components of microbes. Members of the halogen family ( especially iodine, chlorine and to
some extent bromine) are used as key components in various antimicrobial chemicals . Iodine is the
oldest microbicidal agent with highest efficiency .Mode of Action:- Halogens act as oxidizing
agents and damage the essential sulfydryl groups of enzymes by oxidation reaction. Iodine is a strong
oxidizing agent and can destroy essential microbial metabolic compounds by oxidation. Iodine
combines with tyrosine amino acid to inactivate enzymes and other proteins.
On adding free chlorine to water , hypochlorous acid (HClO) is formed , which is responsible for
the antimicrobial activity of chlorine and it’s compounds.
Cl2 + H2O → HCl + HClO
Hydrochloric Hypochlorous
Acid Acid
In water hypochlorites and chloramines hydrolyses into hypochlorous acid which further gives
nascent oxygen [ O] :
HClO → HCl + [O]

Nascent
Oxygen
Nascent oxygen being a strong oxidizing agent damages the vital cellular substances of microbes.
Chlorine combines directly with the cellular proteins of microbes and block their biological activity.
Applications.

1
1) Iodine acts as a germicidal agent , highly effective against all types of bacteria .
2) It also acts as a sporicidal , fungicidal , virucidal and amoebicidal .
3) Tincture of Iodine made of 2% iodine in 70 % alcohol acts as an antiseptic.
4) Iodine with neutral carrier polymers ( polyvinyl pyrrolidone ) forms iodophores ( eg.
Povidone – iodine ) which allow slow release and reduce the irritation of antiseptic .
Iodophores diluted with 50 % alcohol are used for preparing hand washes .
5) 10 % Povidone Iodine in undiluted form is used as a pre and post operative skin
disinfectant.
6) Iodine preparations are mainly used as skin disinfectants .
7) Iodine is also used for disinfecting small quantities of water and for sanitizing food utensils.
8) Sometimes iodine vapour is used for disinfecting air.
9) Liquefied chlorine gas is preferred for disinfecting drinking water and swimming pool water.
It is also used for treating effluents from sewage treatment plants.
10) Products having calcium hypochlorite are used to sanitize utensils in restaurants and
equipment in dairy plants.
11) 1% Sodium hypochlorite solution is used for personal hygiene and for affordable household
disinfection.
12) Higher concentration ( around 5-12 % ) of sodium hypochlorite are used as household
bleaches , disinfectants and as sanitizers in dairy and food processing plants.
Heavy metals:- The term heavy metals is used for denoting mercury, lead, silver and copper. In
ancient times , water storage containers of silver and copper were used ,as it was noticed that these
metal vessels kept water safe to drink. In the early 20 th century, mercuric chloride had been widely
used as a general disinfectant; but later it was replaced with other less toxic and corrosive agents.
Other than mercuric chloride, silver nitrate, copper sulphate and organic mercury salts
( mercurochrome, merthiolate) are egs of other heavy metal compounds.
Mode of action:- Heavy metals bind to the cellular proteins and inactivate them, for eg. mercuric
chloride combines with sulphydryl( SH) groups that inactivate enzymes.
Applications:-
1. Certain organic mercury containing compounds show high antimicrobial activity with low
toxicity in comparison to inorganic mercury compounds. Eg. of such organic compounds are
merbromin(Mercurochrome), thimerosal ( Merthiolate) and niromersol (Metphen) , which
are used for trating minor cuts, wounds and skin infections.
2. Earlier a 1% silver nitrate solution was used for disinfecting the eyes of infants at birth to
prevent the gonoconnal infections.
3. 0.5% silver nitrate solution is used in sponges or layers of gauze for preventing infections in
burns.
4. Silver sulphadiazine is applied topically for preventing colonization and infection of burned
tissues.
5. Copper salts are used as fungicides. For eg. copper sulphate is used as algicide for
disinfecting open water bodies like reservoirs and pools.

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6. 6.It also acts as a fungicide and is a constituent of Bordeaux mixture used as a garden spray
to protect plants against fungal infections.
7. Zinc compounds are fungicidals and are used in formulation of ointments and powders used
for treating athlete’s foot.
Oxidising Agents:- Oxidising agents oxidize the cell membrane of microorganisms , resulting in
the loss of cellular structure and cell lysis or death of microbes. The key elements in oxidizing
agents are chlorine and oxygen. Hydrogen peroxide is a chief oxidizing agent which in 3-6%
concentration is effective against almost all organisms . At higher concentration ( 10-25%) it
kills all the organisms including spores.
Mode of action :- Hydrogen peroxide releases nascent oxygen, therefore is used as an

antimicrobial agent.It also gives hydroxyl free radical which denatures proteins and DNA
molecules.
Applications:-
1. At 6% concentration , it is used for decontaminating the instruments and equipments

eg.ventilators.
2. At 3%concentration,it is used for disinfecting the skin and deodorizing wounds and ulcers.
3. Strong solutions of Hydrogen peroxide is used as sporicidal.
Surface Active Agents:-Soaps or detergents are the egs. of surface active agents. Detergents
carrying negatively charged long chain hydrocarbons are anionic detergents eg. soap and bile
salts and detergents having the fat soluble part is made to have a positive charge by combining
with a quaternary nitrogen atoms are cationic detergents ( eg. cetrimide and banzalkonium
chloride ) . Cationic detergents are also known as Quaternary ammonium compounds or quat.
Mode of action:-Surface active agents aligin themselves at the interfaces between lipid layer of
bacterial cell membrane and surrounding aqueous medium . These compounds carry long
chain of fat soluble hydrocarbons and water soulble charged ions. Under the influence of this
dual nture of fat and water solubility , they concentrate on the membrane surfaces and disrupt
the membrane , thus resulting in the leakage of cell constituents . Quaternary ammonium
compounds act by the denaturation of cell proteins , interference with metabolic processes and
damage to cytoplasmic membrane.
Applications:-
1) They inhibit the growth of vegetative cells , Mycobacteria and enveloped viruses .
2) Solutions of 1-2% of dilution are widely used as disinfectants for domestic and hospital
purposes .
3) The Quaternary ammonium compounds are germicidal in nature with low toxicity , high
water solubility , high stability in solution and with less corrosive properties , hence preferred
as useful antiseptic , disinfectants and sanitizing agents.
4) They are used for disinfecting floors, walls and other surfaces in hospitals , nursing homes
and other public places .
5) They are also used for sanitizing food and beverage utensils in restaurants , surfaces and
equipment in food processing industry.
Dyes:
Aniline dyes include crystal violet, malachite green and brilliant green , while acridine dyes are
acriflavin and aminacrine. Acriflavin is a mixture of proflavine and euflavine: in which only

1
euflavine has effective antimicrobial properties. Dyes shows better antimicrobial property for
gram positive bacteria than gram negative bacteria and are more bacteriostatic in nature.
Mode of action:-
Both aniline and acridine dyes have low bactericidal action and are bacteriostatic in high
dilution. Aniline dyes interrupt the synthesis of peptidoglycan ( a compound of bacterial cell
wall) and acridine dyes interrupt the synthesis of cellular nucleic acids and proteins of bacteria.
Applications:-
1. They have better antibacterial action for gram positive bacteria.

2. They are topically applied as antiseptics for treating mild burns.
3. Gentian violet and acriflavine are applied as paint over the skin to treat bacterial infections .
4. Malachite green is incorporated in medium for inhibiting the growth of all bacteria except
M.tuburculosis.
Factors influencing Disinfection.

Extent of antimicrobial action, rate of disinfection, and potency of a disinfectant depends on the
following factors.
1)Concentration of Disinfectant : Concentration of the disinfectant directly influences the rate of
killing microorganisms . The curve obtained between the efficiency and concentration of
disinfectant is generally exponential rather than linear.
2) Temperature: Normally , on increasing the temperature the rate of disinfection increases .
Temperature coefficient (Q10) is used for quantitative representation of temperature effect on
bacterial activity . Thus
Time required to kill at T0
Q10 = -------------------------------
Time required to kill at (T + 10)0.
t1
Q10( T2 – T1) = ----
t2
Where T2 and T1 = Two temperatures differing by 100C.
t2 and t1 = corresponding lethal times.
3) Time of Exposure : Each disinfectant takes sufficient time of contact to produce its
action and to explain this first order kinetics . The velocity or rate constant (K) is the
measurement of the efficiency of the disinfectant .
1
K= --- log N0
t -----
Nt
Where t = Time for the viable count to fall from N 0 to Nt
N0 = Initial number of microorganisms .
Nt = Final number of microorganisms .
The survivor / time curve is not constant and shape of the curve mainly depends on the
concentration of the disinfectant .
.

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4) The pH of Environment : the rate of growth of inoculum and the potency and ability of
the disinfectant to combine with the cell surface during the disinfection process depends
on the pH changes .
5) Surface Tension : The surface properties influence the contact between the aqueous
solutions of disinfectants. These surface properties help in adsorption of surface active
disinfectants on the surfaces of cell , and also in spreading and wetting properties of the
solution.
6) Formulation of the Disinfectant : Formulation of a disinfectant is an important factor as it
influences the effective use of disinfectant . For example , quaternary ammonium
compounds and chlorhexidine are efficient in 70 % alcohol than in aqueous solution .
7) Chemical structure of Disinfectant : Any change in the molecular structure of the chemical
compound may alter the activity and efficiency of the disinfectant . For example para
substitution of an alkyl chain up to 6 – carbons in length increases antimicrobial activity ,
but greater than 6- carbons in length decreases water solubility and disinfectant activity.
8) Type and Number of Microorganisms Present : Number and nature of the contaminating
microorganisms directly influence the efficiency of disinfection. The presence or absence
of bacterial spores directly affects the efficiency of disinfectant , as most of the disinfecting
agents are ineffective on bacterial spores and viruses .
9) Interfering substances in the Environment : Presence of many compounds ( eg. Body

fluids , food residues , blood , milk , pus , or colloidal proteins ), even in small amount may
decreases the effectiveness of many disinfecting agents.
10) Synergism ,Antagonism and Potentiation of Disinfectants : Synergisms or synergistic
effects are often shown by two antimicrobial agents which gives an increased activity .
Antagonism or antagonistic effects result in decreased antimicrobial activity and is used in
the elimination of antimicrobial properties of materials ( tested for sterility ) eg. Sodium
thiosulphate , lubrol W+ lecithin etc. Potentiation of a disinfectant leads to enhanced
antimicrobial activity
eg. polysorbate 80
Evaluation for Bacteriostatic and Bactericidal Actions .
For the evaluation of efficiency , potency, and reactivity of disinfectants following

techniques are used.
1) Tube dilution and agar plate method.
2) Cup plate , filter paper and agar plate method .
3) Ditch plate or giant colony method .
4) Kelsey-Sykes Test.
5) In use dilution test .
6) Phenol coefficient test – Rideal Walker Test and
7) Chick Martin test.

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Tube Dilution and Agar plate method:-

The chemical disinfecting agent is introduced into the agar medium or nutrient broth medium. After
that the medium are inoculated with the test microorganisms. The test tubes containing medium and
microorganisms is incubated at 30- 35 oC for 2-3 days and then the results are observed in the form of
turbidity or colonies. These turbid colonies are recorded and the activity of given disinfectant is
compared as shown in figure.
a) Agar plate method.
Cup -plate, Filter Paper and Cylinder Plate Method
In this method heating is performed for melting the agar and cooled up to 45 0c. The agar medium is
inoculated with the test microorganisms and poured into a sterile petri dish. In the cup plate method ,
when the inoculated agar has been solidified , holes of about 9mm in diameter are made by cutting
the medium with sterile cork borer and the antimicrobial agents are directly placed in these holes.
Evaluation of disinfectant by cup plate method.

In the filter paper and cylinder plate method, the antimicrobial agents are applied on the surface of
solidified , inoculated agar medium, by using filter paper disc and cylinder respectively. On
incubation at the temperature of 30-35oC for 2-3 days ,a zone of inhibition is observed. The diameter
of this zone gives an indication of the relative activities of different antimicrobial substances against
the test microbes.
Ditch –Plate or Giant Colony Method: -A ditch is prepared in agar plate and the solution of
antimicrobial substance is carefully poured into the ditch. On the Agar surface, a loopful of each test
organism is streaked outwards from the ditch. Microbes resistant to the antimicrobial agent grow on

1
right of the ditch, where as susceptible microorganisms show a zone of inhibition adjacent to the
ditch or centre of plate.
Evaluation of Disinfectants by Ditch -Plate or Giant Colony Method.
The width of the zone of inhibition indicates the relative activity of antimicrobial substances
against
various microbes used during test .
Kelsey- Sykes ( KS) Test .

This test is the measurement of the capacity of a disinfectant to retain its activity , when it is used
repeatedly in different microbiological operations . This test is also known as capacity test .
In this test , standard microbes ( eg. Esch. Coli , Ps.aeruginosa and Staphylococcus aureus are added
to the disinfectant in three continuous lots at a time difference of 0. 10, and 20 minutes . These
three lots are kept in contact with disinfectant for 8 minutes and samples are transferred at the
difference of 8, 18 and 28 minutes respectively to a recovery medium . Thus , efficiency of
disinfectant is determined by its ability to kill bacteria , and not by comparing with phenol. This
test is performed both in dirty and clean conditions ; therefore it also measures the effectiveness of
a disinfectant in presence of n organic matter.
The selection of the test organism ( S. aureus, P.aeruginosa, P .vulgaris and E. coli) depends on the
type of disinfectant used for testing procedure . For carrying the test under ‘clean ’ conditions , the
dilutions of the disinfectant are made in hard water ; where as t run the test under ‘ dirty’ conditions
yeast suspension is used for making dilutions of the disinfectants. Test organism with yeast alone
is added at time intervals of 0, 10, and 20 minutes. The test organism and the disinfectant are kept in
contact for 8 minutes . Three sets of five replicate cultures are made corresponding to each
condition and are incubated at 32 0C for 48 hours and growth is assessed by turbidity . Evaluation
of the disinfectant is performed on the basis of ability of disinfectant to kill microbes or lack of it ,
results obtained after this are regarded as a fail or pass.
All the tubes are incubated at 320C for 48 Hours.
Results will be negative , if two or more negative cultures are obtained in a single set. In case of
negative results, the disinfectant passes the dilution test , after the first and second conditions .
The third condition is not considered in fail /pass criterion but positive cultures serve as inbuilt

1
controls. A lower concentration of the disinfectant may be tested , if negative results are obtained
after the third condition.
In- Use Dilution Test:- This test is used for examining the quantities of viable organisms , and also
for testing the liquid disinfectants used in hospitals . the efficiency of a disinfectant is determined on
the basis of it’s capacity to kill the known number of a standard strain of pathogenic Staphylo
coccus, within the given time and surface. Results obtained by this test are practically more useful
than the results obtained from Rideal –Walker Test and its modifications.
In this test disinfectants are used in diluted form and the dilution at which the efficiency and potency
of the disinfectant is determined. The current standards for in use test are provided by the American
Official Analytical chemists (AOAC) that mentioned the use of three bacterial strains.
1.Staphylococcus aureus2. Salmonella choleraesuis 3. Pseudomonas aeruginosa.
Standardised cultures of the test organisms are grown in liquid media. This media is standardized and
metal carrier rings are dipped into it and removed and dried at 37 0C for a short time . After that , the
dried cultures are placed into a disinfectant solution , made at a concentration specified by the
manufactures. The culture is left for 10 minutes at 20 0C. In the next step , the carrier rings are
transferred to a culture medium that allows the growth of any surviving organisms. Number of
organisms in the resulting culture is calculated showing the effectiveness of disinfectants.
Phenol Coefficient Test- Rideal Walker (RWC )Test :-
In phenol coefficient or Rideal- walker test , a suspension with similar quantities of organisms is used
in the estimation of efficiency and action of different concentration of phenol and the disinfectant to
be tested . A solution o f the test disinfectant that sterilizes the suspension in a given time is divided
by the corresponding dilutions of phenol; this gives the phenol coefficient . however , phenol
coefficient is not the measurement of the practical functioning of the test disinfectant in the presence
of organic matters.
By using Rideal –Walker test ( that uses nutrient broth and Salmonella typhi) ,the phenol coefficient
of test disinfectant can be calculated.
By using the testing disinfectant and phenol, different dilutions are prepared and 5ml of each dilution
are inoculated with 0.5ml of the 24 hours broth culture of the organism. All incubated tubes
( disinfectant+ organisms and phenol+ organisms) are kept at the temperature of 17.5 0C.(AC room).
Subculture s of each reaction mixture is prepared and transferred to 5ml sterile nutrient broth, at time
intervals of 2.5, 5,7.5 and 10 minutes.
The nutrient broth tubes are incubated at the temperature of 37 0C for 48- 72 hours and examined for
the absence or presence of the growth of microorganisms . A typical eg. of test result is shown below
. The Rideal –Walker coefficient of the test disinfectant is calculated according to the data obtained.
Determination of Rideal –Walker coefficient
R.W. Coefficient = Dilution of disinfectant killing in 10 minutes but not in 5 minutes 2000
-------------------------------------------------------------------------------- = ------ =
20
Dilution of phenol killing in 10 minutes but not in 5 minutes 100
Conclusion .

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1.If the Rideal- walker or phenol coefficient for a given test disinfectant is 1 ,the disinfectant has
the
same effectiveness as phenol.
2.If phenol coefficient of test disinfectant is 20 , the disinfectant is 20 times more active than
phenol.
3. If phenol coefficient of test disinfectant is less than 1, it is less effective and if more than 1, it is
more effective as compared to phenol.
Chick Martin Test:- The phenol coefficient test is performed on laboratory level to study the
bactericidal action of compound with respect to phenol. In 1908, Chick and Martin suggested that
the disinfectants are essential to act in the presence of organic matter . so they recommended the use
of dried human faeces in the test system. As a substitute to human faeces, Garrod suggested to use
dried yeast.
In this test Salmonella typhi (test organism) is inoculated into solutions having graded concentrations
of test substnces ( or phenol in case of standard) along with dried yeast. The components are left for
30 minutes at 200C to interact and lastly duplicate subcultures are made into nutrient broth. These
subculture tubes are incubated for 48hours at 37 0C. Observations are made regarding the presence or
absence of growth of microorganisms . The phenol concentration that prevents growth in both
systems is determined and the mean value is calculated. The same value would be obtained for the
unknown . For calculating the coefficient, the value obtained for phenol is divided by the value found
for the unknown.
Chapter 7.Immunology- Immunity, Definition, Classification, General

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principles of natural immunity, Phagocytosis, Acquired immunity ( Active

and passive). Antigens, Chemical nature of Antigens , Structure and
formation of Antibodies , Antigen – Antibody reactions . Bacterial
exotoxins and Endotoxins . Significance of toxoids in active immunity ,
Immunization programme and importance of booster dose.
Immunology is the scientific study of immune system. As immune responses constitute the
principal means of natural defence against infections by pathogenic microorganism, they are
essential for survival. Immune responses are a set of adaptive processes that enable the individual
organism to produce, on demand an immense variety of specifically reactive proteins and cells that
can recognize and cause destruction of an almost infinitive variety of foreign substances.
Mechanism of Defence against diseases.The survival of humans and lower animals on earth for
millions of years is attributed to many built in mechanisms of defence against harmful
microorganisms and the infections caused by them.Humans have been gifted with three lines of
defence against bacteria, fungi, virus and other parasitic organisms.
The first two lines of defence are nonspecific in the sense that the body attempts to destroy all types
of substance that are foreign to it. As a first line of defence the body protects itself against invasion
by foreign microorganism and other substances by defending the body openings to the respiratory,
digestive, urinary and reproductive systems with mucous membranes that entrap the invaders.The
nonspecific cellular and chemical responses to microbial invasion are considered the second line of
defence.The third line of defence is very specific. Antibodies are formed in response to the presence
of particular foreign substance.
Although the lymphoid system is the site and source of most immune activity, the immune system
encompasses the whole body.
The cells involved in the immune responses have their origin in bone marrow . At least two lines of
lymphocytes B cells and T cells and probably NK cells ( Natural killer cells) derive from the
lymphoid stem cells of bone marrow . About 50 % of these stem cells migrate to the thymus gland
where they differentiate in to T cells ( T for thymus) for the helper, suppressor , or cytotoxic type.
Thymus processing of T cells begins shortly before birth. T cells are small lymphocytes found in

1
the blood , lymph and lymphoid tissue. These T cells do not produce humoral antibodies but help in
the control of antibody production and are involved in the cell mediated immune responses .Other
lymphocytic stem cells differentiate into B cells. B cells migrate to the lymphoid tissue where they
will produce antibodies to be circulated through out the lymph and blood to protect the individual.
Thousands of B cells exist in the body even though these cells live only about 1 to 2 weeks. Each B
cell is capable of producing specific antibodies when stimulated by an antigen. The key features of
immune system are specificity ( the ability to focus on specific pathogens) and memory ( the ability
to recognize and respond rapidly to previously encountered infections).
Immune response .
Many chemicals, cells and reactions are involved in the immune responses. Some chemicals and
complexes are nonspecific yet depend on the antigen – antibody complex for activation, as does
complement. Complement comprises a group of heat labile serum proteins which when activated are
associated with the destruction of bacteria in the body in a variety of ways. The specific humoral
response always depends on the presence of specific antibodies for each antigen or antigenic
determinant. However, some types of cell mediated responses occur in the presence of antibodies ,
but others do not involve the antigen- antibody complex. About 1% of the body cells are white blood
cells .
The body cells which are most important to immune responses are :
1. B cells : Lymphocytes which produce antibodies( antibody mediated

immune response).
2. Macrophages : Phagocytic cells which alert helper T cells of the presence of
pathogens.
3. Helper T cells : Master switches of the immune system which stimulate the
rapid division of both killer T cells and B cells.
4. Suppressor T Cells : Lymphocytes with regulatory functions ( Slow down or prevent
Immune response).
5. killer T cells & : Lymphocytes that directly destroys body cells which already
NK cells have been infected by pathogens or cancer cells.
6. Memory cells : A group of the T cell and B cell population, which were
produced during the primary encounter with a pathogen but
which were not used in the combat. These circulate through the
Body, ready to respond rapidly to later attacks by the same
organisms .
Immunity is generally classified as given below

Types of Immunity
Natural Immunity Acquired Immunity

(Innate immunity) (adaptive immunity)
1
Active Passive
Species Racial Individual
Natural Artificial Natural Artificial

Clinical or Vaccines Congenital Antiserum
Subclinical Dead or extract Colostrum Antitoxin
Diseases Attenuated ɤ-globulin
Toxoids
Natural Immunity:- It is the resistance to disease attributable to inherent constitutional make

up of an individual. It is not acquired due to the use of biologicals .
Levels of natural resistance to certain infectious diseases vary considerably among species, races
and even among individuals as to age and sex.
1.Species: This types of natural resistance varies in species . However, Some diseases like
Tuberculosis, Anthrax, Rabies etc. occur both in animals and man alike. Man is susceptible to Plague
but birds are not. Mice are not affected by Typhoid fever but it is a serious disease in humans . These
differences in natural immunity are attributed to differences in species.
2. Race: Certain races of man are more susceptible to Tuberculosis than others. Negroes ( a member
of a dark skinned group of peoples originally native to South Africa) posses a high resistance to
yellow fever than white men. Negroes or white Indians ( Anglo Indians) are more susceptible to
Tuberculosis than the Caucasian race (A race of human kind native to Europe, North Africa and
southwest Asia).
1.Individual : Some people are more resistant to colds and skin infections than others. Most of the
children in the age group of 2 to 5 years are susceptible to Diphtheria where as most adults are
immune to it. The type and nutritional value of food as well as the living environment are
important in individual immunity.
Key points:- Natural (Innate) immunity shows the following features.
It is due to the genetic and constitutional make up of an individual. Prior contact with
microorganisms or their products is not essential.
It acts as the first line of defence of the host immune system .
The mechanism s involved in innate immunity are present in place even before exposure to the
foreign agent . They are not specific to any infectious agent and do not seem to improve response
on repeated exposures. Phagocytic cells ( eg. Macrophages and neutrophils ) , barriers ( eg. skin and
mucous membrane) , and a variety of antimicrobial compounds synthesized by the host , all play
important roles in innate immunity.
Acquired Immunity.
Acquired immunity is developed in response to a specific stimulus. Such immunity is needed
because the natural immunity is inadequate for protection against many microbial diseases.
It is developed due to the introduction of foreign substances or the immune substances produced in
another individual or in another species.
Thus acquired immunity may be actively acquired due to the individual’s antibody producing cells
or passively acquired eg. when the antibodies from another person or animal are introduced.

1
Active immunity: It may be acquired either naturally or artificially .
a) Naturally acquired active immunity: It is acquired as a result of natural infection by

pathogens ; either in a recognizable clinical presentation or a subclinical non recognizable
form. It is also acquired when a person recovers from certain diseases eg. after suffering
from diphtheria, small pox or poliomyelitis; and persist for life. Where as the immunity
acquired after suffering from influenza, pneumonia and Gonorrhoea is short lived. This type
of immunity may also be acquired after subclinical infections due to smaller number of
invading organisms. A person becomes immune because his antibody producing cells have
received an adequate stimulus . Thus children and adults staying in slums develop
naturally acquired active immunity to a variety of diseases as they are frequently exposed
to sub infections.
b)Artificially acquired active immunity : This type of immunity is acquired by the

administration of antigens usually by injection. The antigens used in this way are known as
vaccines and may be live or dead microorganisms or their products .Administration of
antigens stimulates formation of antibodies and in the process , immunity is acquired .
2. Passive Immunity :
Passive immunity may also be acquired either naturally or artificially .
a)Naturally acquired passive immunity : Antibodies formed in a mother in response to a

disease may be transferred to the foetus through the placental blood . This provides
immunity to the infant for several months. This also explains why babies are highly
resistant for about six months to diphtheria , measles , chicken pox .
b) Artificially acquired passive immunity :

This type of immunity is acquired by injecting the preparations known as antisera, sera or
immune sera. These products are the antibodies produced in an animal , usually a horse
and occasionally in human beings. Passive immunity is not long lasting. Antibodies are
formed in response to the antigens in the body as a defence mechanism , which is in
addition to phagocytosis. They are specific in nature and react with antigens in the body by
various mechanisms and neutralize them.
Key points .
Adaptive ( Acquired) immunity shows the following features.
It is the resistance acquired by an individual during the life. It occurs after exposure to an
agent and is mediated by antibodies as well as T lymphocytes ( Helper T cells and cytotoxic T
cells). .It has immunologic memory and a remarkable capability of discriminating between self
and non-self agents. Once an antigen has been recognized by the cells of acquired immune
system, the response to it is specific and can be repeated . In most cases , the acquired immune
response improves with repeated exposure. The immune response to the second challenge occurs
more quickly than the first , is stronger , and is often more effective in neutralizing and clearing the
pathogen.
Antigens.
Substances which can behave as antigens (Ags), are almost limitless.
Chemical nature: Virtually all proteins , many polysaccharides, lipoproteins ,

1
synthetic polypeptides and an enormous number of small molecules that can be

suitably linked to proteins ; can act as antigens. All antigens have two properties.
Immunogenicity ie. the capacity to stimulate the formation of the corresponding
antibodies and Specificity ie. the ability to react specifically with those
antibodies . But the two properties are not always associated . Thus antigens
which have the capacity to stimulate the formation of corresponding antibodies are
known as immunogens .
COMPLETE ANTIGEN
Where as haptens have ability to react specifically with the appropriate antibodies but are not
immunogenic .
HAPTEN MOLECULE
Specific means that the antigen ( or hapten ) combines, in a highly selective fashion,
with the corresponding antibodies and not with the multitude of other antibodies
evoked by other antigens. Haptens are not immunogenic because they cannot
activate helper T cells . Failure of hapten to activate helper T cells is due to their
inability to bind to MHC proteins. Major histocompatibility complex (MHC), group
of genes that code for proteins found on the surfaces of cells that help the immune
system recognize foreign substances. MHC proteins are found in all higher
vertebrates. In human beings the complex is also called the human leukocyte antigen
(HLA) system.

1
major histocompatibility complex Protein images comparing the MHC I (left) and MHC II (right)
molecules. The orange segments represent the protein chains that attaches the MHC molecules to the
surfaces of cells, and the shorter pink chains represent the proteins that stabilize the structures. The
colour red represents peptides bound by MHC for the purpose of T-cell recognition. The term
immunogenicity means the ability of an antigen to elicit an Immune reaction in the form of a B –cell
or T- cell response. Based on Immunogenicity:(Classification of Antigens)
Complete antigen : Substances which can induce antibody formation
by themselves & can react specifically with
these antibodies.
Incomplete antigen (haptens) – Substances unable to induce antibody formation on it’s own but
can become immunogenic when covalently linked to proteins , called carrier proteins.
HAPTEN MOLECULE
The term Antigenicity means just the ability to combine specifically with the products of the above
responses. All molecules that are immunogenic are antigenic too, but all antigenic Molecules cannot
be considered immunogenic. Thus haptens can be said to lack immunogenicity. Immunogenicity is
not an inherent property of a macromolecule, eg. rabbit serum albumin (RSA) isolated from rabbits
is not immunogenic in this species , but it can elicit copious amounts of anti RSA antibodies in
virtually any other species of vertebrate. Responses to antigens differ markedly depending on the
quantity injected and the frequency and route of administration.
An important prerequisite is that the immunogen must be recognized as foreign ( ie.
non – self) by the responding organism . Those restricted parts of the antigen that are

1
recognized by antibody and determine the specificity of Ag- Ab reactions, are known
as antigenic determinants or Epitopes.
Q. Write a note on antigenic determinants.( 4 marks)
Determination of Antigenicity. A number of factors have been identified that make a Substance
immunogenic .
Some of the important determinants of antigenicity include
1. Molecular size.
2. Foreignness.
3. Chemical structure complexity.
4. Stability.
5. Other factors.
Molecular Size: In general protein molecules with a large molecular weight are
highly antigenic.
Substances with molecular weights of about 100,000 Dalton and more are highly
Immunogenic, while substances with molecular weights of less than 5000 Dalton are
generally not immunogenic. This property has been exploited in experimental studies by
using high molecular proteins like bovine gamma globulin( MW 150,000 Da) to induce an
immune reaction . Substances with low molecular weight may be made antigenic by
adsorbing these on carrier particles, such as bentonite , kaolin and other inert particles.
Foreignness: To be immunogenic , a molecule must be recognized as non-self, ie. Foreign. The

molecule is considered self or non-self by the immune system depending on whether or not the
molecule was exposed to the immune system during fetal development. Foreignness implies ability
of the host to tolerate self antigens. Tolerance to self antigens develop by contact with them in the
initial phases of development of immune system, particularly during the development of
lymphocytes. In general , the more distantly related two species are, the greater the immunogenicity
of a molecule from one species will be when exposed to the other. For example , the bovine serum
albumin is more immunogenic in a chicken than in a goat.
Chemical Structural Complexity:- Proteins are the most potent immunogens followed by
polysaccharides. Nucleic acids and lipids are not efficient in eliciting a good immune reaction,
although they may act as haptens. Structural complexity of a protein contributes to it’s
immunogenicity. Chains of single amino acids or single sugars are poorly immunogenic, but if
different aminoacids or sugars are combined in the same molecule, the immunogenicity is greatly
enhanced.
Stability: Highly stable and non-degradable substances ( eg. Some plastics , metals or chains of D-
amino acids (Dextro rotatory)) are not immunogenic. This is because internalization , processing and
presentation by antigen presenting cells (APCs) are always essential to mount an immune response .
(Antigen-presenting cells (APCs) are a heterogeneous group of immune cells that mediate the cellular
immune response by processing and presenting antigens for recognition by certain lymphocytes such
as T cells. Classical APCs include dendritic cells, macrophages, Langerhans cells and B cells).
(Dendritic cells (DCs) are antigen-presenting cells (also known as accessory cells) of the mammalian
immune system. Their main function is to process antigen material and present it on the cell surface to
the T cells of the immune system.
Macrophages - a large phagocytic cell found in stationary form in the tissues or as a mobile white
blood cell, especially at sites of infection.
Langerhans cells (LC) are members of the dendritic cells family, residing in the basal and suprabasal
layers of the epidermis and in the epithelia of the respiratory, digestive and urogenital tracts. They
specialize in antigen presentation and belong to the skin immune system (SIS). Therefore , very
stable substances ( such as silicon) have been successful as non-immunogenic materials for
reconstructive surgeries ,such as breast implants.

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On the other hand , if a substance is very unstable , it may break up before an APC c an be
internalized ,and hence becomes immunogenic .In addition , large , insoluble complexes are
more immunogenic than smaller , soluble ones .This is because macrophages find it easier to
phagocytose, degrade, and present the insoluble complexes than the soluble complexes.
Other Factors:-
Biological systems :- Biological systems also plays an important role in determining the
immunological efficiency of an antigen. Some substances are immunogenic in one individual but
not in others.This is due to the fact that individuals may lack or have altered genes that code for the
receptors for antigen on B cells and T cells , or they may not have the appropriate genes needed for
the APC to present antigen to the helper T(TH) cells.
Dosage and route of the antigen: The dose of antigen and the route by which it comes into contact
with the immune system also influence immunogenicity of the antigen. Very low doses of antigen
do not stimulate the immune responses, either because too few lymphocytes are contacted or because
a nonresponsive state is elicited, Conversely , an extremely high dose also fails to elicit tolerance.
Repeated administration of antigens (Booster doses) may be required to enhance Immune response
of the host to certain antigens. This is particularly important in case of vaccines where a prerequisite
immune level needs to be attained. Hence the booster doses of vaccines ,such as DPT (Diphtheria,
Pertussis,Tetanus),DT (Diphtheria, Tetanus) etc, are given to ensure good protective levels of
antibodies.Generally , antigens are administered by the parenteral route to produce good level of
antibodies.
The antigen can be given by a) Intravenous, b) subcutaneous, c) intradermal,d) intramuscular ,
e) intraperitoneal and f) mucosal routes. Usually the subcutaneous route of administration proves to
be better than intravenous routes at eliciting an immune response.
Antigenic Specificity:- Antigenic Specificity of the antigen depends on antigenic

determinants or epitopes.
Epitopes :- An epitope is defined as the immunologically active region of an immunogen that
binds to antigen specific membrane receptors on lymphocytes or secreted antibodies. The interaction
between cells of the immune system and antigens takes place at many levels and the complexity of
any antigen is mirrored by it’s epitope.
There are two types of epitopes: B- cell epitopes and T – cell epitopes.
B- cell epitopes :- B- cell epitopes are antigenic determinants recognized by B cells .B- cell epitopes
can combine with it’s receptors only if the antigen molecule is in it’s native state. B- cell epitope is
about six or seven sugar residues or amino acids long. B- cell epitopes tend to be hydrophilic and are
often located at bends in the protein structure. They are also found in regions of proteins which have
a higher mobility.
T- cell epitopes: T cell recognise amino acids in proteins but do not recognize polysaccharides or
Nucleic acid antigens .
This is the reason why polysaccharides are considered as T- independent antigens and proteins as T-
dependent antigens .The primary sequence of amino acids in proteins determines the antigenic
determinants Recognized by T cells .
Free peptides are not recognized by T cells , while the complex of MHC molecules and peptide are
recognized by T cells.
Key points
Processing of an antigen by APC is absolutely essential for a T cell to recognize it. Two different
types of processing can prepare a protein antigen for antigenic presentation . These include :
Externally derived antigen’ s processing : In this process, phagocytosed bacteria are Killed and lysis
by phagocytic cells , such as macrophages . Pieces of bacteria are then processed and presented in the
context of class II MHC Molecules. Endogenously derived antigens processing :In this process ,
virus proteins synthesized in a cell are processed and then presented In the context of class I
MHC molecules.

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Species Specificity : Tissues of all individuals in a species possess certain species – specific
antigens . However some degree of cross reactions occurs between antigens from related Species
Isospecificity :- Isospecificity is determined by the presence of isoantigens or isohistocompatibility

Antigens Isoantigens : Isoantigens are antigens found in some , but not all , members of a species.
A species may be grouped depending on the presence of different isoantigens in its Members . These
are genetically determined Human erythrocyte antigens based on which individuals are classified
into different Blood groups , are the best examples of isoantigens in humans .
The blood groups are of primary importance in:Transfusion of blood and blood products.
Isoimmunization during pregnancy and providing valuable evidence in paternity disputes, the
results of which are supplemented by More recent DNA finger print tests.
Histocompatibility Antigens :- Histocompatibility Antigens are the cellular
determinants Specific for each individual of a species. These antigens are associated
with the plasma membrane of tissue cells . Human leukocyte antigen (HLA) is the
major histocompatibility antigen that determines the homograft rejection. Therefore
, HLA typing is absolutely essential before carrying out transplantation of
tissue or organ from one individual to another.
Autospecificity: - Self antigens are generally nonantigenic . Sequestrated antigens ( such as eye lens
proteins and sperm) are , however This is because corneal tissue and sperm are never encountered by
the immune system during the development of tolerance to self antigens . Therefore these tissues
becomes immunogenic if accidentally or experimentally released into the blood or tissues.
Organ Specificity :- Antigens characteristic of an organ or tissue are called organ specific antigens .
These antigens found in the brain , kidney and lens tissues , even of different animal species ,share the
same antigen specificity . Organ specific antigens such as brain –specific antigens , shared by human
and sheep Brain are one such example . The anti-rabies vaccines prepared from sheep brain , when
given , may induce immune response in some humans , causing damage to neutral tissues of the
recipient . This may result in neuroparalytic complications in some individuals .
Heterophile Specificity :- Heterophile specificity is determined by the presence of heterophile
antigens . The same or closely related antigens , sometimes present in tissues of different biological
Species, classes or kingdoms are known as heterophile antigens. Antibodies against the
heterophile antigens produced by one of the species cross react with the antigens of the other
species .
Super antigens :- Superantigens are a class of molecules that can interact with APCs and T –
lymphocytes In a non specific way . The superantigens act differently by interacting with MHC
class II molecules of the APC and the Vb domain of the T- lymphocyte receptor . This interaction
results in the activation of a larger number of T cells ( 10 %) than conventional antigens (1%),
leading to massive cytokine expression and immunomodulation. Examples of superantigens are
staphylo coccal enterotoxins , toxic shock syndrome toxin and some viral proteins .
Q. what are different types of antigens. Write the chemical nature of antigen and antobody
Write a note on antigenic determinants.(2+4+4)
Q. What are haptens (2 mark)
Q.What are Epitopes and Paratopes ( 2 marks).
Paratopes and epitopes are the unique binding regions of an antibody and antigen, respectively.
More specifically, antigens are known to contain specific antigenic determinants (which are
epitopes), while antibodies contain antigen-binding sites (which are paratopes).
The binding of an antibody to a foreign body occurs across a specific region that is a few amino
acids long and does not represent the whole protein. This region, which is recognized by the
antibody, is called the antigen. A protein may often contain several epitopes to which antibodies may
bind.

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Antibodies
Antibodies are globular proteins ( immunoglobulins ) that are synthesized in serum and
tissue fluids, which react specifically with the antigen that stimulated their production. Three
types of globulins are present in the blood : alpha, beta and gamma . The antibodies are the
gamma globulins. The term antibody refers to the protein or set of proteins formed in
response to an antigen, which react specifically with that antigen or related substances.
Those antibodies found in serum are called humoral antibodies or circulating antibodies. All
antibodies belong to a family of proteins known as immunoglobulins.
Immunoglobulins have been identified as five classes , designated as IgG, IgM, IgA, IgD and IgE.
Antibodies are one of the major plasma proteins ,and against infection often referred to as “ first
line of defence”. The most important function of antibodies confer protection against
microbial pathogens . Antibodies confer protection in the following ways.
1.They prevent attachment of microbes to mucosal surfaces of the host.

2.They reduce virulence of microbes by neutralizing toxins and viruses .
3. They facilitate phagocytosis by opsonisation (Opsonisation is the process of making pathogens
easier for phagocytes to grab onto and phagocytize by decorating their surface with molecules to
which the phagocytes receptors can bind) of microbes. They activate complement, leading to
complement- mediated activities against microbes. (The complement system is a part of the
immune system that enhances (complements). The ability of antibodies and phagocytic cells to clear
microbes and damaged cells from an organism, promotes inflammation, and attacks the pathogen's
plasma membrane). The WHO in 1964 coined the term “ immunoglobulin(Ig)” for the term antibody.
Structure of Immunoglobulins. Immunoglobulins show the following properties :

They are glycoproteins .They are complex structure of four polypeptide chains: Two identical
heavy
( typically 55 kDa each) chains and two identical light chains ( 25kDa each).
This give Immunoglobulin an overall ‘ Y’ or ‘T’ shape , which is the most widely
recognized.
Feature of Immunoglobulin structure.
The term “ heavy” and “light” chain refer to the molecular weights of the chains . The heavy chains
have a molecular weight of 50,000 to 70,000 Da, while light chains have a molecular weight of
25,000 Da. The heavy chains are longer and light chains are shorter.
Heavy chains :
An immunoglobulin molecule has two heavy chains .

Each heavy chain is made up of 420- 440 amino acids .
The two heavy chains are held together by one to five disulphide (S-S) bonds.

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1. IgG( Immunoglobulin G) is the smallest ( Mol.Wt.150,000 ) but most abundant of the serum
Antibodies. It binds to antigens in the serum as well as in the lymph or intercellular fluids. It can
cross the placenta and move freely in the intercellular fluids as well as in the blood. IgG consists of
four polypeptides ( amino acid chains), two identical light weight (short) chains and two heavy (
longer ) chains held together by disulphide (S-S) bonds. It has two ends ( bipolar) that bind
specifically to the antigen that stimulated its production.
The middle portion of this macromolecules will bind nonspecifically to complement,
Phagocytes or tissue cells. The molecular weight of the heavy chain is about 50,000 and
that of the light chain is about 25,000. An Ig producing cell usually makes , at any one time,
only one type of L chain and one type of H chain, and hence IgGs are symmetric.
2.Ig M ( Immunoglobulin M) is the largest ( Mol.wt. 900,000 ) of the gamma globulins and
consists of five IgG type molecules held together by disulphide bonds .The typical star shape
of IgM accounts for it’s binding with only five identical antigens .The IgM antibodies
appear to be the first antibodies formed in response to infections , especially caused by
Gram(-)ve bacteria. The human IgM molecule is a pentamer made up of five
immunoglobulin subunits and one molecule of J chain.( The joining (J) chain is a small
polypeptide, expressed by mucosal and glandular plasma cells, which regulates polymer
formation of immunoglobulin (Ig)A and IgM). Each monomeric IgM is composed of two
light chains and two heavy chains.
3.IgA ( Immunoglobulin A) are found in saliva, tears, colostrum and other body secretions , as
well as in the blood stream. They are also known as secretory antibodies . They consists of two
IgG sized antibodies held together by a secretory piece and serve to protect the external
openings and mucous membranes from invasion with pathogens .
4. IgD( Immunoglobulin D) is a serum antibody whose function is not clearly understood
. Because it is found on the surface of B- lymphocytes, it is probably involved in
the determination as to which plasma cells produce which antibody (IgG type).
5. IgE ( Immunoglobulin E) accounts for only 0.002% of gamma globulins present in the serum.
These antibodies are present in persons with allergy , drug sensitivity or anaphylactic shock .
IgE are mostly bound to target cells in tissues , and cause the rashes seen in various allergies,The
runny nose and eyes of hay fever and asthma , the local reaction in immediate hypersensitivity skin
tests and anaphylactic shock (an extreme, often life-threatening allergic reaction to an antigen to
which the body has become hypersensitive) reaction.
Immunoassays:

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Immunoassays by definition are procedures that measures antigens or antibodies

to determine whether the individual is infected or responding to immunization.
The diagnosis of infectious diseases with immunoassays involve testing for
1) detection of specific microbial antigens.
2) detection of microbial antigen- specific antibodies .
Immunoassays are of two types – liquid phase immunoassay (LPIA) and Solid – phase
immunoassay (SPIA). liquid phase immunoassay (LPIA) are useful in the field of chemistry . Solid
– phase immunoassay (SPIA) is used in microbiology for detection of antigens and antibodies.
Important tests for antigen- antibody reactions include
 Precipitation
 Agglutination
 Complement fixation
 Neutralisation
 Opsonization.
 Immunofluorescence
 Enzyme immune assay
 Radio immune assay.
 Western blotting.
 Chemiluminescence assay.
 Immunoelectron microscopic assay.
Characteristics of Ag – Ab ( Antigen – Antibody) reactions.
1. Reactions is highly specific . However , possible cross reactions

between related Antigens may occur in some cases .
2. The antigenic determinant ( epitope ) makes contact with an area on the
hypervariable region of the antibody ( called paratope) . The molecules are held
together in lock and key arrangement .
3. During combination only surface antigens participate .
4. Combination is firm but reversible .Firmness of binding is dependent on

affinity for one another and avidity of antibody. Affinity denotes intensity of
attraction for one another , usually monovalent binding to an epitope and
avidity is the binding strength of individual antibody with its specific
antigenic determinants as a result of multivalent binding of antibody to
antigen
Measurement of antigen and antibody .

There are several methods available for the purpose eg. measurement in terms of
mass
( eg. mg nitrogen) or more frequently as units or titre.
Antibody titre . The term titre is used to denote the highest dilution of the serum at which
antibody activity is demonstrable , which is usually expressed as the reciprocals of the dilution of the
serum. Eg. 64 , when antibody was detected at a final serum dilution of 1 in 64 .
Stages of Antigen –Antibody interactions :-

The union of antigen and antibody occurs in two stages .
Primary interaction: In primary or initial interaction there is no visible effect
and the reaction is rapid . However , microscopic localisation of antibody can be
observed in a component of particular microorganism when an antiserum is
labeled with a fluorescent dye.
It is rapid and reversible, but with out any visible effects. The ionic bonds, hydrogen bonds, van der
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waals forces and hydrophobic interactions are the weaker intermolecular forces that bind antigen
and antibody together in this primary Stage.
Secondary stage : Secondary stage is an irreversible interaction between antigen and
antibody with visible effects, such as agglutination, precipitation, neutralization,
complement fixation and immobilization of motile organisms . The binding between
antigen and antibody during this stage occurs by covalent binding.
Precipitation :-
Precipitation test shows the following features .It is a type of antigen – antibody reaction in which
the antigen occurs in a soluble form. It is a test in which antibody interacts with the
soluble antigen in the presence of electrolyte(NaCl) at a specified pH (7.4) and
temperature(370C) to produce a precipitate .
A lattice is formed between the antigens and antibodies in certain cases , it is visible as an
insoluble precipitate . Antibodies that aggregate soluble antigens are called as precipitins
. When instead of sedimenting, the precipitate remains suspended as floccules (a
small clump of material), the reaction is known as flocculation. The formation of an
antigen- antibody lattice depends on the valency of both antibody and antigen. The
antibody must be bivalent , a precipitate will not form monovalent Fab fragments. The
antigen must be either bivalent or polyvalent ie. it must have at least two copies of the
same epitope , or have different epitopes that react with different antibodies present in
polyclonal antisera.
Prozone Phenomenon:-
Antigen and antibody reaction occurs optimally only when the proportion of the
antigen antibody in the reaction mixture is equivalent ( zone of equivalence).
On either side of the equivalence zone, precipitation is actually prevented because of
an excess of either antigen or antibody .
The zone of antibody excess is known as the prozone phenomenon and the
zone of antigen excess is known as postzone phenomenon.

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Marrack in 1934 proposed the lattice hypothesis to explain the prozone phenomenon.
Marrack’s hypothesis is based on the assumption that each antibody molecule must
have at least two binding sites and antigen must be multivalent .
In the zone of equivalence where optimum precipitation occurs, the number of
multivalent sites of antigen and antibody are approximately equal.
In this zone, precipitation occurs as a result of random, reversible reactions where by
each antibody binds to more than one antigen and vice versa, forming a stable network of
lattice.
Main zone of antigen – antibody precipitate curve

Quantitative precipitation test showing : A- Antibody excess, B- optimal proportion
of Ag- Ab( zone of equivalence) C- Antigen excess.
As they combine , it results in a multimolecular lattice that increases in
size until it precipitates out of solution .

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Mechanism of precipitation by lattice formation

Antibody excess (left up column) Equivalence (right up column)
Antigen excess ( left down)Monovalent hapten (right down).
Types of precipitation Reaction .

There are three principal types of
precipitation. 1.Precipitation in solution.
2.Precipitation in agar.
3.Precipitaion in agar with an electric field.
1.Precipitation in solution - Ring Test and Flocculation Test are examples of precipitation in
solution.
2.precipitation in agar /Gel diffusion test (immunodiffusion).
Single diffusion one dimension.
Single diffusion in two dimensions.
Double diffusion in one direction
Double diffusion in two dimensions
3.Precipitaion in agar with an electric field/Immunoelectrophoresis .
Counter current immunoelectrophoresis Rocket Electrophoresis.
1.Precipitation in solution.
Ring Test : In this test , antigen solution is layered over antiserum in a test tube. Precipitation
between antigen and antibodies in antiserum solution is marked by the appearance of a ring of
precipitation at the junction of two liquid layers. C- reactive protein (CRP) and streptococcal
grouping by the Lancefield methods are the examples of the ring test.
Flocculation Test : Flocculation Test is done on a slide (VDRL test for syphilis ) and in tube (kahn
test).

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Slide Flocculation Test, a drop of VDRL antigen solution is added to a drop of patient’s
serum on a cavity slide , and the result is recorded after shaking the slide on a VDRL
shaker. Visible clumps appear in positive cases .
Tube flocculation test (kahn test) was done previously in diagnosis of syphilis .
Tube precipitation test is done in standardization of toxins and toxoids .In tube test a fairly
large volume of antiserum is necessary and the test is not very Sensitive.
Tube precipitation test. Markings from down to up –Antibody in antiserum, Line of
precipitation , Soluble antigen in fluid.

Precipitation in agar (immunodiffusion test) .
The precipitation test in agar gel is termed as immunodiffusion test. In this test reactants are added
to the gel and antigen – antibody combination occurs by means of diffusion. The rate of the diffusion
is affected by the size of the particles, temperature , gel viscosity, amount of hydration and
interaction between matrix and reactants. An agar concentration of 0.3 to 1.5% allows diffusion of
most reactants.
Agarose (a substance which is the main constituent of agar and is used especially in gels for
electrophoresis. It is a polysaccharide mainly containing galactose residues) is often preferred to agar
because agar has a strong negative charge,while agarose has almost none, so that interactions
between the gel and reactants are minimized .
Types of immunodiffusion reactions :
Immunodiffusion reactions are classified based on the

a)Number of reactants diffusing and
b)Direction of diffusion as follows.
Single diffusion in one dimension :

Single diffusion in one dimension as the name suggests , is the single
diffusion of antigen in agar in one dimension. It is otherwise called as Oudin
procedure because this technique was pioneered by Oudin who for the first
time used gels for precipitation reactions .In this method antibody is
incorporated into agar gel in a test tube and the antigen is poured over
it .During the course of time , the antigen diffuses downward toward the
antibody in agar gel and a line of precipitation is formed .The number of
precipitate bands shows the number of different antigens present in the
antigen solution.

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Single diffusion in one dimension. Broad line precipitation bands.
Single diffusion in two dimensions: Single diffusion in two dimensions

is also called radial immunodiffusion . In this method antiserum solution
containing antibody is incorporated in agar gel on a slide or petri dish.
The wells are cut on the surface of gel. The antigen is then applied to a
well cut into the gel .
When antibody already present in the gel reacts with the antigen,
which diffuses out of the well, a ring of precipitation is formed
around the wells.
The diameter of the ring is directly proportional to the concentration of antigen .
Radial Immunodiffusion.
Double diffusion in one direction : This method is also called

Oakley –Fulthrope Procedure. In this method the antibody
is incorporated in agar gel in a test tube , above which a layer of plain agar is
placed . The antigen is then layered on top of this plain agar .During the course of
time , the antigen and antibody move towards each other through the intervening
layer of the plain agar. In this zone of plain agar , both antigen and antibody react
with each other to form a band of precipitation at their optimum concentration .

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Arrows show direction of diffusion . Broad line – Precipitation bands.
Double diffusion in two dimensions: The method is also called the

Ouchterlony Procedure. In this method , both the antigen and
antibody diffuse independently through agar gel in two dimensions ,
horizontally and vertically . The test is performed by cutting wells in
the agar gel poured on a glass slide or in a petri dish . The
antiserum consisting of antibodies is placed in the central well ,
and different antigens are added to the wells surrounding the
centre well. After an incubation period of 12- 48 hours in a moist
chamber , the lines of precipitins are formed at the site of
combination of antigens and antibodies .
Three types of reactions can be demonstrated

1.Line of precipitations at their junction forming an arc represents serological
Identity or the presence of a common epitope or antigens.
2.A pattern of crossed lines demonstrates two separate reactions and indicates
that the compared antigens are unrelated and share no common epitopes .
3.Fusion of two lines with a spur indicates cross reaction or partial identity. In
this case , the two antigens share a common epitope, but some antibody
molecules are not captured by the antigen and traverse through the initial
precipitin line to combine with additional epitopes found in the more complex
antigen.

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Precipitation in agar in an electric field .

Immunoelectrophoresis: Immunoelectrophoresis is a process of
combination of Immunodiffusion and electrophoresis .It is a method in which
different antigens in serum are separated according to their charge under an
electric field.
In this method a drop of antigen is placed into a well in agar on a glass slide .
An electric current is then passed through the agar. During electrophoresis, Antigens move in the
electric field according to their charge and size. Following electrophoresis , a trough is cut into the
agar and is filled with the antibody and diffusion is allowed to occur. The antigen and antibody
diffuse toward each other, they form a series of lines of precipitation . The main advantage of
immunoelectrophoresis is that a number of antigens can be identified in serum . The method is used
to detect normal as well as abnormal proteins , such as myeloma Proteins in human serum.
Counter current immunoelectrophoresis :- Counter current immunoelectrophoresis

depends on movement of antigen towards the anode and of antibody towards the
cathode through the agar under electric field . The test is performed on a glass slide with
agarose in which a pair of wells is punched out. One well is filled with antigen and the
other with antibody. Electric current is then passed through the gel . The migration of
antigen and antibody is greatly facilitated under electric field , and a line of precipitation
is made visible in 30- 60 minutes .
Rocket Electrophoresis :- This technique is an adaptation of radial

immunodiffusion Developed by Laurell. It is called so due to the appearance of the
precipitin bands in the shape of cone like structures ( rocket appearance ) at the end
of the reaction . In this method , antibody is incorporated in the gel and antigen is
placed in wells cut in the gel . Electric current is then passed through the gel, which
facilitates the migration of antigen into the agar. This results in formation of a
precipitin line that is conical in shape resembling a rocket. The height of the rocket ,
measured from the well to the apex, is directly proportional to the amount of antigen
in the sample.

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Application:-
Precipitation reactions are very sensitive and can detect as little as 1µg protein antigen .
The test may be done either as qualitative or quantitative test. The qualitative test is much more
widely used to detect the presence of antigen and is of considerable value in
1.Identification of bacteria, eg. detection of group specific polysaccharide of strepto

coccus.
2.Identification of bacterial components in infected tissues eg. Bacillus anthracis .
3.Detection of unknown antibody eg. VDRL and KT in syphilis.
4.Medico-legal identification of human blood or seminal fluid.
5.Standardization of toxins and antitoxins .
6.Testing for food adulteration .
2.Agglutination
Agglutination is an antigen antibody reaction , in which an antibody combines with a particulate

Antigen in presence of electrolytes at optimal pH and temperature resulting in visible
clumping (aggregation ) of the particles . Agglutination occurs optimally when antigens and
antibodies react in equivalent proportions.
Agglutination reactions are mostly similar to precipitation reactions in their fundamentals
and share similar features . This reaction is analogous to the precipitation reactions in that
antibodies act as a bridge to form a lattice network of antibodies and the cells that carry
antigen on their surface. Because cells are so much larger than a soluble antigen, the result is
more visible when the cells aggregate into clumps.
Agglutination differs from precipitation reaction in that since the former reaction takes Place
at the surface of the particle involved, the antigen must be exposed and be able to bind with
the antibody to produce visible clumps.
Types of agglutination reactions .

Agglutination reactions where the antigens are found naturally on a particle are known as
direct Agglutination .
This is different from passive agglutination , which employs particles that are coated with
Antigens not normally found on their surfaces .
Direct agglutination .
Direct agglutination reactions can be broadly of the following types .
a) Slide agglutination .

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b) Tube agglutination.
c) Heterophile agglutination.
d) Antiglobulin (Coomb’s ) test.
Passive agglutination reaction , depending on the carrier particles used , can be of the
following types.
i) latex agglutination test

ii) hemagglutination test and
iii) coagglutination test.
Slide agglutination test : It is a basic type of agglutination reaction that is performed on a slide
.Identification of bacterial types represents a classic example of a direct slide
agglutination that is still used today.In this test , a suspension of bacteria is prepared and is
added to a drop of standardized Antiserum.
A positive reaction is indicated by clumping of bacteria and clearing of the background

Solution.
Clumping occurs instantly or within seconds in a positive test . A control consisting of antigen
suspension in saline without adding antiserum is included on the same slide .It is used to
validate the results and also to detect possible false positives due to autoagglutination of the
antigen.
Tube agglutination test:- Tube agglutination test, as the name suggests , is performed in glass
Tubes . Typically , in these tests , patients serum is diluted in a series of tubes and bacterial
Antigen specific for the suspected disease are added to it. Antigens and antibody reactions are
demonstrated by demonstration of visible clumps of agglutination . It is a standard method used
for quantitative estimation of antibodies in the serum.
Tube agglutination tests are routinely used for demonstration of antibodies in the serum for
serodiagnosis of enteric fever and brucellosis (An infection spread from animals to people,
mostly by unpasteurised dairy products.Brucellosis is a bacterial infection that affects
thousands of people worldwide. Avoiding unpasteurised dairy products and taking precautions
when working with animals or in a laboratory can help prevent brucellosis. Symptoms may
include joint and muscle pain, fever, weight loss and fatigue. Some people develop stomach
pain and cough. Treatment includes antibiotics).
Widal test is used to diagnose enteric fever (Enteric fever is a bloodstream infection caused by
the bacteria Salmonella Typhi or Salmonella Paratyphi A, B, or C. It is common in low resource
settings and linked to poor water quality and sanitation. Symptoms: Abdominal pain; Fever;
Headache) and uses different Salmonella antigens to detect the presence of antibodies to
Salmonella typhi, S.paratyphi A and S. paratyhpi B) in patient’s serum.
Heterophile agglutination test : This test depends on demonstration of

heterophilic Antibodies (antibodies produced against poorly defined antigens and that
they are generally “weak” antibodies ) in Serum present in certain bacterial
infections .
Weil Felix test is an example of heterophilie agglutination reaction for serodiagnosis of
rickettsial Infections.( Rickettsial infections and related infections are caused by an unusual
type of bacteria that can live only inside the cells of another organism. Most of these
infections are spread through ticks, mites, fleas, or lice.( Ticks and mites are tiny animals that
are found all over the world. They are related to spiders.
Many ticks and mites are parasites. This means that they live on or inside other animals, which
are called hosts). Symptoms - fever, a severe headache, and usually a rash develop, and people

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feel generally ill).

In this test, the cross reacting antibodies produced against rickettsial pathogen are
detected by using cross reacting related antigens. Streptococcus MG agglutination
test is a similar test used for detection of antibodies to Mycoplasma pneumonia
causing primary atypical pneumonia .
Antiglobulin ( Coomb’s Test ) – Coomb’s test was devised originally by Coomb’s

Mourant, and Race for detection of incomplete anti –Rh antibodies that do not
agglutinate Rh+ erythrocytes in saline. When serum containing in complete anti- Rh
antibodies are mixed with Rh+ erythrocytes in saline , incomplete antibody antiglobulin
coats the surface of erythrocytes but does not cause any agglutination.
When such erythrocytes are treated with antiglobulin or Coomb’s serum ( rabbit
antiserum against human ϒ globulin), then the cells are agglutinated .
Commb’s test is of two types.

a) Direct coomb’s test and
b) Indirect Coomb’s test .
Direct coomb’s test – In this test, sensitization of red blood cells ( RBC) with
incomplete antibodies Takes place in vivo. The cell bound antibodies can be detected
by this test in which antiserum against human immunoglobulin is used to
agglutinate patient’s red cells
In Direct coomb’s Test : In this test , the sensitization of RBCs with incomplete
antibodies takes place in vitro . In this test , the patient’s serum is mixed with normal
red cells and antiserum to human immunoglobulin is added. Agglutination occurs if
antibodies are present in the patient’s serum . Coomb’s tests are used for detection of a) anti-
Rh antibodies and b) incomplete antibodies in Brucellosis and other diseases .

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Passive agglutination: The agglutination employs carrier particles that are coated with
soluble antigens . This is usually done to convert precipitation reactions into
agglutination reactions , since the latter are easier to perform and interpret and are
more sensitive than precipitation reactions for detection of antibodies .When the
antibody instead of antigens is adsorbed on the carrier particle for detection of
antigens, it is called reverse passive agglutination . Until the 1970s erythrocytes were
the major carrier particles used for coating of antigens .
Recently , however a variety of other particles including polystyrene latex, bentonite ,
and charcoal are used for this purpose.
Passive agglutination reaction , depending on the carrier particles used , can be of
the following types.
1.latex agglutination test

2.hemagglutination test and
3. coagglutination test.
Latex agglutination Test :- It is a test that employes latex particles as carrier of antigens or
antibodies. In 1955, Singer and Plotz accidently found that IgG was naturally adsorbed to the
surface of Polystyrene particles. Latex particles are inexpensive, relatively stable and are not
subject to cross reactivity with other antibodies. These particles can be coated with antibodies
to detect antigen in the serum and other body fluids.
Additionally , the large particle size of the latex facilitates better visualization of antigen-
antibody reactions by the naked eye observation. The latex agglutination tests are used for
rapid identification of antigens of group B streptococcus, Staphylo coccus aureus etc. The tests
have also been found to be useful for detection of soluble microbial antigens in urine, Spinal
fluid and serum for diagnosis of a variety of infectious diseases .

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Hemagglutination Test - RBCs are used as carrier particles in hemagglutination tests. RBC’s
of Sheep, human, chick etc. are commonly used in the test . When RBCs are coated with
antigen to detect antibodies in the serum, the test is called indirect hemagglutination (IHA) test.
IHA is almost commonly used test for serodiagnosis of many parasitic diseases
including amoebiasis.
When antibodies are attached to the RBCs to detect microbial antigen it is known as
Reverse Passive hemagglutination (RPHA). The RPHA has been used extensively in the past
to detect viral antigens , such as in HBsAg in the serum for diagnosis of hepatitis B infection.
The test has also been used for detection of antigens in many other viral and parasitic
infections .
Viral hemagglutination . Many viruses including influenza, mumps and measles have the ability
to agglutinate RBCs with out antigen- antibody reactions . This process is called Viral
hemagglutination .
This hemagglutination can be inhibited by antibody specifically directed against the virus
and this phenomenon is called hemagglutination inhibition. This forms the basis of the viral
hemagglutination inhibition test , which is used to detect antibodies in patient’ s sera that
neutralize the agglutinating viruses .To perform this test , patient’s serum is first incubated with
a viral preparation . Then RBCs that the virus is known to agglutinate are added to the
mixture. If antibody is present , this will combine with viral particles and prevent
agglutination, and a lack of or reduction in agglutination indicates presence of antibody in
patient’s serum.

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Coagglutination Test :- Coagglutination is a type of agglutination reaction in which Cowan I

strain of S. aureus is used as a carrier particle to coat antibodies .
Cowan I strain of S. aureus contains protein A, an anti- antibody, that combines with the Fc
portion of the immunoglobulin, IgG, leaving the Fab region free to react with the antigen
present in the specimens .
In a positive test , protein A bearing S. aureus coated with antibodies will be agglutinated if mixed
with specific antigen.
The advantage of the test is that these particles show greater Stability than latex particles and
are more refractory to changes in ionic strength .
Coagglutination test has been used for Detection of cryptococcal antigen in the CSF
(cerebro spinal fluid) for diagnosis of cryptococcal meningitis. Grouping of streptococci and

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mycobacteria and for typing of Neisseria gonorrhoeae.
3. Complement- dependent Serological Tests

The complement system is a group of serum proteins that is present in normal serum. The
system consists of 20 or more serum proteins that interact with one another and with cell
membrane. It is a biochemical cascade that helps to clear pathogens from the body.
It aids the antibodies in lysing bacteria, promoting phagocytosis and in immune
adherence . The complement –dependent serological tests may be of the following types.
1. Complement fixation test .
2. Immune adherence test .
3. Immobilization test .
4. Cytolytic or cytocidal reactions .
Complement Fixation Test . The principle of the complement fixation test is that when antigen
and antibodies of the IgM or the IgG classes are mixed , complement is fixed to the antigen –
antibody complex .
If this occurs on the surface of RBCs the complement cascade will be activated and
Haemolysis will occur.
The complement fixation test consists of two antigen –antibody complement systems
a) An indicator system
b) A test system.
Indicator system: It consists of RBCs that have been pre-incubated with a specific anti RBC
antibody, in concentrations that do not cause agglutination , and no haemolysis of RBCs occurs in the

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absence of complement. Such RBCs are designated as sensitized red cells .

Test system :
In the test system, patient’s serum is first heated to 560C to inactivate the native complement .
Then the inactivated serum is adsorbed with washed sheep RBC to eliminate broadly cross
reactive anti –RBC antibodies ( also known as Forssman type antibodies), which could
interfere with the assay. The serum is then mixed with purified antigen and with a dilution of
fresh guinea pig serum, used as source of complement. The mixture is incubated for 30
minutes at 370C to allow antibody in the patient’s serum to form complexes with the antigen
and to fix complement . In complement fixation test , sensitized red cells are then added to the
mixture . If the red cells are lysed, it indicates that there were no antibodies specific to the
antigen in the Serum of the patient. The complement therefore was not consumed in the test
system and was available to be used by the anti –RBC antibodies, resulting in hemolysis . This
reaction is considered negative. The test is considered positive if the red cells are not lysed .
Nonlysis of the cells indicates that patient’s serum had antibodies specific to the antigen,
which have fixed complement. Hence , no complement was available to be activated by the
indicator stystem.
Indirect complement fixation test : - Indirect complement fixation test is carried out to test
the sera that cannot fix guinea pig complement . These include avian sera ( eg. parrot,
Duck) and mammalian sera ( eg .cat, horse ). The test is carried out in duplicate and after
the First test, the standard antiserum known to fix the complement is added to one set .
Hemolysis indicates a positive test . In a positive test , if the serum contains antibody , the
antigen would have been used up in the first test, standard antiserum added subsequently
would fail to fix the complement ,therefore causing hemolysis.
Immune adherence Test:- Immune adherence test is a test in which certain pathogens
( eg. Vibrio cholera, Treponema palladium etc, ) react with specific antibodies in the
presence of complement and adhere to erythrocytes or platelets.The adherence of
cells to bacteria is known as immune adherence ,which facilitates
phagocytosis of the bacteria .
Immobilization test :-
Immobilization test is a complement dependent test in which certain live bacteria , such as
Treponema palladium are immobilized when mixed with patient’s serum in the presence of
complement . This forms the basis of Treponema palladium immobilization test . A positive test
shows serum to contain treponemal antibodies.
Cytolytic or cytocidal reactions :-

When a live bacterium such as Vibrio cholera, is mixed with its specific
antibody in the presence of complement , the bacterium is killed and lysed . This forms the
basis of test used to measure antichlorea antibodies in the serum.
4.Neutralization test
Neutralization is an antigen- antibody reaction in which the biological effects of viruses and
toxins are neutralized by homologous antibodies known as neutralizing antibodies .

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Neutralization test
These tests are broadly of two types.
a)Virus Neutralization Tests

b) Toxin Neutralization Tests.
Virus Neutralization Tests: Neutralization of viruses by their specific antibodies are called virus
neutralization tests. Inoculation of viruses in cell cultures, eggs ,and animal results in the
replication and growth of viruses . When virus specific neutralizing antibodies are injected
into these systems ,replication and growth of viruses is inhibited . This forms the basis of virus
neutralization tests. Eg.Viral hemagglutination inhibition test is an example of virus
neutralization test Frequently used in the diagnosis of viral infections, such as influenza,
mumps, and measles. If patient’s serum contains antibodies against certain viruses that have
the property of agglutinating the red blood cells , these antibodies react with the viruses and
inhibit the agglutination of the red blood cells .
Toxin neutralization Test :- Toxin neutralization Tests are based on the principle that biological
action of toxin is neutralized on reacting with specific neutralizing antibodies called antitoxins .
Examples of neutralization tests include : In – vivo – Shick test to demonstrate immunity against
diphtheria .
Erythematic -Redness of the skin caused by dilatation and congestion of the capillaries .
In- vitro- Nagler reaction used for rapid detection of Clostridium welchii .
5.Opsonization
Opsonization is a process by which a particular antigen becomes more susceptible to

Phagocytosis when it combines with opsonin. The opsonin is a heat labile substance
present in fresh normal sera. Unlike opsonin , bacteriotropin is heat stable substance present
in the serum but with similar activities . The term opsonic index is defined as the ratio of
the phagocytic activity of patient’s blood from a normal individual . It is used to study the
progress of resistance during the course of disease .
It is measured by incubating fresh citrated blood with the suspension of bacteria at 370C
for 15 minutes and estimating the average number of phagocytic bacteria from the stained
blood films .
6.Immunofluorescence :-
The property of certain dyes absorbing light rays at one particular wavelength (
ultra violet light) and emitting them at a different wavelength ( visible light ) is
known as fluorescence. Fluorescent dyes , such as fluorescein isothiocynate and
lissamine rhodamine , can be tagged with antibody molecules . They emit blue green

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and orange -red fluorescence , respectively under ultraviolet rays in the

fluorescence microscope. This forms the basis of immunologicaltest.
Immunofluorescene tests have wide application.
In research and diagnosis .
These tests are broadly of two types .
1. Direct immunofluorescence.
2. Indirect immunofluorescence.
Direct immunofluorescence test .

Direct immunofluorescence test is used to detect unknown antigen in a cell or tissue by
employing a known labeled antibody that interacts directly with unknown antigen . If
antigen is present , it reacts with labeled antibody and the antibody – coated antigen is
observed under UV light of the fluorescence microscope . Direct immunofluorescence test
is widely used for detection of bacteria , parasites, viruses Fungi or other antigens in
CSF(Cerebrospinal fluid) , blood , stool , urine , tissues and other specimens .
Direct immunofluorescence test for antemorteum diagnosis of rabies . The test is used for
detection of rabies virus antigen in the skin smear collected from the Nape of the neck (is
the back of the neck) in humans and in the saliva of the dogs . Also used for
detection of N.gonorrhea (Neisseria gonorrhoeae), C.diphtheria
(Corynebacterium diphtheria) , T. pallidum (Treponema pallidum) etc. directly in appropriate
clinical specimens.

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Indirect Immunofluorescence Test :The indirect immunofluorescence test is used for detection of
specific antibodies in the serum and other body fluids for serological diagnosis of many infectious
diseases .
Indirect immunofluorescene is a two stage process. In the first stage , a known antigen is fixed on a
slide . Then the patient’s serum to be tested is applied to the slide , followed by careful washing. If
the patient’s serum contains antibody against the antigen , it will combine with antigen on the slide .
In the second stage , the combination of antibody with antigen can be detected by addition of
a fluorescence dye labeled antibody to human IgG, which is examined by a fluorescence
Microscope. The first step in the indirect immunofluorescence test is the incubation of a fixed
Antigen (eg. in a cell or tissue ) with unlabeled antibody, which becomes associated with the
antigen. Next , after careful washings , a fluorescent antibody ( eg. Fluorescent labeled anti-
IgG) is added to the smear . This second antibody will become associated to the first , and the
antigen- antibody complex can be visualized on the fluorescence microscope. The indirect
method has the advantage of using a single labeled antiglobulin ( antibody to IgG ) as a
universal reagent to detect many different specific antigen- antibody reactions.The test is often
more sensitive than the direct immunofluorescence test.
Indirect Immunofluorescence test is used widely to :( applications)
Detect specific antibodies for serodignosis of syphilis ,leptospirosis (is a rare bacterial infection
we get from animals. It's spread through their urine, especially from dogs, rodents, and farm
animals. They may not have any symptoms, but they can be carriers) , amoebiasis, and many
other infectious diseases. Identify the class of a given antibody by using fluorescent antibodies
specific for different immunoglobulin isotopes. Detect autoantibodies such as antinuclear
antibodies in auto immunodiseases.
The major limitation of immunofluorescene is that the technique requires.

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a) Expensive fluorescence microscope and reagents.

b) Trained personnel .
7.Enzyme Immunoassays:-
ELISA is an immunoassay that detects either antigen or antibodies in the specimen,
by using enzyme- substrate- chromogen system for detection.
Principle of ELISA.
ELISA is so named because of two components:
Immunosorbent : Here an absorbing material is used ( eg. polystyrene , poly vinyl ) that
specifically absorbes the antigen or antibody present in serum.
Enzyme is used to label one of the components of immunoassay ( ie . antigen or antibody).
Substrate chromogen system : A substrate chromogen system is added at the final step of ELISA.
The enzyme reacts with the substrate , which in turn activates the chromogen to produce the
colour. The classical example is horseradish peroxidase used as enzyme which reacts with it’s
substrate ( hydrogen peroxide), that in turn activates the chromogen ( tetramethyl benzidine) to
produce a colour.
The colour change is detected by spectrophotometry in an ELISA reader . Intensity of the colour is
directly proportional to the amount of detection molecule( Ag or Ab) present in test serum.
Procedure of ELISA.
ELISA is performed on a microtiter plate containing 96 wells made up of
polystyrene, polyvinyl or polycarbonate material.
microtitre plates 96 well Elisa Reader

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Types of ELISA
Direct ELISA
Indirect ELISA
Sandwitch ELISA
Competitive ELISA
Direct ELISA :
It is used for detecting the antigen in test serum. Here the primary antibody (targeted against
the serum antigen) is labeled with the enzyme.
Step 1 : Wells of microtitre plate are empty , not precoated with antigen or antibody.
Step 2 : Test serum ( containing antigen ) is added into the wells . Antigen becomes attached to the
solid phase by passive adsorption .
Step 3: After washing , the enzyme – labeled primary antibodies ( raised in rabbits) are added.
Step 4: After washing , a substrate- chromogen system is added and colour is measured .
Indirect ELISA : It is used for detection of antibody or less commonly antigen in the serum.
It differs from the direct ELISA in that the secondary antibody is labeled with enzyme
instead of primary antibody. The secondary anti body is an anti- species antibody eg.

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antihuman Ig ( an antibody targeted to Fc region of any human Ig).
Step 1: The solid phase of the wells of microtiter plates are precoated with the Ag.
Step 2: Test serum (containing primary Ab specific to the Ag) is added to the wells . Ab
gets attached to the Ag coated on the well.
Step 3: After washing , enzyme labeled secondary Ab ( Antihuman immunoglobulin ) is

added.
Step 4: After washing , a substrate - chromogen system added and colour is developed .
Applications .
The test is extensively used for determination of serum antibodies for diagnosis of Human
immunodeficiency virus (HIV) infection, Japanese encephalitis (An infection found in Asia
and the west Pacific that can cause brain swelling. Japanese encephalitis is a virus spread by the
bite of infected mosquitoes. It's more common in rural and agricultural areas. Most cases are
mild. Rarely, it causes serious brain swelling with a sudden headache, high fever and
disorientation) , dengue and many other viral infections .
Sand witch ELISA: It detects the antigen in test serum .

It is named because the antigens gets sand witched between a capture antibody and a
detector antibody.
Step 1: The microtiter well is precoated with the capture antibody ( monoclonal Ab raised in
rabbit) targeted against the test antigen.
Step 2 : The test serum ( containing antigen) is added to the wells .Ag gets attached to the
capture antibody coated on the well.
Step 3: After washing , an enzyme labeled primary detector antibody specific for the antigen is
added . The detector antibody can be the same as the capture antibody.
Step 4 : After washing , a substrate – chromogen system is added and colour is developed .

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Sandwitch ELISA
Application :-
The sandwitch Elisa is used to detect rotavirus and entero toxin of Escherichia coli in feces.
Competitive ELISA
Competitive ELISA is so named because , antigen in the test serum competes with another
antigen of the same type coated on well to bind to the primary antibody.
Step 1: Primary antibody is first incubated in a solution with a serum sample containing the test
antigen.
Step 2: This antigen – antibody mixture is then added to the microtiter well precoated with the
same type of antigen.
Step 3: The free antibodies bind to the antigen coated on the well. More the test antigens
present in the sample, lesser free antibodies will be available to bind to the antigens coated on
to well.
Step 4: After washing ( to remove free antibodies and antigens), enzyme conjugated
secondary antibody is added.
Step 5 : After washing , a substrate – chromogen system is added and colour is developed .
Intensity of the colour is inversely proportional to the amount of antigen present in the test
serum.
Application .
Competitive ELISA is the most commonly used test for detection of HIV antibodies in

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serum in patients with HIV.
8.Radioimmunoassay :-
When radioisotopes instead of enzymes are used as labels to be conjugated with antigen or
antibodies, the technique of detection of the antigen – antibody complex is called as Radio
immunoassay (RIA).
The classical RIA methods are based on the principle of competitive binding.
In this method Unlabeled antigen competes with radiolabeled antigen for binding to antibody
with the appropriate specificity . Thus, when mixtures of radiolabeled and unlabeled antigen
are incubated with the corresponding antibody , the amount of free ( not bound to antibody )
radiolabeled antigen is directly proportional to the quantity of unlabeled antigen in the
mixture .
In the test , mixtures of known variable amounts of cold antigen and fixed amounts of labeled
Antigen and mixtures of samples with unknown concentrations of antigen with identical
amounts of labeled antigen are prepared in the first step . Identical amounts of antibody are
added to the mixtures.Antigen – antibody complexes are precipitated either by cross linking
with a second antibody or by means of the addition of reagents that promote the precipitation
of Antigen – antibody complexes .
Counting radio activity in the precipitation allows the determination of the amount of
radiolabeled antigen precipitated with the antibody .A standard curve is constructed by plotting
the percentage of antibody - bound radiolabeled antigen against known concentrations of a
standardized unlabeled antigen , and the concentrations of antigen in patient samples are
extrapolated from that curve . The extremely high sensitivity of RIA is it’s major advantage.
Application :- The test can be used to determine very small quantities ( eg. nanogram ) of antigen
and antibodies in the serum. The test is used for quantitation of hormones , drugs, HBs
Ag(Hepatitis B surface antigen) and other viral antigens.
The main drawbacks of the RIA include

a) The cost of equipment and reagents.
b) Short shelf – life of the radiolabeled compounds and
c) The problems associated with the disposal of radioactive waste.
9. Western Blotting :
Western blotting is done for the detection of proteins . Western blotting is usually done on a tissue
homogenate or extract . It uses SDS- PAGE ( Sodium dodecyl sulphate polyacryl amide gel
electrophoresis ) , a type of gel electrophoresis to first separate various proteins in a mixture on the
basis of their shape and size . The proteins band thus obtained are transferred onto a nitrocellulose or
nylon membrane where they are probed with antibodies specific to the protein to be detected.The
antigen –antibody complexes that form on the band containing the protein recognized by the
antibody can be visualized in a variety of ways. If the protein of interest was bound by a
radioactive antibody, its position on the blot can be determined by exposing the membrane to a
sheet of X-ray film, a procedure called autoradiography. However , the most generally used detection
procedures employ enzyme – linked antibodies against the protein .After binding of the enzyme –
antibody conjugate , addition of a chromogenic substrate that produces a highly coloured and
insoluble product causes the appearance of a coloured band at the site of the target antigen. The

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site of the protein of interest can be determined with much higher sensitivity.
If a chemiluminescent (Energy can be transferred into (and out of) matter in many different
ways, as heat, light, or by chemical reactions. When energy in the form of light is released from
matter because of a chemical reaction the process is called chemiluminescence) compound
along with suitable enhancing agents is used to produce light at the antigen .
Immunoblotting (“Western Blotting”).
Western blotting allows simultaneous but independent detection of antibodies

against a number of different proteins present in a particular virus preparation.
There are four key steps to western blotting. First, concentrated virus is solubilized and the constituent
proteins separated into discrete bands according to molecular mass (Mr) by sodium dodecylsulfate-
polyacrylamide gel electrophoresis (SDS-PAGE).
Second, the separated proteins are electrophoretically transferred (“blotted”) onto a
nitrocellulose sheet in order to immobilize the separated polypeptides and to make these
available for reaction with antibodies.
Third, the test serum is allowed to react with the viral proteins on the nitrocellulose sheet and
unbound components are washed away, Finally, any bound antibody is demonstrated using
an enzyme-labeled anti-species antibody.
Thus, immunoblotting permits the demonstration of antibodies to some or all of the proteins
of any given virus preparation, and can be used to monitor the development of antibodies to
different antigens at different stages of infection. The technique has played a crucial role as

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the ultimate confirmatory test for samples giving an initial reaction in screening EIAs for
anti-HIV antibody; experience has taught which western blotting patterns are likely to
represent true anti-HIV antibodies, and which represent likely false-positive reactions in the
initial EIA.
Principles of western blotting for identification of antibodies or antigens.
1.A virus-containing sample is digested with the anionic detergent sodium dodecylsulfate
(SDS) and electrophoresed on a polyacrylamide slab gel (PAGE), which separates the different
viral proteins according to Mr. (2)
2.The bands of viral protein are then transferred (“blotted”) onto a nitrocellulose membrane
by capillary transfer or usually by electrophoresis in a different plane to immobilize the
polypeptides.
3. The unoccupied areas of the membrane are blocked (“quenched”) by saturation with a
suitable protein, then washed, dried, and cut into strips which can be used to test individual
patient’s sera.
4. Each test serum or plasma (“primary” antibody) is then incubated with one strip to
enable antibodies to bind to the individual viral proteins.
5.Following rinsing, bound antibody is detected by the addition of enzyme-labeled anti-

human immunoglobulin (“secondary” antibody).
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6.Following another wash, the bands are revealed by the addition of a substrate chosen to
produce an insoluble colored product.
Applications .
The Western blot test is most widely used as a confirmatory test for diagnosis of
HIV , where this procedure is used to determine whether the patient has antibodies
that react with one or more viral proteins or not.
The western blotting is also used for the demonstration of specific antibodies in
the serum for diagnosis of tubercular meningitis.
10. Chemiluminescence Assay:-

The Chemiluminescence Assay uses Chemiluminescent compounds that emit energy in
the form of light during the antigen – antibody reactions. The emitted lights are measured
and the concentration of the analyze is calculated . The assay is a fully automated
method, which is used commonly for drug sensitivity testing of Mycobacterium
tuberculosis.
11.Immunoelectromicroscopic Tests :-
These are the type of antigen- antibody reactions that are visualized directly
by electron microscopy.
These are of the following types .
Immunoelectromicrosco
py
Immunoenzyme test
Immunoferritin Test
Immunoelectromicroscopy : This is a test used to detect rotavirus (Rotavirus is a genus
of double-stranded RNA viruses in the family Reoviridae. Rotaviruses are the most
common cause of diarrhoeal disease among infants and young children) and hepatitis A
virus directly in feces .
In this test , viral particles are mixed with specific antisera and are demonstrated as
clumps of virion particles under the electron microscope .
Immunoenzyme test :- This test is used to detect antigen directly in tissue specimens ,in
which tissue sections are treated with peroxidase – labeled antisera to detect corresponding
antigen . The peroxidase bound to the antigen is visualized under the electron microscope.
Immunoferritin Test :- Electron dense substances such as ferritin are conjugated

with antibody and such labeled antibodies reacting with antigen can be
visualized under the electron microscope .
Immunization Products.

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Active immunization - is the process of increasing resistance to infection where by

microorganisms or products of their activity acts as antigens and stimulate certain body
cells to produce antibodies with a specific protective capacity . It is a slow process
dependent up on the rate at which the antibody formation takes place. Biological products
comprising vaccines and toxoids confer active immunity .
Passive immunization :- Which results in immediate protection of short duration , may be

achieved by the administration of the antibodies themselves .Biological products
comprising human immune sera ( homologous sera ) and animal immune sera
(heterologous immune sera ) confer passive immunity .
Immunization products.
Vaccines : According to Indian Pharmacopoeia , vaccines are preparations of antigenic
materials , which are administrated with the object of inducing in the recipient a specific and
active immunity against the infectious agent or toxin produced by it. They may contain
living or killed microorganisms , bacterial toxoids or antigenic material from particular
parts of the bacterium , rickettsia or virus. Edward Jenner developed the first vaccine in
1798. The term vaccine is derived from a Latin term vaccinia meaning cowpox. Vaccines
are the most important of all the biological products .They are prepared from bacteria,
viruses, rickettsia or toxins and recently through the application of biotechnology.
Vaccines may be single component or mixed component vaccines .
Single vaccines are prepared from a single species of microorganisms . Mixed or compound
vaccines are prepared from two or more species . Simple vaccines containing only one
strain of a species are univalent and those containing two or more strains of the same
species are called polyvalent.
Stock vaccines are prepared from standard strains of bacteria where as autogenous
vaccines are prepared from a patient’s own organisms ( a vaccine prepared from
cultures of microorganisms obtained from an individual and then used to immunize that
same individual against further spread and progress of the same microorganisms).
Vaccines are used for active immunization as a prophylactic (intended to prevent disease)
measure against some infectious diseases . They provide partial or complete protection for
months or years .
For inactivated Vaccines a slight and rather slow antibody response of primary
immunoglobulin M( IgM) ( the primary response ) is produced after the first or second dose
but, when a further dose is given after a suitable interval , a prompt antibody response
follows and high concentration of IgG occurs in the body ( the secondary response ).
Though the antibody concentration may fall later, a further dose of vaccine promptly
restores it.
For most live vaccines only one dose is required although 3 doses of live ( oral )
poliomyelitis vaccines are needed to achieve complete immunization .
Some inactivated vaccines contain an adjuvant such as aluminium hydroxide or aluminium
phosphate to enhance the immune response .
There are many types of vaccines, categorized by the antigen used in their preparation.
Their formulations affect how they are used, how they are stored, and how they are
administered.
The globally recommended vaccines are of four main types.
Types of Vaccine
Live attenuated (LAV)
Inactivated (killed antigen)
Subunit (purified antigen)

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Toxoid (inactivated toxins)
Types of vaccines in common uses ( human ) are listed below
Type Live vaccine Killed vaccine

Bacterial Tuberculosis (BCG) Cholera
Typhoid
Whooping cough
Viral Small pox Influenza

Rubelia Rabies
Measles Poliomyelitis (
Yellow fever injection)
Mumps
Poliomyelitis (oral)
(OPV)
Rickettsial Typhus
Toxoids Diphtheria
Tetanus
Mono and polyvalent vaccines.

Vaccines may be monovalent or polyvalent. A monovalent vaccine contains a single strain
of a single antigen (e.g. Measles vaccine), whereas a polyvalent vaccine contains two or
more strains/serotypes of the same antigen (e.g. OPV).
Combination vaccines.
Some of the antigens above can be combined in a single injection that can prevent different
diseases or that protect against multiple strains of infectious agents causing the same disease
(e.g. combination vaccine DPT combining diphtheria, pertussis and tetanus antigens).
Combination vaccines can be useful to overcome logistic constraints of multiple injections,
and accommodate for children’s fear of needles and pain.
Live attenuated vaccines (LAV).

Available since the 1950s, live attenuated vaccines (LAV) are derived from disease-
causing pathogens (virus or bacteria) that have been weakened under laboratory
conditions. They will grow in a vaccinated individual, but because they are weak,
they will cause no or very mild disease.
Immune response of LAV

LAVs stimulate an excellent immune response that is nearly as good as compared to an
infection with the wild-type pathogen. Live microorganisms provide continual antigenic
stimulation giving sufficient time for memory cell production.
In the case of viruses or intracellular microorganisms where cell-mediated immunity is
usually desired, attenuated pathogens are capable of replicating within host cells.
Safety and stability.
Since LAVs contain living organisms, there is a degree of unpredictability raising some
safety and stability concerns. Attenuated pathogens have the very rare potential to revert to
a pathogenic form and cause disease in vaccinees or their contacts. Examples for this are the
very rare, serious adverse events of: vaccine-associated paralytic poliomyelitis (VAPP) and
disease-causing vaccine-derived poliovirus (VDPV) associated with oral polio vaccine
(OPV). Functional immune systems eliminate attenuated pathogens in their immune
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response. Individuals with compromised immune systems, such as HIV-infected patients

may not be able to respond adequately to the attenuated antigens. Sustained infection, for
example tuberculosis (BCG) vaccination can result in local lymphadenitis ( is the
inflammation of lymph nodes).
If the vaccine is grown in a contaminated tissue culture it can be contaminated by other
viruses (e.g. retro viruses with measles vaccine). As a precaution, LAVs tend not to be
administered during pregnancy. However, the actual potential for fetal damage remains
theoretical. For example, numerous studies have demonstrated that accidental rubella
vaccination during pregnancy did not result in an increased risk of birth defects. LAVs can
have increased potential for immunization errors:
Some LAVs come in lyophilized (powder) form. They must be reconstituted with a specific
diluent before administration, which carries the potential for programmatic errors if the
wrong diluent or a drug is used.
Many LAVs require strict attention to the cold chain for the vaccine to be active and are
subject to programme failure when this is not adhered to virus Oral polio vaccine (OPV).
Inactivated whole-cell vaccines.
Inactivated vaccines are made from microorganisms (viruses, bacteria, other) that
have been killed through physical or chemical processes. These killed organisms
cannot cause disease.
Immune response
Inactivated whole-cell vaccines may not always induce an immune response and the
response may not be long lived. Several doses of inactivated whole-cell vaccines may be
required to evoke a sufficient immune response.
Safety and stability

Inactivated whole-cell vaccines have no risk of inducing the disease they are given
against as they do not contain live components. They are considered more stable than LAV
vaccines.
Subunit vaccines.
Immune response Subunit vaccines, like inactivated whole-cell vaccines do not contain live
components of the pathogen. They differ from inactivated whole-cell vaccines, by containing only the
antigenic parts of the pathogen. These parts are necessary to elicit a protective immune response. This
precision comes at a cost, as antigenic properties of the various potential subunits of a pathogen must
be examined in detail to determine which particular combinations will produce an effective
immune response within the correct pathway. Often a response can be elicited (evoke or draw
out a reaction) but there is no guarantee that immunological memory will be formed in the correct
manner.

Like inactivated vaccines, subunit vaccines do not contain live components and are
considered as very safe.
Protein-based subunit vaccines.
Protein based subunit vaccines present an antigen to the immune system without viral
particles, using a specific, isolated protein of the pathogen. A weakness of this technique
is that isolated proteins, if denatured, may bind to different antibodies than the protein of
the pathogen.
Commonly used protein-based subunit vaccines are the following:
Acellular pertussis (aP) vaccines contain inactivated pertussis toxin (protein) and may
contain one or more other bacterial components. Pertussis or Whooping cough is
particularly dangerous for infants. Besides a cough that sounds like "whoop", symptoms
include a runny nose, nasal congestion and sneezing.

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The pertussis toxin is detoxified either by treatment with a chemical or by using

molecular genetic techniques. Hepatitis B vaccines are composed of the hepatitis B virus
surface antigen (HBsAg), a protein produced by hepatitis B virus. Earlier vaccine products
were produced using purified plasma of infected individuals. This production method has
been replaced by recombinant technology that can produce HBsAg without requiring human
plasma increasing the safety of the vaccine by excluding the risk from potential
contamination of human plasma.
Polysaccharide vaccines.
Some bacteria when infecting humans are often protected by a polysaccharide (sugar)
capsule that helps the organism evade the human defense systems especially in infants and
young children. Polysaccharide vaccines create a response against the molecules in the
pathogen’s capsule. These molecules are small, and often not very immunogenic. As a
consequence they tend to:
1.Not be effective in infants and young children (under 18–24 months),
2.Induce only short-term immunity (slow immune response, slow rise of antibody levels,
no immune memory).
Examples of polysaccharide vaccines include Meningococcal disease caused by

Neisseria meningitides groups A, C, W135 and Y, as well as Pneumococcal disease.
Conjugate subunit vaccines.
Conjugate subunit vaccines also create a response against the molecules in the pathogen’s
capsule. In comparison to plain polysaccharide vaccines, they benefit from a technology that
binds the polysaccharide to a carrier protein that can induce a long-term protective response
even in infants. Various protein carriers are used for conjugation, including diphtheria and
tetanus toxoid.
Conjugate subunit vaccines, can therefore prevent common bacterial infections for which
plain polysaccharide vaccines are either ineffective in those most at risk (infants) or
provide only short-term protection (everyone else).
The advent of conjugate subunit vaccines heralded a new age for immunization against
diseases caused by encapsulated organisms such as meningococcus, Haemophilus
influenzae type b (Hib) and pneumococcus.
WHO recommends that children receive Haemophilus influenzae type b (Hib) and
pneumococcal conjugate vaccines. In addition, the meningococcal A vaccine introduced in
Africa is also a conjugated subunit vaccine.
Toxoid vaccines.
Toxoid vaccines are based on the toxin produced by certain bacteria (e.g. tetanus or
diphtheria). Tetanus is a serious disease caused by a bacterial toxin that affects your
nervous system. Tetanus causes painful muscle contractions, particularly in the jaw and
neck.It can interfere with the ability to breathe, eventually causing death.
Diphtheria an acute and highly contagious bacterial disease causing inflammation of the
mucous membranes, formation of a false membrane in the throat which hinders breathing
and swallowing, and potentially fatal heart and nerve damage by a bacterial toxin in the
blood. The toxin invades the bloodstream and is largely responsible for the symptoms of
the disease. The protein-based toxin is rendered harmless (toxoid) and used as the antigen in
the vaccine to elicit immunity. To increase the immune response, the toxoid is adsorbed to
aluminium or calcium salts, which serve as adjuvants.

Toxoid vaccines are safe because they cannot cause the disease they prevent and there
is no possibility of reversion to virulence. The vaccine antigens are not actively multiplying

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and do not spread to unimmunized individuals. They are stable, as they are less susceptible
to changes in temperature, humidity and light.
Combination vaccines.
Licensed combination vaccines undergo extensive testing before approval by national
regulatory authorities to assure that the products are safe, effective, and of acceptable
quality. Combination vaccines consist of two or more antigens in the same preparation. This
approach has been used for over 50 years in many vaccines such as DTP and MMR.
Combination products simplify vaccine administration and allow for the introduction of new
vaccines without requiring additional health clinic visit and injections.
Potential advantages of combination vaccines include
Reducing the cost of stocking and administering separate vaccines, Reducing the cost of extra health
care visits. Improving timeliness of vaccination (some parents and health-care providers object to
administering more than two or three injectable vaccines during a single visit because of a child’s fear
of needles and pain, and because of concerns regarding safety),
Facilitating the addition of new vaccines into immunization programmes. It is very important,
however, that combination vaccines are carefully tested before introduction.
For instance, adjuvants in a combination vaccine could reduce the activity of one antigen
and excessively increase the reactivity of another antigen. There could also be interactions
with other vaccine components such as buffers, stabilizers and preservatives. With all
combinations, manufacturers must therefore evaluate the potency of each antigenic
component, the effectiveness of the vaccine components when combined to induce
immunity, risk of possible reversion to toxicity, and reaction with other vaccine
components.
Key point.
No evidence exists that the administration of several antigens in combined vaccines
overwhelms the immune system, which has the capability of responding to many millions of
antigens at a time. Combining antigens usually does not increase the risk of adverse
reactions. In fact, it can lead to an overall reduction in adverse reactions.
With all combinations, manufacturers must, however, evaluate the potency of each antigenic
component, the effectiveness of the vaccine components when combined to induce
immunity, risk of possible reversion to toxicity, and reaction with other vaccine
components.
Components of a vaccine.
Vaccines include a variety of ingredients including antigens, stabilizers, adjuvants,
antibiotics, and preservatives. They may also contain residual by-products from the
production process. Knowing precisely what is in each vaccine can be helpful when
investigating adverse events following immunization (AEFIs) and for choosing alternative
products for those who have allergies or have had an adverse event known or suspected to
be related to a vaccine component.
Antigens
Antigens are the components derived from the structure of disease-causing organisms,
which are recognized as ‘foreign’ by the immune system and trigger a protective immune
response to the vaccine.
Stabilizers -Stabilizers are used to help the vaccine maintain its effectiveness during storage.
Vaccine stability is essential, particularly where the cold chain is unreliable. Instability can
cause loss of antigenicity and decreased infectivity of LAV.
Factors affecting stability are temperature and acidity or alkalinity of the vaccine (pH).
Bacterial vaccines can become unstable due to hydrolysis and aggregation of protein and
carbohydrate molecules. Stabilizing agents include MgCl2 (for OPV), MgSO4 (for
measles), lactose-sorbitol and sorbitol-gelatine.

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Adjuvants -Adjuvants are added to vaccines to stimulate the production of antibodies

against the vaccine to make it more effective. Adjuvants have been used for decades to
improve the immune response to vaccine antigens, most often in inactivated (killed)
vaccines.
In conventional vaccines, adding adjuvants into vaccine formulations is aimed at enhancing,

accelerating and prolonging the specific immune response to vaccine antigens. Newly
developed purified subunit or synthetic vaccines using biosynthetic, recombinant, and other
modern technology are poor vaccine antigens and require adjuvants to provoke the desired
immune response. Chemically, adjuvants are a highly heterogeneous group of compounds
with only one thing in common:
Their ability to enhance the immune response. They are highly variable in terms of how they
affect the immune system and how serious their adverse reactions are, due to the resulting
hyperactivation of the immune system.
Today there are several hundred different types of adjuvants that are being used or studied
in vaccine technology.
Aluminium salts example
Aluminium salts are among the oldest adjuvants that are commonly used.
They slow the escape of the antigen from the site of injection thereby lengthening the
duration of contact between the antigen and the immune system (i.e. macrophages and other
antigen-receptive cells). Aluminium salts are generally recognized as safe, however, they
can cause sterile abscesses and nodules at the site of injection.
The formation of a small granuloma (a mass of granulation tissue, typically produced in
response to infection, inflammation, or the presence of a foreign substance) is inevitable
with alum-precipitated vaccines.
To ensure safe vaccination it is important that aluminium salts are administered
intramuscularly and not subcutaneously.
Subcutaneous administration can result in necrotic breakdown ( is a form of cell injury
which results in the premature death of cells in living tissue by autolysis ) and cyst (an
abnormal, usually noncancerous growth filled with liquid or a semisolid substance,
sometimes causing pain) and abscess formation.
To ensure the proper handling of intramuscular injections, it is critical to ensure that
vaccination staff has been well trained.
Antibiotics.
Antibiotics (in trace amounts) are used during the manufacturing phase to prevent bacterial
contamination of the tissue culture cells in which the viruses are grown.
Usually only trace amounts appear in vaccines, for example, MMR vaccine and IPV each
contain less than 25 micrograms of neomycin per dose (less than 0.000025 g).
Used during the manufacturing phase to prevent bacterial contamination of tissue culture
cells in which viruses are grown, Usually only trace amounts appear in vaccines, for
example, MMR (measles, mumps and rubella) and IPV vaccines (inactivated polio vaccine)
each contain less that 25 micrograms of neomycin per dose,
Persons known to be allergic to neomycin should be closely observed after vaccination so
any allergic reaction can be immediately treated.
Preservatives.
Preservatives are added to multidose vaccines to prevent bacterial and fungal growth. They
include a variety of substances, for example Thiomersal, Formaldehyde, or Phenol
derivatives.
Thiomersal - Very commonly used preservative. Thiomersal is an ethyl mercury- containing

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compound. It has been in use since the 1930 s and no harmful effects have been reported for
doses used in vaccination except for minor reactions (e.g. redness, swelling at injection
site). It is used in multidose vials and for single dose vials in many countries as it helps
reduce storage requirements costs. Thiomersal has been subjected to intense scrutiny, as it
contains ethyl mercury. The Global Advisory Committee on Vaccine Safety continuously
review the safety aspects of Thiomersal.
So far, there is no evidence of toxicity when exposed to Thiomersal in vaccines. Even trace
amounts of Thiomersal seem to have no impact on the neurological development of infants.
Formaldehyde.
Used to inactivate viruses (e.g. IPV) and to detoxify bacterial toxins, such as the toxins used to make
diphtheria and tetanus vaccines, During production, a purification process removes almost all
formaldehyde in vaccines. The amount of formaldehyde in vaccines is several hundred times lower
than the amount known to do harm to humans, even infants. Eg., DTP-HepB + Hib “5-in-1” vaccine
contains less than 0.02% formaldehyde per dose, or less than 200 parts per million.(Pentavalent
vaccine frequently refers to the 5-in-1 vaccine protecting against diphtheria, tetanus, whooping cough,
hepatitis B and Haemophilus influenzae type B, which is generally used in middle- and low-
income countries, where polio vaccine is given separately).
Route of administration.
The route of administration is the path by which a vaccine (or drug) is brought into contact
with the body. This is a critical factor for success of the immunization. A substance must be
transported from the site of entry to the part of the body where its action is desired to take
place. Using the body’s transport mechanisms for this purpose, however, is not trivial.
Intramuscular (IM) injection administers the vaccine into the muscle mass. Vaccines
containing adjuvants should be injected IM to reduce adverse local effects.
Subcutaneous (SC) injection administers the vaccine into the subcutaneous layer above the
muscle and below the skin.
Intradermal (ID) injection administers the vaccine in the topmost layer of the skin. BCG is
the only vaccine with this route of administration. Intradermal injection of BCG vaccine
reduces the risk of neurovascular injury.
Health workers say that BCG is the most difficult vaccine to administer due to the small size
of newborns’ arms. A short narrow needle (15 mm, 26 gauge) is needed for BCG vaccine.
All other vaccines are given with a longer, wider needle (commonly 25 mm, 23 gauge),
either SC or IM.
Oral administration of vaccine makes immunization easier by eliminating the need for a
needle and syringe. Intranasal spray application of a vaccine offers a needle free approach
through the nasal mucosa of the Vaccine .

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Bacterial Vaccines.
Bacterial vaccines are defined as either sterile suspensions of live or killed bacteria or
sterile extracts of derivatives of bacteria .
Preparations : - Bacterial vaccines are generally prepared from carefully selected strains of
the bacteria . Thus bacteria are first tested for purity and identity from other bacteria. The
selected strain is then cultivated on solid medium usually for 24 to 48 hrs and then
washed with sterile normal saline . The suspension is evenly distributed by shaking. If
fragments of the medium are present, the same may be removed by centrifugation or
sedimentation . It is then sterilized either by minimal heat treatment or by means of alcohol
or other bactericide .
Vaccines prepared from cultures of non sporing bacteria may be sterilized in a vaccine bath
at 56 to 600 C for about 1 hr . This kills all the bacteria. Heat treatment should be enough to
kill the microorganisms with out affecting their antigenic properties .
The bacterial concentration of the suspension is determined , desired dilutions are made and
a suitable bacteriostatic agent is added as preservative .
The product should be filled asepetically into previously sterilized containers and sealed to
exclude contaminating organisms .
Vaccines are standardized by determining the number of microorganisms or the dry weight
of the organisms in 1ml or the number of organisms or the dry weight of the organisms
used to prepare 1ml of the product . As a measure of safety against deterioration such
products are not used 18 to 30 months after the preparation . All vaccines should be stored
at temperature as low as possible above 00C; the temperature of storage must not exceed
100C.
The label on the containers must state

Name of the preparation.
The number of microorganism per ml or the dry weight of the microorganism in 1ml.
The nature and strength of substances other than the diluent with which the vaccine
is combined.
The name , address and license number of the manufacturer .
The date of preparation .The date up to which the potency is expected to be
retained on proper storage . The name and percentage of antiseptic added.
Precautions necessary for storage of the product . Batch number .
All vaccines are generally used for active immunization .
Description of some official bacterial vaccines follows .
BCG Vaccine ( Freeze Dried).

It is a freeze dried preparation containing live culture of the Bacillus of Calmette and
Guerin Strain of Mycobacterium tuberculosis var. bovis. The vaccine is produced on the
basis of the seed lot system . The strain which is of uniform composition is selected and
maintained so as to preserve its stability , it’s power to sensitize man to tuberculosis , it’s

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ability to protect laboratory animals against tuberculosis and to retain its relative non
pathogenicity for man and laboratory animals .
The seed lot is maintained in a freeze dried form at a temperature not exceeding 20 0C
and is revived by transplanting on to a suitable medium and growing for not more than 7
days . The culture for harvesting are done on liquid medium and the harvested growth is
separated by filtration in the form of a cake .The cake is homogenized in a grinding flask
and suspended in a suitable sterile liquid medium designed to preserve the antigenicity and
the viability of the vaccine as determined by an appropriate counting.
Method . The suspension is distributed into it’s final sterile containers and freeze dried
under conditions designed to prevent microbial contamination , and finally sealed .The
vaccine contains no antimicrobial agent.
It is available as white pellet or powder , which when reconstituted , yields an opalescent
and homogenous suspension . The vaccine complies with the test for absence of virulent
mycobacteria , test for sterility , skin reactivity , toxicity and potency as specified in IP.
Labeling :- The label states

1.The number of viable particles .
2.The name and volume of the liquid to be used for reconstituting the vaccine .
3.The storage conditions.
4.The expiry date.
5.That any portion of the reconstituted vaccine should not be exposed to light before or
after
reconstitution .
Storage :- BCG vaccine should be stored in hermetically (completely air tight) sealed light
resistant glass containers at a temperature between 2 0 and 80 C . The reconstituted vaccine
should be used immediately after Preparation.
Dose – Prophylatic , by intra-cutaneous injection , as a single dose 0.1ml.
Use- Active Immunizing agent .
Viral vaccines.
Viruses are very minute non cellular organisms capable of self replication . They contain only one
kind of nucleic acid, RNA or DNA but never both . They range in size from 25 to 300 nm and may
pass through bacteria proof filters . Viruses require a living medium for growth .
Some of the important viral diseases are common cold , dengue, influenza , measles ,
mumps, Poliomyelitis , rabies, small pox , yellow fever etc. For preparation of viral
vaccines, viruses are usually grown in the chorioallantoic membrane of fertile hen eggs or in
whole animals . The striking feature of the immunity following recovery from virus
diseases is that very often it is life long eg. after measles , mumps , small pox and yellow
fever. This may be due in larger part to the way in which many viruses infect the body.
They enter through mucous membrane , often of the respiratory tract and are transported to
the reticulo-endothelial system (The reticuloendothelial system (RES) removes immune
complexes from the circulation in healthy persons, and is formed of phagocytic cells that are
found in the circulation and in tissues.) by wandering phagocytes which although they ingest
the viruses, can destroy only those of low virulence . The long incubation period (
commonly two to three weeks) that is characteristic of viral diseases follows and during
this time the viruses are providing a continuous and strong antigenic stimulus in the system
that actually produces antibodies . This could explain the long lasting immunity.
Eventually , the viruses are freed into the blood stream and carried to the sites in which the

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disease becomes manifest ( appearance). The immunity is further strengthened because the
antibodies circulating in the blood are excellently situated to inactivate viruses as they
travel through the blood stream on their way to or from the reticulo-endothelial system.
In many bacterial infections the organisms remain outside the blood stream where they
are less vulnerable to antibody attack.
It has been suggested that the solidity (the quality) of viral immunity might be the result of
persistence (the action of perseverance) in the tissues of viruses in non- - infective form ,
after the host has recovered from the disease. For a number of viral diseases, however
immunity is very short lived .The common cold and influenza are well known examples .
In these infections the viruses are usually confined (restricted in area) to the epithelial cells
of the respiratory tract and are carried from one cell to another by mucus . As a result, the
antigenic stimulus is less direct and much weaker.
After development of immunity the presence of antibodies in the blood serum is relatively
unimportant ( although they may prevent penetration of viruses into deeper tissues ) ; they
must pass out into the mucus to be effective .
As the appropriate host cells can be reached easily by the invading viruses the incubation
period and , therefore , the duration of the antigenic stimulus is usually short. However it
is possible that inadequate production or non- persistence (no preservence) of antibodies is
less responsible for the short immunity in respiratory infections than the ability of the
viruses to change into antigenically different variants.
While the viruses of diseases that are followed by long immunity are antigenically stable,
respiratory viruses frequently give rise to new antigenic types against which the
antibodies of other types are ineffective.
This is reflected in the preparation of viral vaccines. Only one strain need be used if the
disease is caused by an antigenically stable organism , such as yellow fever , but where
more than one strain can promote infection all must be included in the vaccine eg. in the
poliomyelitis vaccine there are three antigenic types.
The problem becomes most acute when , as in influenza , the organism undergoes frequent
variation ; the strains responsible for succeeding epidemics are slightly different from
each other and it is extremely difficult to produce an entirely satisfactory vaccine before
an epidemic arrives .
Living and Dead Vaccines .

The earliest recorded example of the production of active immunity by artificial means is
the ancient practice of variolation which was used to give protection against small pox (
variola). It involved scratching virus from mild cases into the skin , the aim being to
produce a harmless form of the disease by employing an unusual route of infection; small
pox virus normally enters the body via the respiratory tract.
Enough fatal cases of the disease developed to give clear warning of the serious risk
involved in using virulent viruses for immunization and since the time Jenner attempts have
been made to induce viral immunity by attenuated or inactivated Organisms.
Compared with inactivated ( dead) vaccines attenuated ( living ) vaccines have important
advantages.
1.The immunity is stronger and more lasting because the virus multiplies in the tissue.
2.As a result of the multiplication a smaller dose can be given.
3.Satisfactory products can be made from viruses with labile antigens that are

1
destroyed by
inactivation processes. Eg. cowpox and yellow fever.
4.Administration by the normal route of infection may be possible . In some cases this
makes Injections
unnecessary . Attenuated poliomyelitis vaccine given by the ( natural oral route can be
taken on a sugar lump.
They also have a number of disadvantages.
1.The virus may spread to other individuals , could become exalted (to enhance the activity
of)
in virulence as a result of transfer from person to person . On the other hand , if the strain
remained stable , this spread could be a useful way of increasing immunity in the
population.
2.It is much more difficult to attenuate than to inactivate an organism. The attenuated
strain must retain its antigenicity but be free from virulence and incapable of regaining it.
However inactivated vaccines are not entirely free from danger since faulty inactivation
could leave fully virulent strains in the preparation.
3.Because there is no inactivation process great care must be taken to prevent
contamination with viruses capable of causing other infectious diseases.
Cultivation of viruses in Vaccine Production .

Since viruses are intracellular parasites they will grow only within other living cells.
These can be provided in free living animals , fertile eggs or tissue cultures .
Free living Animals :-
Now a days very few vaccines are made from Free living Animals
Fertile Eggs.
Many viruses can be grown in some part of the chick embryo. The advantages of this
method over the use of free living animals include .
a) It is much easier to keep the product free from contaminating
microorganisms.
b) At the age of use the embryo cannot produce antiviral antibodies , which
might affect the yield.
In using eggs a number of precautions must be taken.
Strict aseptic technique must be maintained throughout , to prevent bacterial contamination

. The yolk sac is an excellent medium for bacterial growth , and although the amniotic and
allantoic fluids are antibacterial they cannot cope with heavy infection.
Repeated passage of virus from egg to egg must be avoided because the virus may
become adapted to embryo tissue and less virulent for its natural host.
To ensure an adequate supply of virus of satisfactory virulence the vaccine strain is

grown in quantity in one batch of eggs and then freeze dried or stored at a low
temperature so that the same virus can be used for many future batches of vaccine .
viruses grown in the yolk sac or embryo are separated by grinding and as a result , traces

1
of egg protein get into the vaccine and may cause reactions in the recipient similar to
those produced by the injection of serum proteins .
When either of these two regions is used the egg must not be more than ten to eleven days
old at harvesting because before this time its proteins are not sufficiently developed to
cause hypersensitivity reactions .
The eggs must be candled to confirm that the embryos are alive . This involves examination
in front of a bright light , when spontaneous movement or well defined blood vessels
indicate a living embryo.
Tissue cultures .
A large number of tissues can be successfully cultivated outside the animal body but,
because certain viruses will grow only in primate cells (the group that contains all the
species commonly related to the monkeys, and apes) , monkey kidney has become the most
widely used (Eg. in the manufacture of both poliomyelitis vaccines and one measles
vaccine).
Chick embryo is more satisfactory in some cases , eg attenuated measles virus and cow
pox ,and the latter has also been successfully grown in calf embryo skin.
Ideally the tissues should be free from living microorganisms but while the absence of
bacterial diseases such as tuberculosis in monkeys is comparatively easy to confirm by
quarantine of the animals and a post mortem before use of the kidney.
Establishment of growth and or maintenance of metabolism under artificial conditions.
After the organ or tissue has been removed from the animal under surgically clean
conditions, it is minced ( Cut to small pieces ) and treated , usually with trypsin , to
disperse the cells .

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The result is a suspension of single cells or small aggregate and this is used in
one or two types of cultures.
a)Suspended cell cultures : In this, the cells are merely suspended in a liquid
medium . The aim is simply to maintain cell metabolism , and since there is very
little or no proliferation , an adequate quantity of cells for virus growth must be
included initially.
a)Fixed cell ( monolayer ) culture . Fewer cells are added to the medium and these
are allowed to settle on to the side of a large flat – sided bottle . During a period of
incubation they become attached to the glass and multiply into a uniform layer
one cell thick .

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When this has spread over the lower side of the bottle the medium is changed
from one that will support growth to another that is adequate to maintain cell
metabolism.
Fixed cell cultures are claimed to give higher yields of virus per cell, possibly
because , as is indicated by the fact that multiplication occurs, the viability of the
cells is higher ,also it is easier to change the medium and as there are fewer
floating cell aggregates to protect the virus, to sterilize the suspension when a
dead vaccine is required.
Extremely complex media are needed to grow and maintain these cultures . They
contain
a)A balanced salt solution to provide the optimal pH and osmotic pressure.
b)Nutrients . The nutritional needs of tissue culture cells necessitate particularly
complicated media .
In addition , in vaccine production , complex materials such as serum and proteins
must be excluded as far as possible because they may cause reactions when the
vaccine is administered .
To satisfy these requirements , many ingredients are included eg. essential amino
acids , growth factors , dextrose , purines , pyrimidines and inorganic salts .
a) A pH indicator , often phenol red , to show the state of the cell
metabolism and when the pH has fallen to a level that necessitates a
change of medium.
b) Antibiotics, both antibacterial and antifungal, to prevent growth
of contaminants .
c) Care is taken largely to inactivate these antibiotics by the end of
processing because it may cause some sensitization in some patients .
Official Viral Vaccines .
From free living Animals .

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Small pox vaccine

When Jenner introduced vaccination he used living cowpox virus which gave good
immunity to small pox because the viruses of the two diseases are closely related .
Since then , small pox vaccines have not always been made from natural cowpox strains;
variola virus attenuated by passage through animals , such as calves or rabbits and known
as vaccinia, has also been used. The vaccine is usually obtained from the skin of suitable
living mammals Eg. calves or sheep.
Selection of animals . Healthy calves or sheep are quarantined and given through
examinations to exclude communicable diseases .
Inoculation . The flanks (the side of an animal's body between the ribs and the hip) and
abdomen ( only the former in sheep because the abdomen is less easy to keep clean ) are
scrubbed , disinfected , shaved , rescrubbed and re- disinfected . Then in special rooms , the
shaved areas are
a) Scarified ie. lightly scratched with a comb like device, without drawing blood .
b) Inoculated , by rubbing seed virus of known potency into the scratches.
Incubation . During the next four to five days , vesicles (a painful swelling on the skin that
contains liquid) containing the virus develop along the lines of scarification, and through
out this period every precaution is taken to keep the inoculated areas aseptically clean.
Harvesting : The animals are killed , exsanguinated ( drain the blood) and washed.
Then the contents of the vesicles (the lymph ) are removed by curettage ( ie . by scraping
with a special (Volkmann’s ) spoon that has a very sharp edge .The pooled material is
homogenized . A post mortem is done on the animal’s carcass to confirm the absence of
infectious diseases.
Purification :- Because it is not possible entirely to prevent contamination with extraneous

microorganisms the lymph must be treated to kill pathogens and to reduce the number
of residual bacteria to a very low level.
For many years this was done by grinding with an equal volume of glycerin and storing
for a long time at – 100C. Now a days a more efficient method is used .
a) The lymph is extracted with a protein solvent eg.

trichlorofluoroethane . Presence of protein lowers the efficiency of
the bactericidal agent.
b) Phenol is added to produce a concentration of 0.4 % and the
material is incubated at 22 Cfor two days or until the bacterial count is low
0
enough .
The viruses are unharmed by this treatment because they are
much more resistant than bacteria to phenol.
Glycerin and peptone are included to give concentrations of 40 % and 1 %
respectively .The glycerin assists the bactericidal action of the phenol during the
subsequent storage at – 100C, which continues until issue . The glycerin also gives
the product a viscosity , a viscous preparation being easier to use for vaccination .
The peptone helps to preserve the viability of the viruses particularly if the
product is to be freeze dried.

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c) Sometimes brilliant green , or another suitable colouring matter , is added to

mark the area of application of the vaccine .
Tests are performed to confirm the absence of Escherichia coli , aerobic
pathogens and anaerobic pathogens and to show that the number of living
extraneous microorganisms not more than the specified limit.
Standardization of Vaccines and sera.
For standardization of vaccines and sera basically three tests are there
1. Toxicity test.
2. Sterility test .
3. Potency test.
Toxicity Tests:
Toxicity in immunological preparations may be caused by .

a) In complete conversion of toxin to toxoid .
b) Incomplete sterilization of a supposedly dead vaccine.
c) Increased virulence in an attenuated organisms.
d) Excessive toxicity in the toxin used for a diagnostic preparation .
The principle underlying most of the tests is that administration of a human ie. a very
large dose , by the usual or by a variety of routes , to a sensitive and small laboratory
animal must not cause , within a specified period , either a serious symptoms or death
When , because of the toxic nature of the preparation itself , injection by any other route
would cause adverse symptoms in laboratory animals the preparation is tested
intracutaneously for example of shick test toxin , injection of 1/25 of the test dose into
guinea pig gives a positive Shick reaction, while 1/50 of the test dose causes no local
effect .
White or pale colored animals are used so that the results can be seen clearly. BCG
Vaccine is also tested in this way; the lesion must not be necrotic (death of a usually
localized area of living tissue).
Shick test
General method of toxicity test.

For sera and vaccines:- Unless otherwise prescribed in the individual monograph inject intra –
peritoneally one human dose but not more than 1.0 ml into each of five healthy mice , weighing
17 to 22 g and , one human dose but not more than 5.0 ml into each of two healthy guinea pigs
weighing 250 to 350 g.
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The human dose is that stated on the label or in the accompanying information leaf
let of the preparation under examination .The preparation passes the test if none of the
animals dies or shows signs of ill health in 7 days following the injection.
If more than one animal dies, the preparation fails the test . If one of the animals die or show signs of
ill health , repeat the test . The preparation passes the test if none of the animals in the second group
dies or show signs of ill health in the time interval specified . Carry out the test also on two healthy
guinea pigs weighing 250 gm to 350 gm . Inject intra-peritoneally into each animal one human dose
but not more than 5.0 ml. The human dose is that stated on the label or in the accompanying
information leaf let of the preparation under examination .
Observe the animals for 7 days. If one of the animals die or show signs of ill health, repeat
the test .The preparation passes the test if none of the animals in the second group dies or
show signs of ill health in the time interval specified. If more than one animal dies , the
preparation fails the test.
2.Sterility Tests.( this is same as the sterility test for bacteria).

Sterility tests shall be performed when ever specified in the requirements for individual
products. Test for sterility are often performed advantageously at various
stages of manufacture, in addition to those given in requirements.
Sampling: The specified number of suitable samples shall be taken from the product.
Such samples shall be taken at least from each final bulk ,as well as from each final lot.
Sterility test for bacteria and fungi.
1. Membrane Filtration .
2. Direct Inoculation .
Culture Medium .
The culture media used for sterility tests for bacteria and fungi shall be those approved by
the national control authority.
Such media shall have been shown to be capable of supporting the growth of a wide
variety of microorganisms with both aerobic and anaerobic growth characteristics .
Medium Incubation
Fluid Thioglycolate Temp(0 C) Condition
30-35 Aerobic
Alternative Thioglycolate 30-35 Anaerobic
Soyabean Casein Digest 20-25 Aerobic
Performance of the test: Prior to conducting a sterility test on any product , it shall have
been determined whether or not the material to be tested itself has the property of killing or
inhibiting the growth of microorganism or contains preservatives or other substances that
have this effect. If such an effect is shown, the sterility test shall be made using a
suitable procedure to counter act the effect.
Inoculum: For sterility test of bulk material, at least 5ml must be used for inoculation into each
culture medium and for each temperature of incubation.
For sterility testing of a final lot from each of the final containers that contain not more than
20ml, an amount of at least 1.0ml shall be inoculated into each culture medium for each
temperature of incubation.
If the volume in each final container is less than 1.0ml, the amount to be inoculated shall be
the entire content of the container for each culture medium and for each temperature of

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incubation.
Incubation : All vessels shall be incubated at the appropriate temperature for the media. The
temperature selected shall be those approved by the national control authority and shall
include 20-250C and 30 -350C.
All vessels shall be incubated for a period of at least 14 days and shall be examined at
regular intervals and on the last day of incubation for evidence of microbial growth.
Membrane filtration of test samples.
Membrane Filtration :-
The membrane filtration method is used for avoiding and

also overcoming the activity of samples for which practically little inactivating agents
exist.
Following conditions should be fulfilled for the membrane filtration method:
1) An exceptional skilled and knowledgeable operators .

2) Rigorous routine usage of positive and negative controls .
Solution of the product under analysis is filtered by membranefilter
which effectively retains the contaminating microbes. Place the membrane in
suitable medium and allow to incubate in the specified temperature.

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Interpretation of test result.

If no evidence of growth is found in any of the vessels inoculated for the test for sterility in
the bulk material of final lot whichever is applicable meets the requirements for this test.
If evidence of growth is found, the preparation tested fails to meet the requirements for the
test for sterility, unless it can be demonstrated to the satisfaction of the national control
authority by retests or by other means .
Sterility test for mycoplasmas (Mycoplasma is a genus of bacteria that lack a cell wall
around their cell membranes. This characteristic make them naturally resistant to antibiotics
that target cell wall synthesis.) for viral vaccines made by growing the virus in animal
tissues or cell cultures , sterility tests for mycoplasmas of virus culture fluid and control
fluid specified for the particular product shall be made by a method approved by the
national control authority.
The sterility tests for the detection of extraneous viruses in products where their presence is

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unacceptable shall be performed as specified in the requirements for the individual products.
The Traditional Vaccine Potency Tests Method.
To evaluate the quality of a vaccine, the potency test of the final product must strictly
enforce the standards of efficacy established by national regulatory authorities.
The universal method for testing the efficacy of vaccines is the animal challenge test.
Inoculate the same type of animal with the recommended immunological dose to the prescribe day of
age after the optimal immune response period, and compare the immunized animal and the control
animal at the same time are capable of causing the animal's pathogenesis, to measure the ability of the
vaccine, and to reduce the incidence of the disease after a certain time according to the protection of
the immunized animal.
The Alternative Methods of Vaccine Potency Tests
The alternative methods include experimental animal substitution and serological

substitution. The first method is to use other animals with similar clinical symptoms to
replace the original animals, and indirectly test the efficacy of this animal vaccine. As for
serological method, the efficacy of vaccine is assessed by detecting the antibody titer level
after immunization. It involves viruses neutralizing experiments and all kinds of ELISA.s
Immunization schedule :
WHO immunization Program.
Following the successful global eradication of small pox in 1975 through effective
vaccination programs and strengthened surveillance, the Expanded programme on
immunizaion (EPI) was launched in India in 1978 to control other vaccine
preventable diseases .
Initially, six were selected ; diphtheria , pertussis, tetanus , poliomyelitis , typhoid
and childhood tuberculosis.The aim was to cover 80% of all infants . Subsequently ,
the programme was universalized and renamed as Universal Immunization Program
(UIP) in 1985.
Measels vaccine was included in the program and typhoid vaccine was
discontinued.
The UIP was introduced in a phased manner from 1985 to cover all districts in the
country by 1990,targeting all infants with the primary immunization schedule and
all pregnant women with tetanus toxoid immunizaion.
In 1992 , the UIP became a part of the Child Survival and Safe Motherhood
Programme (CSSM) , and in 1997, it became an important component of the
Reproductive and Child Health Programme (RCH).
The Polio Eradication Programme was set up with the assistance of the National
Polio Surveillance Project.
Universal Immunization schedule in India.
Primary Vaccination. Route of

administration.

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At Birth BCG Intradermal

OPV-zero dose Oral
6 Weeks OPV- 1 st dose Oral

DPT- 1 st dose Intramuscular
10 Weeks OPV-2 nd dose Oral

DPT-2 nd dose Intramuscular
14 Weeks OPV- 3 rd dose Oral

DPV-3 rd dose Intramuscular
9-12 months Measles Vaccine Subcutaneous
Booster doses
16-24 months OPV Oral

DPT Intramuscular
5 years DT Intramuscular
10 years TT Intramuscular
16 years TT Intramuscular
Pregnant Women
Gestational period
As early as possible during TT -1 st dose Intramuscular

Pregnancy( first contact)
1 month after 1st dose TT- 2nd dose Intramuscular
If previously vaccinated TT- booster Intramuscular

Within 3 years.
BCG- Bacille Calmette- Guerin, OPV- Oral polio vaccine , DPT- Diphtheria, pertussis, tetanus
EPI’s Immunization Schedule
Age Disease Vaccine
At birth Tuberculosis , polio BCG, OPV
6 weeks Diphtheria, pertussis, tetanus , polio DPT-1, OPV-1
9 months Measles Measles vaccine

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Q.What is antiserum
Antiserum, blood serum that contains specific antibodies against an infective organism or
poisonous substance. Antiserums are produced in animals (e.g., horse, sheep, ox, rabbit).
Antiserum is human or nonhuman blood serum containing monoclonal or polyclonal
antibodies that is used to spread passive immunity to many diseases via blood donation.
Antibody containing preparations ( Sera) :-

Q. What is anti toxin – 2 marks.
The plasma of an immune person or animal contains a large number of antibodies which
, if the blood is withdrawn and allowed to clot , are found to contain antitoxic, antibacterial
or antiviral antibodies and accordingly , is called an antitoxic , antibacterial or antiviral
serum . Antitoxic sera , however , are more often known as antitoxins.
Storage of Immunological Products .
Preservation of the potency of immunological products involves maintaining the viability of
living cells or preventing the denaturation of proteins ( ie. antigens or antibodies).
The causes of denaturation and loss of viability are largely chemical and , since the rate
of a chemical reaction is influenced by temperature , the storage at low temperature is
essential.
For most products the optimum storage temperature is between 2 and 100C is directed for
most vaccines, all antitoxins and the tuberculins.
There is an important difference between the storage of bacterial vaccines and antitoxins.
A number of viral vaccines eg. small pox and oral poliomyelitis are more stable at or
below their freezing points, serious deterioration results if bacterial vaccines or
antitoxins are allowed to freeze .
This may be due to mechanical damage by ice crystals or the adverse effects of high ice
concentrations of inorganic salts and preservative bactericides .
Q. Differentiate between killed and live vaccine ?
Live vaccines ,as the name suggests , contain live attenuated organisms that have lost their
pathogenicity but have antigenicity.
The attenuated organisms are the suspensions of live organism with reduced virulence.
These organisms multiply in the body and there by provide a continuous antigenic stimulus,
resulting in production of protective antibodies.
Bacille Calmette –Guerin (BCG). Small pox vaccine, oral polio vaccine (OVP), mumps,
measles and rubella (MMR) vaccine and yellow fever vaccine are some of the examples of
live vaccines.
Killed Inactivated Vaccines- These vaccines contain killed pathogens , hence do not
replicate in body. At least three doses of killed vaccines followed by a booster dose are
essential to confer protective immunity.
Typhoid, cholera , pertussis, pneumococcal , rabies, hepatitis B and influenza vaccines are
the examples of killed attenuated vaccines.
Q. What are Toxoids?
Toxoids are modified toxins, which have retained their antigenicity but have lost their
toxicity.
Adsorption of toxoid to a mineral carrier, such as aluminium hydroxide or aluminium
phosphate enhances antigenicity of toxoids.
This is because these adsorbed toxoids remain longer in a depot after injection and
continue to stimulate immune system of host for a longer period of time .
These are usually prepared by treating toxins with formalin. Toxoids are given to confer protection
against diseases that are caused by production of toxins .

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Two toxoids most widelyused for immunization are tetanus toxoids and diphtheria
toxoids. DPT ( diphtheria , pertussis, tetanus) vaccine is a triple vaccine ,which consists
of tetanus and diphtheria toxoids and pertussis killed vaccine.
Q.Enumerate the differences between endotoxin and exotoxin? ( 5 marks)

Toxin produced by bacteria are generally classified into two groups : exotoxins and endotoxins
Exotoxins : these are heat labile proteins that are produced by several gram positive and gram
negative bacteria . These are bacterial products ,which are secreted into tissues and directly harm
tissues or trigger destructive biological activities. The genes coding for these proteins are frequently
encoded on plasmid or on bacteriophage DNA. Some important toxins encoded by plasmids are
tetanus toxin of C. tetani and heat labile and heat stable toxins of enterotoxigenic E.coli.
Toxins encoded by bacteriophage DNA are cholera toxins , diphtheria toxins and botulism toxin.
Endotoxins :
The endotoxins are the class of toxic substances released after lysis of bacteria from the toxic
substances ( exotoxins ) secreted by bacteria .They are produced by Gram negative bacteria ,
but not by gram positive bacteria . They are lipopolysaccharide (LPS) components of the outer
membrane of gram negative bacteria . These forms an integral part of the cell wall unlike
exotoxins, which are actively released from the cells.
The genes that encode the enzymes that produce the LPS are present on the bacterial
chromosome , but not on plasmids or bacteriophage DNA, which usually encodes the exotoxins
. They are heat stable , and they are released from the bacterial cell surface by disintegration of
the cell wall. They are weakly antigenic and do not induce or poorly induce protective
antibodies . Hence their action is not neutralized by the protective antibodies.
Chapter 8 Diagnostic Tests. Schick’s Test, Elisa Test, Western Blot

Test, Southern Blot , PCR Widal, QBC, Mantaux Peripheral
smear, Study of malarial parasites.
Polymerase chain reaction (PCR technique) .

PCR is a way to make many copies of a sequence of DNA (this is sometimes called 'amplifying' the
DNA). It is done in a lab, using an enzyme called DNA polymerase.
It is called chain reaction because the result of one cycle is used immediately for the next cycle.
It is a method used widely in molecular biology to make millions to billions of copies of a specific
DNA sample rapidly, allowing scientists to take a very small sample of DNA and amplify it to a

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large enough amount to study in detail.
PCR requirements.
1. A target DNA.
In general the shorter the sequence of target DNA, the better the efficiency of the PCR.
2.Primers (which are short single strand DNA fragments known as oligonucleotides that are a
complementary sequence to the target DNA region).
3.Nucleotides.
4.DNA polymerase enzyme.
Taq polymerase preferred as it can withstand high temperature. Taq DNA polymerase is the most
common enzyme used for PCR amplification.This enzyme is extremely heat resistant with a half-life
of 40 minutes at 95°C. which is obtained from Thermus aquaticus a thermophilic organism.
PCR involve repeated cycle for amplification (amplification methods can create millions of identical
copies of a DNA )or RNA "target" sequence in a matter of hours) of target DNA .
Each cycle has three stages.
Denaturation.
It is the first step of PCR, the two strands of the DNA double helix are physically separated into
two strands at a high temperature.
Renaturation or annealing .
In the second step, the temperature is lowered and the primers bind to the complementary
sequences of DNA.The two DNA strands then become templates for DNA polymerase to
enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA.
Synthesis (extension).
The initiation of DNA synthesis occurs at 3’hydroxyl end of each primer. The primers extended by
joining the bases complementary to DNA strands. The synthetic process in PCR in similar to the
DNA replication.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an
enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the polymerase
used was heat-susceptible, it would denature under the high temperatures of the denaturation step.
Before the use of Taq polymerase, DNA polymerase had to be manually added every cycle, which
was a tedious and costly process. After the first cycle, there are 4 DNA strands. The process repeats
with the 4 DNA strands, which will go on to make 8 strands, then repeat it again to make 16
strands.In this way, PCR doubles the amount of DNA in a sample after each cycle, making it
possible to obtain millions of copies of a DNA strand overnight.

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1.The DNA double helix is melted apart at Temperature 94- 96 0C and its strands separate .
2.The temperature is decreased to slightly below the Temperature of both the

primers being used . Both primers bind to the available strands . These primers are supplied in
excess to ensure that the strands do not only come back and reanneal to one another .
3.Polymerization ( extension ) occurs via DNA Polymerase in the 5’ to 3’ direction on each

strand.
4.Incorporated additional nucleotides give rise to new strands that extend past the sequence of
interest .
5. The previously polymerized strands acts as template for the other primer ( if forward primer
bound first , reverse primer now binds and vice versa ).
6. Polymerization occurs via DNA polymerase in the 5’ to 3’ direction on each strand, this
time ending at the end of the sequence of interest .
7.Incorporated additional nucleotides give rise to new strands that only encode the sequence of
interest .
8.The synthesized strands encoding the sequence of interest anneal to one another to form the
end product.
Simple version of PCR
Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer, a
short sequence of nucleotides that provides a starting point for DNA synthesis. In a PCR reaction,
the experimenter determines the region of DNA that will be copied, or amplified, by the primers she
or he chooses. PCR primers are short pieces of single-stranded DNA, usually around
20 nucleotides in length. Two primers are used in each PCR reaction, and they are designed so that
they flank the target region (region that should be copied).
That is, they are given sequences that will make them bind to opposite strands of the template DNA,
just at the edges of the region to be copied.

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The primers bind to the template by complementary base pairing.
Template DNA:
When the primers are bound to the template, they can be extended by the polymerase, and the region
that lies between them will get copied.
The steps of PCR.
The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides
(DNA building blocks).
The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put
through repeated cycles of heating and cooling that allow DNA to be synthesized.
The basic steps are:
1. Denaturation (94 to 96°): Heat the reaction strongly to separate, or denature, the DNA
strands. This provides single-stranded template for the next step.
2. Annealing (68°C): Cool the reaction so the primers can bind to their complementary sequences
on the single-stranded template DNA.
3. Extension (72 °C): Raise the reaction temperatures so Taq polymerase extends the primers,
synthesizing new strands of DNA.

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Result after 1 cycle of DNA molecule doubled
This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours,
depending on the length of the DNA region being copied.
If the reaction is efficient (works well), the target region can go from just one or a few copies to
billions.
That’s because it’s not just the original DNA that’s used as a template each time.
Instead, the new DNA that’s made in one round can serve as a template in the next round of DNA
synthesis.
There are many copies of the primers and many molecules of Taq polymerase floating around in the
reaction, so the number of DNA molecules can roughly double in each round of cycling.
This pattern of exponential growth is shown in the image below.

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Application of PCR technique.
1.PCR in clinical diagnosis: PCR allows isolation of DNA fragments from genomic DNA by
selective amplification of a specific region of DNA. PCR supplies these techniques with high
amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting
material.
2.Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to study these
mutations, therapy regimens can sometimes be individually customized to a patient. PCR permits
early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the
highest-developed in cancer research and is already being used routinely.
3.Detection of HIV: Infections can be detected earlier, donated blood can be screened directly for the
virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can
be quantified.
4.Some disease organisms, such as that for tuberculosis, are difficult to sample from patients and
slow to be grown in the laboratory. PCR-based tests have allowed detection of small numbers of
disease organisms (both live or dead), in convenient samples.
Detailed genetic analysis can also be used to detect antibiotic resistance, allowing immediate and
effective therapy. The effects of therapy can also be immediately evaluated.
5.The spread of a disease organism through populations of domestic or wild animals can be
monitored by PCR testing. In many cases, the appearance of new virulent sub- types can be
detected and monitored. The sub-types of an organism that were responsible for earlier epidemics
can also be determined by PCR analysis.
6.Viral DNA can be detected by PCR. The primers used must be specific to the targeted sequences
in the DNA of a virus, and PCR can be used for diagnostic analyses or DNA sequencing of the
viral genome.The high sensitivity of PCR permits virus detection soon after infection and even
before the onset of disease. Such early detection may give physicians a significant lead time in
treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA
quantitation techniques.
7.PCR in forensic medicine: a single molecule of DNA from any source ( Blood stain, hair, semen
etc) of an individual is adequate for amplification by PCR. Thus, PCR is very important for
identification of criminals.
Immunoblotting
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A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA onto a
carrier (for example, a nitrocellulose, polyvinylidene fluoride or nylon membrane).
In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel
onto the blotting membrane, and other times adding the samples directly onto the membrane.
After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining
(for example, silver staining of proteins), autoradiographic visualization of radiolabelled
molecules (performed before the blot), or specific labelling of some proteins or nucleic acids.
The latter is done with antibodies or hybridization probes that bind only to some molecules of the
blot and have an enzyme joined to them.
After proper washing, this enzymatic activity (and so, the molecules we search in the blot) is
visualized by incubation with proper reactive, rendering either a colored deposit on the blot or a
chemiluminescent reaction which is registered by photographic film.
Southern blotting.
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA
sequence in DNA samples.
Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter
membrane and subsequent fragment detection by probe hybridization.
Western blot.
A western blot is used for the detection of specific proteins in complex samples. Proteins are first
separated by size using electrophoresis before being transferred to an appropriate blotting matrix
(usually polyvinyldiene fluoride or nitrocellulose) and subsequent detection with antibodies.
Northern blotting.
The northern blot is for the detection of specific RNA sequences in complex samples.Northern
blotting first separates samples by size via gel electrophoresis before they are transferred to a blotting
matrix and detected with labelled RNA probes.
Application of blotting
1.Blotting is used for separation and identification of DNA, RNA or protein.
2.Used in scientific research.
3.Used in diagnostic practice.

4.Used for identification of abnormal genes.
5.Used in clinical research.
6.Used in genetic counselling (Genetic counseling provides information and support to people who
have, or may be at risk for, genetic disorders).
7.Used in molecular biology.
8.It is a semi quantitative or quantitative technique for estimation of RNA, DNA or protein.

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Western Blotting
Principles of western blotting for identification of antibodies or antigens.
1.Avirus-containing sample is digested with the anionic detergent sodium dodecylsulfate (SDS) and
electrophoresed on a polyacrylamide slab gel (PAGE), which separates the different viral proteins
according to Mr. (2)
2.The bands of viral protein are then transferred (“blotted”) onto a nitrocellulose membrane by
capillary transfer or usually by electrophoresis in a different plane to immobilize the polypeptides.
3.The unoccupied areas of the membrane are blocked (“quenched”) by saturation with a suitable
protein, then washed, dried, and cut into strips which can be used to test individual patient’s sera.
4.Each test serum or plasma (“primary” antibody) is then incubated with one strip to enable
antibodies to bind to the individual viral proteins.
5.Following rinsing, bound antibody is detected by the addition of enzyme-labeled antihuman
immunoglobulin (“secondary” antibody).
6.Following another wash, the bands are revealed by the addition of a substrate chosen to produce an
insoluble colored product.
Applications .
The Western blot test is most widely used as a confirmatory test for diagnosis of HIV ,

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where this procedure is used to determine whether the patient has antibodies that react with
one or more viral proteins or not.
The western blotting is also used for the demonstration of specific antibodies in the serum
for diagnosis of tubercular meningitis.
Southern Blotting .
Principle:
Southern blotting is an example of RFLP (restriction fragment length polymorphism).It was

developed by Edward M. Southern (1975). Southern blotting is a hybridization technique for
identification of particular size of DNA from the mixture of other similar molecules.
This technique is based on the principle of separation of DNA fragments by gel electrophoresis and
identified by labelled probe hybridization. Basically, the DNA fragments are separated on the
basis of size and charge during electrophoresis.
Separated DNA fragments after transferring on nylon membrane, the desired DNA is
detected using specific DNA probe that is complementary to the desired DNA. A hybridization
probe is a short (100-500bp), single stranded DNA. The probes are labeled with a marker so that
they can be detected after hybridization.
Procedure/ Steps
1.Restriction digest: by RE enzyme and amplification by PCR.
2.Gel electrophoresis: SDS gel electrophoresis.
3.Denaturation: Treating with HCl and NaOH.
4.Blotting.
5.Baking and Blocking with casein in BSA (Bovine serum albumin).
6.Hybridization using labelled probes.
7.Visualization by autoradiogram.

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Step I: Restriction digest
The DNA is fragmentized by using suitable restriction enzyme. RE(Restriction endonuclease)

cuts the DNA at specific site generating fragments. The number of fragments of DNA obtained by
restriction digest is amplified by PCR .
Step II: Gel electrophoresis
The desired DNA fragments is separated by gel electrophoresis
Step III: Denaturation
The SDS gel after electrophoresis is then soaked in alkali (NaOH) or acid (HCl) to denature
the double stranded DNA fragments. DNA strands get separated
Step IV: Blotting
The separated strands of DNA is then transferred to positively charged membrane nylon
membrane (Nitrocellulose paper) by the process of blotting.
Step V: Baking and blocking
After the DNA of interest bound on the membrane, it is baked on autoclave to fix in the
membrane.
The membrane is then treated with casein in Bovine serum albumin (BSA) which saturates all
the binding site of membrane.

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Step VI: Hybridization with labelled probes.

The DNA bound to membrane is then treated with labelled probe.The labelled probe contains the
complementary sequences to the gene of interest.The probe bind with complementary DNA on
the membrane since all other nonspecific binding site on the membrane has been blocked by BSA
or casein.
Step VII: Visualization by Autoradiogram.

The membrane bound DNA labelled with probe can be visualized under
autoradiogram which give pattern of bands.
Application of Southern blotting:
1.Southern blotting technique is used to detect DNA in given sample.
2.DNA finger printing is an example of southern blotting.
3.Used for paternity testing, criminal identification, victim identification.
4.To isolate and identify desire gene of interest.
5.Used in restriction fragment length polymorphism.
6.To identify mutation or gene rearrangement in the sequence of DNA.
7.Used in diagnosis of disease caused by genetic defects.
8.Used to identify infectious agents
Chapter 9
Microbial culture sensitivity Testing: Interpretation of results. Principles and

methods of different microbiological assays , Microbiological assays of

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Penicillin, Streptomycin and Vitamin B2 and B12. Standardization of vaccines

and sera.
Microbiological Assays.
Introduction :- A microbiological assay may be defined as qualitative or quantitative determination

of any chemical compound from a simple or even complex material with the use of
microorganisms. It is necessary to assay antimicrobial agents for determination of potency , for
determining the pharmacokinetics of a drug in animals or man and for monitoring and controlling
antimicrobial chemotherapy . Quantitative chemical or physical methods can assay most of the
currently employed therapeutic agents. Many therapeutic agents, which either inhibit the growth of
microorganisms (antibiotics ) or are essential for their growth ( vitamins and amino acids ) can be
standardised by microbiological assays.
Many therapeutic agents which either inhibit the growth of microorganisms ( antibiotics) or are
essential for their growth( Vitamins and Amino acids) can be standardized by microbiological assay.
Microbiological assays measure the activity of antibiotics ( extent of ability to inhibit the growth of
microorganisms) or vitamins or amino acids ( extent of ability to support the growth of
microorganisms) where as chemical assays of such substances estimate only their
potency( concentration or the amount).Hence microbiological assays are of greater value in case of
antibiotics , amino acids and vitamins . Trace elements in soil samples and biological materials can
be estimated with the help of Aspergillus niger. As with all bioassays the validity of the method
should be confirmed and the error of the result should invariably be indicated.
Microbiological Assay of Antibiotics:- The inhibition of microbial growth under standardised

conditions may be utilized for demonstrating the therapeutic efficacy of antibiotics. The
microbiological assay is based upon a comparison of the inhibition of growth of microorganisms
by measured concentration of the antibiotics to be examined with that produced by known
concentration of a standard preparation of the antibiotic having a known activity. The
microbiological assay is based upon a comparison of the inhibition of growth of bacteria by
measured concentration of the antibiotic under test with that produced by known concentration of
a standard preparation of the antibiotic having a known activity.
The microbiological assays of antibiotics are based on either i) Serial dilution method or ii)
diffusion method . These methods are also employed in the evaluation of disinfectants.
Serial Dilution Method:- The dilution of the antibiotic under test that will inhibit the growth of a
susceptible organism is compared with the dilution of a standard preparation that has identical effect
. From knowledge of the potency of the standard , the strength of the unknown can be calculated .
Dilution in broth is most suitable but in some cases dilution in nutrient agar may be more
satisfactory.
Diffusion method : This method is preferred over serial dilution method because of the ease with
which quantitative results can be obtained. However the method cannot be used when the antibiotics
does not diffuse freely due to adsorption or incompatibility with the medium.
Dilutions of the antibiotic under test and the standard antibiotic preparation are made in geometric
proportions. Plates are prepared by pouring agar, seeding them with test organism, and allowing to
set on a perfectly horizontal surface so that the agar occupies a constant depth throughout the dish.
The test organism may be mixed with the agar before pouring or may be applied to the surface of the
medium, which is already set.
Antibiotic solutions are applied in :-

1. Cups cut in medium using a sterile cork borer about 10mm in diameter . The cut agar disc is
removed by a vacuum device .

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2. Cylinders of stainless steel, glazed porcelain , Pyrex glass or sterilizable plastic having an
external diameter of about 8 mm and a height of about 10 mm. These are slightly warmed
so that they sink to a constant depth when placed on the agar.
3. Filter paper or cellulose discs , which absorb a fixed volume of solution .
4. Standard ceramic insulation beads ( also known as fish spine beads) which attract a definite
volume when touched on the surface of the solution .The surface of the agar medium must
be dry if this method is to be used.
Plates are left at room temperature for 2 hours to allow diffusion of the antibiotic solution through
out the medium to get ahead of growth of the organism.
After incubation , inhibition of growth can be observed as a clear zone of inhibition around each
plate. The diameter of this zone of inhibition is proportional to the log of the concentration of
antibiotic. The diameters of zone of inhibition are best measured in an ‘ antibiotic zone reader’
where in an optical system projects an image of the plate on to a large grid . Two diameters at
right angles are used as a check on ellipticity( the degree of deviation from a circle or a sphere
shape) of the zone.
A standard curve is plotted between the log concentration of the standard against zone of
inhibition, and the log concentration of the test against zone of inhibition is also plotted on the
same graph. If the two lines are parallel, the relative potencies of the standard and test are
represented by horizontal distance between the two lines. Similar results can also be obtained by
calculation.
Official Assay Methods
Pharmacopoeia generally recommends two methods for antibiotics assay: the cylinder plate ( Cup
plate ) method and the turbidimetric ( or tube assay ) method.
The following considerations are common to the two methods of microbiological assays of
antibiotics :-
Standard preparation and units of activity: A standard preparation is an authentic sample of the
appropriate antibiotic for which the potency has been precisely determined by reference to the
appropriate international standard . The potency of the standard preparation may be expressed in
International Units or µg per mg of the pure antibiotic.
A Description of the standard preparation of the antibiotics and their activity is given in table below.
Standard Preparation
Antibiotic Standard preparation mg containing 1 Unit
Amphotericin Amphotericin B 0.001064
Ampicillin Ampicillin 0.0011764
Bacitracin Zinc Bacitracin 0.01351
Cephalexin Cephalexin 0.0010707
Chloramphenicol Chloramphenicol 0.001
Doxycycline Doxycycline Hcl 0.0011494
hemiethanolate,
hemihydrates
Penicillin Benzyl Penicillin 0.0005988
Streptomycin Streptomycin sulphate 0.001282
Test Organisms and Assay conditions for common Antibiotics

Antibiotic Test Organism Assay Medium Incubation
Method Temp0C
Ampicillin Micrococcus luteus A D 32-35

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Chloramphenicol Escherichia coli B C 35-37

Doxycycline Staphylococcus aureus B C 35-37
Erythromycin Micrococcus luteus A D 32-35
Oxytetracycline Bacillus cereus Var A F 32-35
Mycoides
Rifampicin Bacillus subtilis A B 32-35
Streptomycin Bacillus subtilis A B 32-35
sulphate
Tetracycline Bacillus cereus A F 32-35
Tetracycline Staphylococcus aureus B C 35-37
Pecillin( Benzyl Staphyo coccus aureus B C 35-37
Pencillin)
Method A- Cylindrical plate method, Method B- Turbidimetic method
Composition of Media
Ingredients Media
A B C D E F G H
Peptone 6.0 6.0 5.0 6.0 6.0 6.0 9.4 -
Pancreatic digest of casein 4.0 - - 4.0 - - - 17.0
Yeast extract 3.0 3.0 1.5 3.0 3.0 3.0 4.7 -
Beef extract 1.5 1.5 1.5 1.5 1.5 1.5 2.4 -
Dextrose 1.0 - 1.0 1.0 - - 10.0 2.5
Agar 15.0 15.0 - 15.0 15.0 15.0 23.5 20.0
Sodium chloride - - 3.5 - - - 10.0 5.0
Dipotassium hydrogen phosphate - - 3.68 - - - - 2.5
Potassium dihyrogen phosphate - - 1.32 - - - -
Final pH 6.5 6.5 - 6.95- 7.8- 7.8- 5.8- 6.0- 7.2-7.3
After sterilization -6.6 6.6 7.05 8.0 8.0 6.0 6.2
Apparatus :
All equipments must be thoroughly cleaned before after each use . Glass ware for holding
transferring test organisms are sterilized by dry heat or by steam. For cylinder plate method
Petridishes (20x100 mm) made of glass or plastic , or rectangular glass trays can be used .
Cylinders made of glass , porcelain , aluminium or stainless steel should have outside diameter
6mm and length 10 mm. Alternatively holes of 5 to 10mm diameters may be bored in the medium
with a sterile borer or paper discs of suitable quality paper may be used .
For turbidometric method either glass or plastic test tubes of uniform length , diameter or
thickness
( 16mmx125 mm or 18 mmx150mm ) can be used and must be substantially free from surface
scratches.
Method A – Cylinder Plate Method :

This method depends on the diffusion of an antibiotic from a vertical cylinder or a cavity , through
the solidified agar layer of a Petri dish or plate , to an extent such that growth of the added
microorganism is prevented entirely in a circular area or zone around the cylinder or cavity
containing a solution of the antibiotic.

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A previously liquefied medium , appropriate to the assay , is inoculated with the requisite quantity
of suspension of the microorganism, the suspension is added to the medium at a temperature
between 40 to 50 0 C and the inoculated medium is poured immediately into petri dishes or large
rectangular plates
to occupy a depth of 3 to 4 mm.The prepared plates or dishes must be stored in such a way that no
significant growth or death of the test microorganism occurs before use , and the surface of the
agar layer is dry at the time of use.
Solutions of known concentrations of the Standard preparation and the test antibiotic are prepared
in appropriate buffer solutions. These solutions are applied to the surface of the solid medium in
sterile
Cylinders or in cavities prepared in the agar .The volume of solution added to each cylinder or
cavity must be uniform and sufficient to fill the holes. When paper discs are used these should be
sterilized by exposure of both sides under a sterilizing lamp and then impregnated with the
solutions and placed on the surface of the medium.
The dishes or plates are left standing for 1 to 4 hours , at room temperature or at 4 0C , as a period
of pre-incubation diffusion to minimize the effects of variation in time between the application of
different solutions . They are then incubated for about 18 hours at about 37 0C and the diameter or
areas of the circular inhibition zones are measured.
Test organisms for microbiological assay of antibiotics

Antibiotics Test Organism ATCC No
Chlortetracycline Bacillus pumilus 14884
Gentamicin Staphylococcus epidermidis 12228
Kanamycin sulphate Bacillus pumilus 14884
Penicillin (Benzyl penicillin) Staphyo coccus aureus 6538
Streptomycin Bacillus subtilis 6633
Tetracycline Bacillus cereus 11778
Comparison of assay of Penicillin and Streptomycin
Properties Penicillin Streptomycin

Form Crystalline sodium salt of Streptomycin sulphate
benzyl penicillin
Units/ mg 1670 780
Potency in units /ml 0.5- 8 5-20
Mg containing 1 unit 0.0005988 0.001282
Units for standard 1,2,3,4,5 and6 10,12,14,16,18 and 20

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Dissolve 35 mg in 100ml.
Preparation of anti-biotic Take 1ml from above Dissolve 256 mg in
Solution solution and dilute to 100ml. 100ml.Take 1ml from the
Each ml of this contains 6 unit above solution and dilute to
activity. 100ml .Each ml of this
contains 20 units of activity.
1ml or as per size of the
Amount to be added in
cavity. 1ml or as per size of the
quantity
cavity.
The volume of solution added to each cavity or cylinder must be uniform and sufficient to fill the
holes. When paper discs are used , these discs should be sterilized first and then dipped in the
standard solutions or the test solutions and placed on the surface of the medium.
The plates are left standing for 1 to 2 hours at room temperature or at 4 0c, as a period of pre
incubation diffusion to minimize the effects of variation in time between the application of the
different solutions . All plates are then incubated for about 18 to 24 hours at a temperature
indicated in table. The diameters or areas of the circular inhibition zones produced by standard
and test antibiotic solutions are accurately measured .
The graph which relates zone diameter to the logarithm of the concentration of antibiotic s is
plotted and the unknown concentration of test antibiotics is calculated.
Standard preparation for Penicillin Assay
Stock solution - Dissolve 35 mg of Benzyl penicillin in 100ml.
Working Standard Solutions-Take 1ml from the stock solution and dilute to 100ml.
Working standards – 1ml,2ml , 3ml
Zone of Inhibition observed in plate ( Assay of Streptomycin)

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Method B-Turbidimetric or tube assay method :

This method depends up on the growth of a microbial culture in a uniform solution of the antibiotic
in a fluid medium that is favourable to its rapid growth in the absence of an antibiotic . The
method has the advantage of a shorter incubation period for the growth of the test organism ( 3 to
4 hours ). However, the presence of solvent residues or other inhibitory substances affects this
assay more than the cylinder plate assay. This method is not recommended for cloudy or turbid
preparations .
Five different concentrations of the standard solution are prepared by diluting the stock solution
for making the standard curve .A medium concentration is selected and the test sample of
antibiotic solution is adjusted by dilution to obtain approximately this concentration . one ml of
each concentration of the standard solution and of the sample solution are placed in each of the
tubes in duplicate .To each tube 9ml of nutrient medium previously seeded with the appropriate
test organism , is added.
Three control tubes are also prepared simultaneously , one containing the inoculated culture
( Culture control ) , another identical with it but treated immediately with 0.5ml of dilute
formaldehyde solution (blank) and a third containing uninoculated culture medium.
All the tubes are placed randomly distributed , in an incubator and specified temperature ( about
370C) is maintained for 3 to 4 hours . After incubation , 0.5ml of dilute formaldehyde solution is
added to each tube. The growth of the test organism is measured by determining the extinction of
each of the solution in the tubes against the blank , at 530 nm.
Microbiological assay of antibiotics by tube assay method.
Microbiological assay of vitamins :-
Vitamins are important growth factors needed for growth and multiplication of microorganisms.

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They are very sensitive to small amounts of growth factors . It is the ability of these test
microorganisms to synthesis the factor being assayed that forms the basis of the microbiological
assay of vitamins and amino acids .
Test microorganisms used for assaying the water soluble vitamins are listed in the table.
Vitamin Test organism Incubation Temp.(0C) Assay pH

Vitamin B12 Lactobacillus leichamanii 37 6.1
Vitamin B6 Saccharomyces uvarum 30 4.5
Riboflavin (B2) Lactobacillus casei 37 6.8
Thiamine Lactobacillus viridescens 30 6.0
Biotin Lactobacillus plantarum 37 6.8
Niacin Lactobacillus plantarum 37 6.8
Pantothenate Lactobacillus plantarum 37 6.7
Microbiological assay of Cyanocobalamin ( Vitamin B12)
Microbiological assay of Cyanocobalamin may be performed by the following methods.

a) Titrimetric method
b) Turbidimetric method.
Titrimetric method:-
Procedure:- Take clean test tubes and add 0.0ml,0.5ml, 1.0ml,1.5ml,2.0ml 2.5ml, 3.0ml,4.0ml,4.5ml
and 5.0ml respectively of standard Cyanocobalamin solution (0.01- 0.04µg/ml). To each test tube
add 5ml Basal medium stock solution. Adjust the final volume ( 10 ml ) by using water. In the
other four test tubes add 1.0ml, 2.0 ml , 3.0 ml 4.0ml respectively of the test solution to be assayed.
To each test tube add 5ml Basal medium stock solution and adjust the final volume ( 10 ml ) by
using water.
Sterilize all test tubes in autoclave at 121 0C for 5 minutes. After sterilization, cool all test tubes up to
room temperature and inoculate with one loopful of organism Lactobacillus leichamanii. Incubate
the tubes for 64 to 72 hours at 370C.
Procedure for microbiological assay of Vitamin B12 by ( Titrimetric method)
Tube Number Std. Cyanocobalamin solution Basal medium stock Volume of sterile water
solution (ml) (ml)
1 0.0 5 5.0
2 0.5 5 4.5
3 1.0 5 4.0
4 1.5 5 3.5
5 2.0 5 3.0
6 2.5 5 2.5
7 3.0 5 2.0
8 4.0 5 1.0
9 4.5 5 0.5
10 5.0 5 0.0
1’ 1.0( Test solution) 5 4.0

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Titrate the contents of each tube with0.05N NaOH, electrometrically or using 0.1%w/v bromothymol
blue as an indicator( converts to green colour). Determine the average of the titration values for each
level of standard and test samples used. Plot the graph by considering the average titration values
expressed in ml of 0.05 N NaOH against the corresponding levels of standard Cyanocobalamin
solution added. Draw a smooth curve and determine the concentration as activity per ml of test
solution by interpolation of Vitamin B12 activity.
Assay of Vitamin B12

Standard cyanocobalamin stock solution : -0.1 µg/ml in 25% alcohol. Store in a refrigerator for not
more than two months.
Basal Medium Stock Solution :- Composition of Basal Medium Stock Solution is as follows.
Name of Ingredient Quantity
L-cystine 0.1gm
DL-Tryptophan 0.1gm
1NHCl 10ml
Adenine-Guanine-Uracil solution 10ml
Xanthine solution 5ml
Riboflavin-Thiamine-Biotin-Nicotinic acid solution 10ml
P-Aminobenzoic acid –Pyridoxine-pyridoxal- 10ml
pyridoxamine solution.
Calcium pantothenate –Folic acid solution 5ml
Salt solution A 5ml
Salt solution B 5ml
Asparagine solution 5ml
Acid-hydrolysed casein solution 25ml
Dextrose 10gm
Sodium acetate 5gm
Ascorbic acid 1gm First dissolve L- cysteine and DL
Sorbitan mono oleate derivative solution 5ml –tryptophan in HCl and add the
other ingredients /reagents in the
order listed in the formula . Dissolve dextrose, sodium acetate and ascorbic acid separately in 100ml
of water and add to the mixture . Adjust the reaction of the medium to pH 6.0 with 1N NaOH .Add
the sorbitan mono-oleate derivative solution and make up the volume to 250ml with distilled water.
Test solution of the material to be assayed : Take accurate amount of the material to be assayed and
dissolve it in water or dilute if necessary .Add dil.Hcl or solution of sodium hydroxide to adjust the
pH(6.0) and add water to make a final volume.

2
Preparation of Inoculum :
Transfer a few cells of Lactobacillus leichamanii from a recent sub culture into two sterile tubes
each containing 10 ml of culture medium ( yeast extract-0.75 gm, peptone-0.75 gm, dextrose -1gm,
potassium dihydrogen phosphate-0.2gm , tomato juice filtrate -10ml, sorbitan mono-oleate derivative
solution -1ml,pH 6.8 and water to make 100ml. Incubate the tubes for 18 to 24 hours at 37 0C. After
incubation centrifuge the culture till the cells settle to the bottom of the tube. Decant off the
supernatant fluid. Repeat this procedure a third time if necessary. Finally suspend the cells
uniformly in 10ml of sterile medium. Aseptically transfer 1ml of the suspension of the cells to
10ml of sterile suspension medium and mix . The resulting cell suspension is the inoculum.
Turbidimetric Method:-
Apparatus reagents and procedure are same as titrimetric method but this test includes two more
test tubes to which neither standard cyanocobalamin solution nor test solution , nor inoculum is
added. Incubate all test tubes at 30 to 370C for 16 to 24 hours.
By using an uninoculated blank tube adjust the transmittance at 640nm to 100% in the photoelectric
colorimeter. Thoroughly mix the contents of each tube and record the transmittance reading. Plot
the graph by considering transmittance value against the corresponding levels of standard
cyanocobalamin solution. Draw a smooth curve and calculate the concentration of the test solution
of cyanocobalamin.
Microbiological Assay of Vitamin B2 Also known as Riboflavin .

Organism used for the test is Lactobacillus casei is used for the assay.
Microbiological assay of Vitamin B2 (Riboflavin) is performed by the following methods.
a) Titrimetric method.
b) Turbidimetric method.
Titrimetric method:-
Procedure:- Take clean test tubes and add 0ml,0.5ml, 1.0ml,1.5ml,2.0ml 2.5ml,
3.0ml,4.0ml,4.5ml and 5.0ml respectively of standard Riboflavin solution .To each test
tube add 5ml Basal medium stock solution. Adjust the final volume (10 ml ) by using
water.
In the other four test tubes add 1.0ml, 2.0 ml , 3.0 ml 4.0ml respectively of the test
solution to be assayed. To each test tube add 5ml Basal medium solution and adjust the
final volume (10 ml ) by using water.Sterilize all test tubes in autoclave at 1210C for 5
minutes.
After sterilization, cool all test tubes up to room temperature and inoculate withone
loopful of organism Lactobacillus casei. Incubate the tubes for 64 to 72 hours at 370C.
Procedure for microbiological assay of Vitamin B2 by ( Titrimetric method)
Tube Std. Riboflavin solution ( Basal medium stock Volume of sterile

Number ml) solution (ml) water (ml)

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1 0.0 5 5.0
2 0.5 5 4.5
3 1.0 5 4.0
4 1.5 5 3.5
5 2.0 5 3.0
6 2.5 5 2.5
7 3.0 5 2.0
8 4.0 5 1.0
9 4.5 5 0.5
10 5.0 5 0.0
Titrate the contents of each tube with0.05N NaOH, electrometrically or using 0.1%w/v
bromothymol blue as an indicator( converts to green colour).Determine the average of the
titration values for each level of standard and test samples used.
Plot the graph by considering the average titration values expressed in ml of 0.05N NaOH against
the corresponding levels of standard Riboflavin solution added.
Draw a smooth curve and determine the concentration as activity per ml of test solution by
interpolation of Vitamin B2 activity.
Assay of Vitamin B2
Stock solution A of Riboflavin : 50 mg . riboflavin , accurately weighed were suspended in

about 350ml of distilled water containing 0.6ml glacial acetic acid .The suspension was
warmed to about 80 0C till the riboflavin dissolved . Then cool the solution and transfer in to
a 500 ml volumetric flask and the volume made up to the mark with distilled water .This
solution was stored under toluene in a dark bottle in the refrigerator .This solution
containing 100 µg , riboflavin per ml can use for a month if preserved in a refrigerator.
Stock solution B of Riboflavin : 10 ml of stock solution A of riboflavin was accurately diluted to

100 ml with distilled water .This was stored under toluene in a dark bottle in the refrigerator .
The solution can use for a week and contains 10 µg riboflavin per ml.
Standard solution of riboflavin : 1ml of stock solution B of riboflavin was accurately
diluted to 100ml with distilled water .This solution containing 0.1 µg riboflavin per ml is
prepared only on the day of use .
Quantity for 100 ml of Basal medium double strength

Name of the Ingredient Quantity
Casein, Tryptic digest 1000mg
2
Casein, acid hydrolysate 1000mg

Glycin 40mg
L-Cystine 40mg
L-Asparginine 20mg
DL-Trptophan 20mg
Adenine 2mg
Guanine 2mg
Uracil 2mg
Dextrose 4000mg
Sodium acetate anhydrous 1200mg
Sodium chloride 1000mg
KH2PO4 100mg
K2HPO4 100mg
MgSO47H2O 40mg
MnSO44 H2O 2mg
FeCl3 0.4mg
Thiamine Hydrochloride 40µgm
Niacin 80µgm
Pyridoxin hydrochloride 160µgm
Calcium panthothenate 80µgm
Para amino benzoic acid 40µgm
Folic acid 2µgm
Biotin 0.4µgm
PH of the solution 6-8
Test solution of the material to be assayed :
Take accurate amount of the material to be assayed and dissolve it in water or dilute if
necessary.Add dil.Hcl or solution of sodium hydroxide to adjust the pH(6.0) and add water to
make a final volume.
Preparation of Inoculum :Transfer a few cells of Lactobacillus casei from a recent sub culture into
two sterile tubes each containing 10 ml of culture medium . Incubate the tubes for 18 to 24 hours at
370C. After incubation centrifuge the culture till the cells settle to the bottom of the tube. Decant off
the supernatant fluid. Repeat this procedure for three times if necessary. Finally suspend the cells
uniformly in 10ml of saline medium. Aseptically transfer1ml of the suspension of the cells to 10ml
of sterile suspension medium and mix .The resulting cell suspension is the inoculum.
Turbidimetric Method:-
Apparatus reagents and procedure are same as titrimetric method but this test includes two more
test tubes to which neither standard Riboflavin solution nor test solution , nor inoculum is added.
Incubate all test tubes at 30 to 370C for 64 to 72 hours. By using an uninoculated blank tube adjust
the transmittance at 440nm to 100% inthe photoelectric colorimeter. Thoroughly mix the contents of
each tube and record the transmittance reading. Plot the graph by considering transmittance value
against the corresponding levelsof standard Riboflavin solution. Draw a smooth curve and calculate
the concentration of the test solution of Riboflavin.
Assessment of New Antibiotic:
Minimum Inhibitory Concentration (MIC):- Minimum Inhibitory Concentration (MIC) is the lowest
concentration of antimicrobial compound found to inhibit the growth of a particular test
microorganism. It may be applied to assess new disinfectants, antiseptics , preservatives and
antibiotics .MIC values are usually expressed in terms of µg/ml or units/ml.Minimum inhibitory
concentration of different anti microbial compounds may be determined by the liquid dilution
method or the solid dilution method.

2
1.Liquid dilution method or test tube method: In the first tube un-inoculated inoculum is not
added) which is used for checking the sterility of the medium.All other test tubes, inoculum ( 3 to 4
drops) is added to reach the final concentration of microorganisms is 105to 106 cells/ml. In all test
tubes, test chemical is added ranging from 0.5 to 5ml except in the uninoculated and control tube.
The second tube ( control ) is used to check the suitability of the medium for growth of the test
microorganism and the viability of the inoculum. The final volume ( 10ml) in all test tubes is
adjusted by using sterile water .The contents of all test tubes are properly mixed and
incubated at 370C for 2 to 3 days . After incubation , all test tubes are examined for the growth
in the form of turbidity and the results are recorded and minimum inhibitory concentration is
calculated. It is also necessary to conduct a preliminary experiment to determine the approximate
range ( test solution ) which would be suitable for the test.
Determination of MIC by liquid dilution method:-
Tube number Volume of double Volume of test Volume of sterile

strength medium chemical (ml) water (ml)
(ml)
( uninoculated) 5 0.0 5
0’( control) 5 0.0 5
1 5 0.5 4.5
2 5 1.0 4.0
3 5 1.5 3.5
4 5 2.0 3.0
5 5 2.5 2.5
6 5 3.0 2.0
7 5 3.5 1.5
8 5 4.0 1.0
9 5 4.5 0.5
10 5 5.0 0.0
Solid Dilution Method :- In this method test chemical is first mixed into molten agar and
then poured into petri plates.
After solidification, the inoculum is spread on the surface of agar medium. All plates are
incubated at 370c for 2 to 3 days.
After incubation, all plates are observed for growth of inoculum and the minimum inhibitory
concentration of the test chemical is calculated.
The advantages of this method are :
i)Several microorganisms can be tested at the same time by use of multipoint inoculation.
ii) Contaminations are easily detected, because colony features on solid media are more
distinctive than turbidity differences in fluid media.
Standardization of Vaccines and sera.
For standardization of vaccines and sera basically three tests are there
1. Toxicity test.
2. Sterility test .
3. Potency test.
Toxicity Tests:

2
Toxicity in immunological preparations may be caused by .
a) n complete conversion of toxin to toxoid .
b) Incomplete sterilization of a supposedly dead vaccine.
c) Increased virulence in an attenuated organisms
d) Excessive toxicity in the toxin used for a diagnostic preparation .
The principle underlying most of the tests is that administration of a human ie. a very large
dose , by the usual or by a variety of routes , to a sensitive and small laboratory animal must
not cause , within a specified period , either a serious symptoms or death.
When , because of the toxic nature of the preparation itself , injection by any other route
would cause adverse symptoms in laboratory animals the preparation is tested
intracutaneously . for example of shick test toxin , injection of 1/25 of the test dose into
guinea pig gives a positive Shick reaction, while 1/50 of the test dose causes no local effect .
White or pale colored animals are used so that the results can be seen clearly. BCG Vaccine
is also tested in this way; the lesion must not be necrotic.
General toxicity test.
General method of toxicity test.
For sera and vaccines:- Unless otherwise prescribed in the individual monograph inject intra –
peritoneally one human dose but not more than 1.0 ml into each of five healthy mice ,
weighing 17 to 22 g and , one human dose but not more than
5.0 ml into each of two healthy guinea pigs weighing 250 to 350 g.
The human dose is that stated on the label or in the accompanying information leaf let of the
preparation under examination .
The preparation passes the test if none of the animals dies or shows signs of ill health in 7 days
following the injection. If more than one animal dies, the preparation fails the test .
If one of the animals die or show signs of ill health , repeat the test .
The preparation passes the test if none of the animals in the second group dies or show signs of
ill health in the time interval specified .
Carry out the test also on two healthy guinea pigs weighing 250 gm to 350 gm . Inject
intraperitoneally into each animal one human dose but not more than 5.0 ml .
The human dose is that stated on the label or in the accompanying information leaf let of the
preparation under examination .
Observe the animals for 7 days. If one of the animals die or show signs of ill health, repeat the
test .
The preparation passes the test if none of the animals in the second group dies or show signs of
ill health in the time interval specified.
The preparation passes the test if none of the animal shows signs of ill health . If more than one
animal dies , the preparation fails the test.
2.Sterility Tests.( this is same as the sterility test for bacteria)
Sterility tests shall be performed when ever specified in the requirements for individual
2
products.
Test for sterility are often performed advantageously at various stages of manufacture, in
addition to those given in requirements.
Sampling: The specified number of suitable samples shall be taken from the product. Such
samples shall be taken at lease from each final bulk ,as well as from each final lot.
Sterility test for bacteria and fungi.
1. Membrane Filtration .
2. Direct Inoculation .
Culture Medium .
The culture media used for sterility tests for bacteria and fungi shall be those approved by the
national control authority. Such media shall have been shown to be capable of supporting the
growth of a wide variety of microorganisms with both aerobic and anaerobic growth
characteristics .
Medium Incubation
Fluid Thioglycolate Temp(0 C) Condition
30-35 Aerobic
Alternative Thioglycolate 30-35 Anaerobic
Soyabean Casein Digest 20-25 Aerobic
Performance of the test: Prior to conducting a sterility test on any product , it shall have been
determined whether or not the material to be tested itself has the property of killing or inhibiting
the growth of microorganism or contains preservatives or other substances that have this effect. If
such an effect is shown, the sterility test shall be made using a suitable procedure to counter act
the effect.
Inoculum: For sterility test of bulk material , at least 5ml must be used for inoculation into each
culture medium and for each temperature of incubation.For sterility testing of a final lot from each
of the final containers that contain not more than 20ml, an amount of atleast 1.0ml shall be
inoculated into each culture medium for each temperature of incubation. If the volume in each final
container is less than 1.0ml, the amount to be inoculated shall be the entire content of the container
for each culture medium and for each temperature of incubation.
Incubation : All vessels shall be incubated at the appropriate temperature for the media. The
temperature selected shall be those approved by the national control authority and shall include 20-
250C and 30 -350C.All vessels shall be incubated for a period of atleast 14 days and shall be
examined at regular intervals and on the last day of incubation for evidence of microbial growth.
Membrane filtration of test samples.
Membrane Filtration :- The membrane filtration method is used for avoiding and also overcoming
the activity of samples for which practically little inactivating agents exist. Following conditions
should be fulfilled for the membrane filtration method:
1.An exceptional skilled and knowledgeable operators .
2.Rigorous routine usage of positive and negative controls .
3.Solution of the product under analysis is filtered via hydrophobic –edged membrane filter
which
effectively retains the contaminating microbes.
4.The resulting membrane is washed in situ to remove traces of samples adhered to the membrane
surface.
5.The segregated microorganisms are aseptically transferred to the culture media.

2
Interpretation of test result.

If no evidence of growth is found in any of the vessels inoculated for the test for sterility in the
bulk material of final lot which ever is applicable meets the requirements for this test. If
evidence of growth is found, the preparation tested fails to meet the requirements for the test
for sterility, unless it can be demonstrated to the satisfaction of the national control authority
by retests or by other means .
Sterility test for mycoplasmas . for viral vaccines made by growing the virus in animal tissues
or cell cultures , sterility tests for mycoplasmas of virus culture fluid and control fluid specified
for the particular product shall be made by a method approved by the national control authority.
The sterility tests for the detection of extraneous viruses in products where their presence is
unacceptable shall be performed as specified in the requirements for the individual products.
3. Potency tests:-
There are few products for which the need for potency tests exceeds that for immunological
products The correlation between effectiveness testing and clinical efficacy is the basis
for vaccine usage. Different methods of testing, like physical chemistry, anti-
pathogenicity, immunity, infection, and protection against infection or disease, are used
to measure efficacy.
Their applicability depends on the nature of the antigen and the purpose of the test.
Physical chemistry and antigenic analysis can be used separately or in parallel, in order
to control the consistency of production and the vaccine formula, but the correlation
between them and the protective effects is often inconclusive.
In most cases, the basic immunological characteristics of the antigen and the
relationship between these characteristics and the mechanisms of the disease and the
mechanism of the immune defense mechanism is very little known.
The Traditional Vaccine Potency Tests Method.
To evaluate the quality of a vaccine, the potency test of the final product must strictly
enforce the standards of efficacy established by national regulatory authorities.
The universal method for testing the efficacy of vaccines is the animal challenge test.
Inoculate the same type of animal with the recommended immunological dose to the
prescribe day of age after the optimal immune response period, and attack the
immunized animal and the control animal at the same time are capable of causing the
animal's pathogenesis, to measure the ability of the vaccine, and to reduce the incidence
of the disease after a certain time according to the protection of the immunized animal.
The Alternative Methods of Vaccine Potency Tests.
The alternative methods include experimental animal substitution and serological

substitution.The first method is to use other animals with similar clinical symptoms
to replace the original animals, and indirectly test the efficacy of this animal vaccine.
As for serological method, the efficacy of vaccine is assessed by detecting the antibody
titer level after immunization. It involves viruses neutralizing experiments and all kinds
of ELISA.

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Chapter 10. Study of infectious diseases : Typhoid, Tuberculosis,

Malaria , Cholera, Hepatitis , Meningitis , Syphilis & Gonorrhea and
HIV.
Typhoid
Salmonella typhi causes Typhoid fever. Salmonella are gram -ve bacilli measuring 1 to 3 µm in
size. They are motile with the presence of peritrichous flagella .
They are aerobic and facultatively anaerobic , they grow at an optimum of 370C in a pH
of 6 -8 on a variety of non selective ( Mueller –Hinton agar) and selective ( Wilson and
Blair’s bismuth sulfite medium ) media.
Cell wall components and Antigenic structures :-
Lipopolysaccharide ( LPS)
The cell wall of the salmonella like any other Gram –ve bacilli contains a complex
lipopolysaccharide structure . The LPS is liberated during lysis of the cell and to some
extent during culture .The LPS moiety functions as an endotoxin and is an important
component of the virulence of the bacteria.
The LPS complex consists of three components
i) an outer O polysaccharide coat

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ii) a middle portion ( the R core)
iii) inner lipid A coat .
The LPS of salmonella is important because of the following reasons .
1.The repeating sugar units in the outer O polysaccharide chain are responsible for “O ”–
antigen specificity .This also helps to determine virulence of the bacteria . Salmonella
strains lacking the complete sequence of “O” sugar repeat units are known as rough
strains . They are so called because of rough appearance of the colonies.
The rough strains are less virulent or avirulent than the smooth strains , which have a
full complement of “O” sugar repeat unit .
The endotoxin component of the cell wall is important in the pathogensis of salmonella
infections . Endotoxins cause fever , activate the serum complement kinin and clotting
systems and depresses myocardiac functions. The circulatory endotoxin is also responsible
in part for development of septic shock that can occur in systemic infections . Antibodies
produced against R core ( common enterobacterial antigen ) are protective against
infection caused by wide variety of Gram –ve bacteria due to sharing of common
core structure .
In some situations , the antibodies against R core mediate the lethal effects of
Gram -ve bacteria .

2
Antigenic properties :
Salmonella possess three major antigens :

1. H or flagellar antigens .
2. O or somatic antigens .
3. Surface antigens ( vi antigen , M and N antigen and F antigens).
H or flagellar antigens : This antigen is present on the flagella and is heat and alcohol labile
.
The antigens are destroyed by boiling or by treatment with alcohols and acid , but they are
preserved in 0.2 -0.4% formaldehyde .
The H antigens of salmonella are genus specific and are not shared by other enterobacteria .
The H antigen is strongly immunogenic and is associated with the formation of antibodies
following infection or immunization .
O or somatic antigen : O antigens occur on the surface of the outer membranes and are
determined by specific sugar sequences on the cell surfaces . O antigen is an LPS complex
and is an integral part of the cell wall . It is heat stable, resistant to boiling up to 2 hours
and 30 minutes.
It is also alcohol stable , resistant to treatment with 96% ethanol at 370C for 4 hours and is
also resistant to 0.2% formaldehyde. This antigen can be extracted from cell wall by

2
treatment with trichloroacetic acid .Treatment with phenol removes the antigenicity but
retains the toxicity of the bacteria .
Antigen is less immunogenic than H antigen. Generally the O antibody titre produced after infection
or immunization is lower than that of H antibodies .
The O antigen is not a single factor but a mosaic of two or more antigenic factors .
Surface antigens : These include a) vi antigen b)M and N antigens and c) F antigens. .
Vi antigen : Vi antigen is a surface antigen overlaying the “O” antigen. The presence of this
antigen on the surface renders these bacteria inagglutinable by their specific O antiserum
but agglutinable by vi antisera.
The vi antigen is heat labile and is destroyed by boiling within 1 hour.Vi antigen is also
destroyed by treatment with phenol hydrochloric acid and 0.5M sodium hydroxide, but the antigen
remains unaffected by 0.25% formaldehyde or alcohol .
M and N antigens : These antigens are present on the surface of bacteria and are
polysaccharide in nature .Boiling for two and half hours destroys these antigens .
F antigen : These antigens are present on the fimbriae .
Antigenic variation :
Salmonella antigens undergo phenotypic and genotypic variation as follows :
1. OH- O variation.
2. V- W variation .
3. S- R variation
4. Phase variation
OH-O variations : Flagellated salmonella ( OH) sometimes give rise to non- flagellated
(O) strains . This variation known as OH- O variation is associated with the loss of
flagella .
When salmonellae are grown on agar containing phenol (1: 800), flagella are not formed.
This change is phenotypic (refers to the observable physical properties of an organism )and
temporary because flagella reappear when the strain is subcultured on media with out
phenol.
The loss of flagella is usually not total but there is only a decrease in the number of flagella
and the quantity of the H antigen. Rarely , salmonellae may lose their flagella by
mutation.
S.Typhi 901-O strain is an example of a stable non motile mutant, which is used for the
preparation of O- agglutinable bacterial antigen suspension for use in Widal test and
other serological tests.
The major differences between O antigen and H antigen include:

2
S.N. Character O Antigen H Antigen

1. Referred to as Somatic Antigen Flagellar antigen
Based on oligosaccharides
2. Determination associated with Based on flagellar proteins.
lipopolysaccharide.
Part of the cell wall
3. Cell wall lipopolysaccharide Not a part of the cell wall.
(LPS).
4. Composition Polysaccharide. Proteinaceous (Flagellin).
Flagellar antigens are
5. Heat sensitivity Somatic antigens are heat stable. heat- labile.
6. Alcohol sensitivity Resistance to alcohol Sensitive to alcohol
Formaldehy
7. de Formaldehyde labile Formaldehyde stable
sensitivity
Trichloro-acetic acid is used
for extraction of O antigens.
8. Extraction Since the property was first Formaldehyde is used
shown by Boivin, O antigen for extraction of H
alternatively referred to as antigens.
boivin antigen.
9. Immunogenicity Less immunogenic Highly immunogenic
Produces antibody formation Induces antibody
10. Antibody levels with low titres. formation with high
titres.
11. Antibody formation Rapid and Early Rapid and Sustained
12. Lifespan Antibody levels fall off quickly. Persists for longer periods.
O antibody appears early, H antibody appears
13. Antibody indicates disappears early: indicates late, disappears late:
recent infection. lndicates convalescent
stage.
Type of Produces compact, chalky Produces cottony,
14. agglutination and granular clumps. fluffy precipitates.
reaction shown
Agglutination takes
15. Reaction time Agglutination takes place slowly place rapidly.
Optimum Optimum temperature Optimum temperature
16. temperature for for agglutination is for agglutination is
reaction 55’°C. 37’°C.
Reaction Round bottom Felix tube are Conical bottom Dreyer’s
17. observed with used to see agglutination. tube is used to see
agglutination.
The most important virulence
factor responsible for endotoxic Makes the bacteria

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18. activity; it protects the bacteria motile, hence

Role as contributing to their
virulence factor from phagocytosis and
bactericidal effect of virulence.
complement.
Flagellar antigens exist in
two alternative phases-
19. Existence in phases No phases Phase I and II.
Most o f them are
biphasic except S.
Typhi which is
monophasic.
In WidaI test, H antigens
20. Widal test In Widal test, O antigen of S.Typhi, S.Paratyphi A
of Salmonella Typhi is used.
and B are used.
Serogroups are
21 Use in classification Serogrouping of differentiated into
salmonellae is based on the serotypes based on H
O antigen. antigen.
V-M variation: Almost all freshly isolated strains of S.Typhi carry a surface layer of vi
antigen that completely masks the O antigen. When fully expressed , such bacilli are
agglutinable with vi antiserum but not with the O antiserum. This is called the V form.
S-R variation – The smooth to rough (S-R) variation occurs due to mutation and is
associated with the
i) change in the colony morphology from smooth to rough,
ii) loss of the O antigen and
iii) loss of virulence.
The rough colonies become large, rough and irregular and are auto agglutinable in saline.
Phase variation : The flagellar or H antigens of most salmonellae occur in two alternate
phases : Phase 1 and phase 2.
Phase 1 antigens are either specific for a serotype or shared by a few species only , hence
known as the “ specific ” or species phase .
Phase 2 antigens are widely shared , hence known as the “non specific” or “group ” phase.
Pathogenesis of enteric bacteria :
The severity of disease in individuals infected with salmonellae is dependent on the

virulence factors of the infecting strains as well as on the human host.
Infective dose:The infection is acquired by ingestion of food or
Water contaminated with salmonellae .

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Ingest contaminated food

↓
Ingested S. typhi invade small intestinal mucosa
↓
Taken up by macrophage and transported to regional lymph node
↓
S. typhi multiply in the intestinal lymphoid tissue
↓
Intact with enterocytes & M cells ( ileal Peyer’s patches) during the 1-3
week of incubation period ( Diarrhoea)
↓
End of incubation period s. typhi enter blood stream( onset of typhoid fever)
↓
Bacteria invade the gall bladder , biliary system and lymphatic tissue of
the bowel and multiply in high number. Then re-infection of the intestine
↓
Then pass into the intestinal tract ( stool)
Enteric fever ( Typhoid fever). Symptoms.
Enteric fever is generally an acute illness manifested by fever , headache and abdominal
symptoms . The incubation period is usually from 7 to 14 days , but may range from 3 to 56

2
days. Onset of disease is usually gradual, with headache, malaise ( a general feeling of
discomfort , illness ), anorexia, a coated tongue , and abdominal discomfort with either
constipation or diarrhea.
A step – ladder pyrexia (fever) with relative bradycardia (wherein an individual has a resting
heart rate of under 60 beats per minute (BPM) in adults. Bradycardia typically does not
cause symptoms until the rate drops below 50 BPM) and toxemia (blood poisoning by
toxins from a local bacterial infection) is the typical feature. Rose spots may appear on the
upper abdomen by the end of the first week.
The condition is associated with a soft , palpable spleen and an enlarged liver.These
Symptoms are present for a week or more and are followed by gastrointestinal symptoms.
This phase corresponds to an initial bacteremic phase which is followed by
colonization of gallbladder and then re-infection of the intestine.
Intestinal perforations ( is a hole in the wall of part of the gastrointestinal tract.) , severe
hemorrhage and circulatory collapse are most important complications .
Toxic encephalopathy (characterized by an altered mental status, memory loss, and visual
problems), cerebral thrombosis (is the presence of a blood clot in the dural venous sinuses,
which drain blood from the brain. Symptoms may include headache, abnormal vision, any
of the symptoms of stroke such as weakness of the face and limbs on one side of the body,
and seizures.) , hepatitis , pancreatitis , arthritis and myocarditis (can affect the heart
muscle and the heart's electrical system, reducing the heart's ability to pump and causing
rapid or abnormal heart rhythms ) are other complications .
Reservoir, source and transmission of infection .
The infected patient and more frequently carriers are important reservoirs of infection for
enteric fever. About 2 -4% of patients become chronic carriers .
The bacilli persist in the gall bladder and are excreted in feces ( fecal carrier) or persist in
the kidney and are secreted in the urine ( urinary carrier). Urinary carriers are less frequent
than the fecal carriers and usually present with some urinary lesion such as calculus.The
carrier state is more common in women than in men and in the older people ( over 40
years ) than in young people.
Food handlers or cooks who become carriers are potentially dangerous to the community .
Mary Mallon ( Typhoid Mary) a New York cook was a classical case of such carrier who ,
over a period of 15 years , caused at least seven outbreaks affecting more than 200 persons.
Food , vegetables and water contaminated with human feces infected by S. Typhi are the
common sources of infection.
Typhi infection occur when food or water contaminated by infected food handlers or due
to poor personal hygiene is ingested .
The infectious dose for S. Typhi infections is low , so person to person spread is common .
The infectious dose is still lower for people at high risk for disease because of age ,
immune-suppression or underlying diseases .
Laboratory Diagnosis :-
Laboratory diagnosis of enteric fever is based on the following methods.

2
1. Isolation of salmonella species by culture.
2.Serodiagnosis by demonstration salmonella antibodies and antigens and
3.molecular diagnosis by DNA probes (DNA probes are stretches of single- stranded
DNA used to detect the presence of complementary nucleic acid sequences (target
sequences) by hybridization. DNA probes are usually labelled, for example with
radioisotopes, fluorophores to enable their detection.) and PCR.
Specimens.
Blood , bloodclot, bone marrow and stool are common specimens used
for isolation of typhoid bacilli for culture .
Other specimens include the cerebrospinal fluid , peritoneal fluid ,lymph nodes,
intestine , pharynx , tonsils , bone and urine .
Culture
Blood culture- Blood culture is a very useful procedure for diagnosis of enteric fever . It is
positive in approximately 90% of the cases in the first week of fever, 75% of cases in the
second week , 60 % in the third week and 25% thereafter till the subsidence of pyrexia
(fever).
Blood cultures however become negative on treatment with antibiotics. In this method approximately
5 -10 ml of blood is collected aseptically by venepuncture and inoculated into a culture bottle
containing 50 -100 ml of 0.5% bile broth .
This 10 fold dilution of blood is achieved by adding5- 10 ml of blood to 50- 100ml of bile
broth which is carried out to neutralize the bactericidal action of many substances that are
present in the blood . The addition of liquid ( sodium polyanethol sulfonate) further
counteracts the bactericidal action of blood .Blood culture bottle is incubated at 37oC for up
to 7 days .
After incubation overnight at 37oC, the bile broth is sub-cultured on Mac Conkey agar.
Castaneda’s biphasic method of blood culture : It is a better method of culture to reduce the
risk of contamination during repeated subcultures.
The method has additional advantage of being more safe and economical .In this method , the
culture bottle has an agar slant in one side ,which is flooded with bile broth.
After inoculation of blood , the bottle is incubated in the upright position so that surface
of the agar remains free without any broth covering the slant.Broth remains only in the
lower part of the agar slope.For subculture , the bottle is simply tilted so that the broth
flows over the surface of the agar slant and is re-incubated in the upright position .If
salmonella are present colonies appear on the agar slant.

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Clot culture : Clot culture is a more sensitive method than the blood culture , because
certain inhibitory substances that are found in the serum are absent in the clot .
Another advantage of this method is that serum eluted from the blood during process of
clotting can be used for demonstration of salmonella antigens or antibodies.
In this method , 5ml of blood collected under strict aseptic conditions , from the patient ,
into a sterile test tube and allowed to clot. The serum is pipette off and used for

2
serological tests.
The clot is broken up with a sterile glass rod and added to a bottle of bile broth containing
streptokinase ( 100 units/ ml).
Streptokinase facilitates lysis of the clot with release of bacteria trapped inside the clot .
The bile broth is incubated and sub-culturedon media in the same way
as described for blood culture.
Bone marrow Culture : - Bone marrow culture is a most sensitive method .

It is positive in most cases even when blood cultures are negative . It is also positive even if
patients have been taking antibiotics for several days , regardless of how long they have been
suffering from enteric fever . This test is recommended for patients whose initial blood culture
results are negative , possibly due to prior antibiotic therapy.
Feces culture : Feces culture is also useful because salmonellae are excreted in feces
through out the disease .The feces is collected from the patient in a sterile container and
sent immediately to the laboratory .
If delay is anticipated , the stool specimens may be collected in a buffered glycerol saline
transport medium . The fecal samples are inoculated directly on Mac Conkey, DCA and
Wilson – Blair media .
Relatively , a heavy inoculation of stool is made on the Wilson – Blair media because it is
highly selective. For enrichment, one tube each of selenite and tetrathionate broth is
inoculated and incubated at for 12 -18 hours before subculture on to selective media .
The plates are incubated at 370C overnight. S. Typhi produces large block colonies ,with
a metallic sheen on the Wilson – Blair media.
Salmonella form pale non lactose fermenting colonies on Mac Conkey and DCA media . If
no growth is observed after 7days , then the culture is declared negative.

2
Identification of bacteria :
Colonies are identified by carrying out motility test , biochemical test and slide
agglutination with specific salmonella antisera .
Slide agglutination test : The test is performed with a loopful of growth
From nutrient agar plate or slope emulsified in two drops of saline on a clean
slide .
If S.Typhi is suspected , that is when no gas is formed from glucose , a loopful of
typhoid O antiserum is added to one drop of bacterial emulsion on the slide . The
slide is rocked( moved gently to and fro or from side to side) gently. Development
of immediate agglutination suggests salmonella .
Salmonella slide agglutination test showed agglutination with O antisera (left), H

antisera (right) and control negative (middle).
Serodiagnosis :
Serodiagnosis of enteric fever is based on detection of specific salmonella antibodies in

the serum , or antigen in the serum and also in urine by various serological tests.
Demonstration of serum antibodies:
Widal test:
Widal test is a serological test which is used for the diagnosis of enteric fever
or typhoid fever. Typhoid or enteric fever is caused by a gram negative bacteria
Salmonella enterica (Salmonella Typhi or Salmonella Paratyphi), found in the
intestine of man. Salmonella paratyphi also causes Typhoid but of a milder form.
Salmonella possess O antigen on their cell wall and H antigen on their flagella.On infection,
These antigen stimulates the body to produce specific antibodies which are released in the blood.
The Widal test is used to detect these specific antibodies in the serum sample of
patients suffering from typhoid using antigen-antibody interactions. These specific
antibodies can be detected in the patient’s serum after 6 days of infection (fever).
Salmonella Typhi possesses O antigen on the cell wall and H antigen on flagella.

2
Salmonella Paratyphi A and S. Paratyphi B also possess O antigen on their cell wall and
but have AH and BH antigen on their flagella respectively.
Principle of Widal test:
Widal test is an agglutination test in which specific typhoid fever antibodies are detected
by mixing the patient’s serum with killed bacterial suspension of Salmonella carrying
specific O, H, AH and BH antigens and observed for clumping ie. Antigen- antibody
reaction.
The main principle of Widal test is that if homologous antibody (The antibody elicited by
a specified antigen )is present in patient’s serum, it will react with respective antigen
in the suspension and gives visible clumping on the test slide or card.
Requirements for widal test:
i) Fresh serum, stored at 2-8° Serum should not be heated or inactivated.
ii) The complete kit containing five vials containing stained Salmonella antigen
 S. Typhi———-O antigen
 S. Tyhhi———- H antigen
 S. Paratyphi —–AH antigen
 S. Paratyphi —–BH antigen
iii) Widal positive control

iv) Widal test card or slide
v) Applicator stick
Procedure of widal Test:-
Widal test can be done in two ways-one is rapid test on slide and another is tube test in
which result may be obtained after one night of incubation.
I. Rapid slide test:
1. Clean the glass slide or test card supplied in the kit well and make it dry.
2. Label the circles (1, 2, 3, 4, 5 and 6) in the test card as O, H, AH, BH,
Negative control and Positive control
3. Place a drop of undiluted test serum in each of the four labelled circle (1, 2, 3
and 4) ie O, H, AH and BH and place a drop of Negative control serum in
circle 5 and Positive control in circle 6.
4. Place a drop of antigen O, H, AH and BH in circle 1, 2, 3, and 4

respectively and no antigen in circle 5 and O/H antigen in circle 6.

2
5. Mix the content of each circle with a separate wooden applicator stick and
spread to fill the whole area of the individual circle.
6. Rock the test card for a minute and observe for agglutination.
If agglutination is visible within 1 minute, proceed for quantitative slide test or tube test for the
quantitative estimation of the titre of the antibody.
II. Quantitative slide test:
1. Clean the test card and make it dry.
2. Put 0.005 ml, 0.001 ml, 0.02ml, 0.04ml and 0.08ml of undiluted serum in 1st, 2nd,
3rd, 4th and 5th circles respectively in the test card.
3. Add a drop of appropriate antigen suspension which showed agglutination in

rapid slide test, to each of the above circles.
4. Mix the contents of each circle with a separate wooden applicator stick.
5. Rock the slide slowly for 1 minute and observe for agglutination.
6. The titre of the antibody is the highest dilution of serum up to which there is
clear agglutination.
7. Repeats steps 1 to 6 with all the antigens, which showed agglutination in rapid
slide test.
The serum volumes in the quantitative slide test corresponds approximately to the tube test
is given below:
Approx. test
Circle Serum volume Antigen drop tube titre
1
st
0.08 ml 1 drop 1 : 20
2
nd
0.04 ml 1 drop 1 : 40

2
3
rd
0.02 ml 1 drop 1 : 80
4
th
0.01 ml 1 drop 1 : 160
5
th
0.005 ml 1drop 1 : 320
III. Quantitative tube test:
1. Take a set of 8 clean dry test tubes (Kahn tubes) and label as 1, 2,3, 4, 5, 6, 7 and 8
for O antibody detection.
2. Similarly, take 3 sets of 8 test tubes and label then as 1, 2…8.
3. Dilute the serum samples as follows:
4.Pipette into the tube No.1 of all sets 1.9 ml of isotonic saline.
5.To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline.
6.To the tube No.1 tube in each row add 0.1 ml of the serum sample to be tested
and mix well.
7.Transfer 1.0 ml of the diluted serum from tube no.1 to tube no.2 and mix well.
8.Transfer 1.0 ml of the diluted sample from tube no.2 to tube no.3 and mix well.
Continue this serial dilution till tube no.7 in each set.
9.Discard 1.0 ml of the diluted serum from tube No.7 of each set.
10.Tube No.8 in all the sets, serves as a saline control. Now the dilution of the
serum sample achieved in each set is as follows: Tube No. : 1 2 3 4 5 6 7 8
(control) Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280.
4. Add a drop of appropriate widal test antigen to all the test tubes
5. Mix well and incubate at 37°C for 16-20 hours and examine for agglutination.
6. Antibody titre is the highest dilution of serum showing clear agglutination.

2
Test
tube 1 2 3 4 5 6 7 8
1 : 1 : 1 : 1 : 1 : 1 : 1 : Control
Dilution 20 40 80 160 320 640 1280 (saline)
Result interpretation of Widal test:
Antibody titre greater than 1 : 80 is considered significant and usually suggests positive test
for Salmonella infection. Low titres are often in normal individuals.
A single positive is less significant than the rising antibody titre, since rising titre is
considered to be a definite evidence of infection.
Applications of Widal test:
Rapid test for screening typhoid fever in endemic areas. When culture facilities is not available, Widal
test is very reliable.Use for both Salmonella Typhi and Salmonella Paratyphi
Limitations of Widal test:
Widal test is time consuming to find antibody titre and often times when diagnosis is
reached it is too late to start an antibiotic regimen.
Widal test may be falsely positive in patients who have had previous vaccination or
infection with S.Typhi. Widal test cannot distinguish between a current infection and a
previous infection or vaccination against typhoid.
Widal test shows cross-reactivity with other Salmonella species.
False positive Widal test results are also known to occur in typhus, acute falciparum
malaria(particularly in children), chronic liver disease associated with raised globulin levels
and disorders such as rheumatoid arthritis, myelomatosis and nephrotic syndrome.
Widal test should be interpreted in the light of baseline titers in a healthy local population.
The antibody levels found in a healthy population however, may vary from time to time and
in different areas, making it difﬁcult to establish a cut off level of baseline antibody in a
deﬁned area and community.
Severe hypoproteinaemia may also prevent a rise in 0 and H antibody titres. False negative
Widal tests may be due to antibody responses being blocked by early
antimicrobial treatment or following a typhoid relapse.
In low typhoid endemic areas, weak and delayed O and H antibody responses limit the
usefulness of the Widal test. Variations also exist between laboratories in the performance
and reading of Widal tests which compromise further the reliability of the test.
The World Health Organization (WHO) has said that due to the various factors that can

2
influence the results of a Widal test, it is best not to rely too much on this test.
Other serological test : Indirect hemagglutination , counter immune

current
electrophoresis , indirect fluorescent Vi antibody , and indirect ELISA for IgM and
IgG antibodies to S. Typhi polysaccharides are available for diagnosis of typhoid
fever with varying sensitivity and specificity
Demonstration of serum antigens : In typhoid fever , circulating S. typhi antigen is

present in the serum as well as in the urine , but absent in serum of a cured case of
typhoid .
Counter current immune-electrophoresis , co –agglutination test and ELISA are frequently

employed for detection of circulating antigen in the serum and also in urine for diagnosis of
typhoid fever with varying sensitivity and specificity.
Treatment :
Chloramphenicol was the antibiotic of choice for treatment of enteric fever since its
introduction in 1948. It acts by binding to 50S bacterial ribosomal subunits and inhibits
bacterial growth by inhibiting protein synthesis . Because of low cost, for sensitive S.typhi
strains , Chloramphenicol is still used to treat typhoid fever.
At present , the fluoroquinolones ( eg. ciprofloxacin, perfloxacin, norfloxacin) and a third

generation cephalosporins ( eg.ceftazidime, ceftriaxone,cefotaxime) are the antibiotics of
choice for treatment of multidrug resistant S.typhi. Cefotaxime prevents bacterial cellwall
synthesis , which inhibits bacterial growth. A 14 day course of Chloramphenicol,
Ampicillin, or Trimethoprim and Sulphamethoxazole is indicated for S. typhi infection .
Prevention and control :
Availability of safe drinking water , proper food hygiene and sanitary disposal of excreta are
the most cost effective strategies for reducing the incidence of typhoid fever in endemic
countries .
Immunization : Immunization with typhoid vaccines at regular intervals also considerably

reduces the incidences of typhoidal salmonella infections . Routine typhoid vaccination is
indicated for
1) persons with intimate exposure ( eg. house hold contacts ) to S. typhi cases or
carrier .
2) travelers to countries associated with an increased risk of exposure to S. typhi and
3) microbiology laboratory personnel working with S.typhi .
The following two types of typhoid vaccines , killed and oral are used.
Killed vaccines .
TAB vaccine : The TAB vaccine is a killed whole cell vaccine that contains heat killed and
0.5% phenol preserved S. typhi , 1000million/ml , and S .paratyphi A and B , 750 million
each per ml .
2
The vaccine is given subcutaneously in two doses of 0.5ml , each at an interval of 4- 6

weeks , followed by a booster dose every 3 years .Field trials have shown overall
efficacy of 70 -90 % in typhoid fever for a period of 3 -7 years .
Fever and pain at the site of injection are the side effects . Injection of a large volume of
antigen is also another concern.
Therefore in India divalent typhoid -paratyphoid. A vaccine with out S .Paratyphi B is used
instead of the trivalent TAB vaccine , because S. Paratyphi B infection is not that
common in the country.
Vi capsular polysaccharide antigen vaccine ( ViCPS) : The Vi CPS antigen vaccine

composed of purified Vi antigen , the capsular polysaccharide produced by S. Typhi
isolated from blood cultures . Primary vaccination with ViCPS is carried out by a single
parenteral dose of 0.5ml ( 25 µg ). Booster doses are needed every 2 years to maintain
protection , if continued or renewed exposure to S. Typhi is expected.
Fever , headache , erythema(superficial reddening of the skin, usually in patches, as a result
of injury or irritation causing dilatation of the blood capillaries). and induration (a
hardened mass or formation) are some of the side effects . The vaccine is not recommended
for children below 2 years.
Oral vaccines :
Ty21a oral vaccine: This is an oral vaccine containing live attenuated S.Typhi Ty21 a
strains in an enteric coated capsule . S.Typhi Ty21a strain is a stable mutant lacking the
enzyme UDP- galactose-4- epimerase . On ingestion , the strain initiates infection , but
after four or five cell divisions causes self destruction , hence lacks the capability to
cause any illness .
Primary vaccination with Ty21a consists of one enteric coated capsule , taken on alternate
days , with a total of four capsules. Booster doses are needed every 5 years to maintain
protection if continued or renewed exposure is expected.
Tuberculosis (TB)
Tuberculosis is a potentially fatal contagious disease (spread from person to person in
several ways. One way is through direct physical contact, like touching a person who has
the infection. Another way is when an infectious microbe travels through the air after
someone nearby sneezes or coughs.) that can affect almost any part of the body but is
mainly an infection of the lungs. Tubercle- Round nodule /swelling Osis – Condition.
Causative Organism - Mycobacterium tuberculosis the human type . Mycobacterium bovis -

in animals.
They are acid fast, they contain mycolic acid. They contain a high ( 61- 71%) guanine and
cytosine content in the DNA. Mycobacterium tuberculosis organisms are straight or
slightly curved rods occurring singly, in pairs or in clumps.
Pathogenesis .
Tuberculosis may be primary or post primary depending on the time of infection and
the type of host immune response .

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Primary tuberculosis - Primary tuberculosis represents the initial infection caused by

Mycobacterium tuberculosis in an infected host.
This condition is usually seen in young children in endemic countries (a

disease which is restricted to a particular location, region, or population.)
like India . On inhalation of the aerosolized bacteria , the bacilli reach the lower
respiratory tract.
Majority of the inhaled bacteria are killed by the natural defensive mechanisms of
the upper respiratory tract . The bacilli that survive these defensive mechanisms
reach the lungs and enter alveolar macrophages (are the primary phagocytes of the
innate immune system, clearing the air spaces of infectious, toxic, or allergic particles
that have evaded the mechanical defenses of the respiratory tract, such as the nasal
passages) .
The phagocytosed bacilli inhibit acidification of the phagosomes and prevent
subsequent fusion of phagosome (a vacuole in the cytoplasm of a cell,
containing a phagocytosed particle enclosed within a part of the cell
membrane) and lysosome (A lysosome is a membrane-bound cell organelle
that contains digestive enzymes.
Lysosomes are involved with various cell processes. They may be used to destroy invading
viruses and bacteria).
This makes the bacteria multiply freely either in the phagosome or

in the cytoplasm. Multiplication inside the cells leads to destruction
of the cells and release of mycobacteria . This is followed by further

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cycles of phagocytosis of bacteria by macrophages , multiplication of

mycobacteria and lysis of macrophages . Some bacilli are transported
by macrophages to the hilar lymph nodes .
These are attracted to this site by the presence of bacilli, cellular components and
chemotactic factors,( Any small molecule that acts as a chemical stimulus along a
concentration gradient, attracting macrophages and other cells–eg, to a site of
inflammation) such as complement C5a of the serum .( The complement system can
be activated by many factors, including immune complexes, leading to the generation of
biologically active split products like C5a anaphylatoxin. This study presents a
technique which may be used for measuring C5a activity in human serum).
This leads to formation of the focus known as Ghon’s focus (A Ghon focus is a primary
lesion usually subpleural, often in the mid to lower zones, caused by Mycobacterium
bacilli (tuberculosis) developed in the lung of a nonimmune host (usually a child). ,
which is formed of multinucleated giant cells known as Langerhans cells .

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Langerhans cells
The focus is commonly found in the lower lobe or in the lower part of the upper lobe
of the lungs. This focus is also associated with enlargement of the hilar lymph nodes .
Both Ghon focus and enlarged lymph nodes constitute the primary complex , which
usually develops in 3 to 8 weeks after infection by tubercle bacilli .The lesion (a
region in an organ or tissue which has suffered damage through injury or disease )in
majority of cases heals spontaneously within 2 to 6 months. If small numbers of bacilli
are present , the bacilli are destroyed by macrophages with minimal tissue damage.
However if many bacilli are present, it leads to development of tissue necrosis (Necrosis
is the death of body tissue). The bacilli may be present dormant in this stage or may
become reactivated in old age .Reactivation of the site causes post primary (
secondary tuberculosis )
Post primary ( secondary ) tuberculosis : Post primary tuberculosis is caused either by

reactivation of latent infection or by exogenous re-infection .Reactivation of primary
lesion occurs more commonly in patients with decreased immunity , such as patients
receiving transplants , those infected with human immunodeficiency virus (HIV) , and
in the elderly patients .

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Clinical syndromes .
The clinical manifestations of tuberculosis depend on the site of infection.
However primary infections is usually pulmonary .
M. tuberculosis produces following clinical syndromes
a) Pulmonary tuberculosis
b) Extra pulmonary tuberculosis .
Pulmonary tuberculosis: Productive cough , fever and weight loss are atypical symptoms of
pulmonary tuberculosis . Hemoptysis or chest pain , night sweats , fatigue and anorexia
(lack or loss of appetite for food) are the other systematic manifestations . The sputum may
be scanty (small or insufficient in quantity or amount )or with blood and as a result, is usually
associated with cavitary lesions in the lungs .
Right upper lobe cavitary lung lesion
Extrapulmonary tuberculosis : Extrapulmonary tuberculosis usually occurs as a result of

spread of the bacilli through blood circulation during the initial stage of multiplication
at the site of primary infection , ie. lung .
Depending on the site of infection , extrapulmonary infection may be
a) genitourinary tuberculosis
b) tubercular meningitis
c) gastro intestinal tuberculosis
d) skeletal tuberculosis
e) tubercular lymphadenitis and

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f) other conditions .
Genitourinary tuberculosis : Genitourinary tuberculosis is one of the most common

extrapulmonary manifestations of tuberculosis . The typical symptoms include dysuria
(painful or difficult urination), increased frequency of urination .In women , the
condition may manifest (clear) as pelvic inflammatory disease.Genitourinary
tuberculosis is responsible for approximately 10 % of sterility in women.
Tubercular meningitis : This is one of the most severe complications of tuberculosis.

The condition may persist as head ache , which is either intermittent or persistent.
Gastro intestinal Tuberculosis : The clinical manifestation (action) of the condition

depends on the site affected in the gastro intestinal tract .
For example , infection of stomach or duodenum manifests abdominal pain mimicking

peptic ulcer disease, where as infection of large intestine manifests as pain in the
abdomen , diarrhea etc.
Skeletal tuberculosis . Spine is the most common site involved in skeletal tuberculosis .
Tubercular lymphadenitis : Most commonly involves the neck along the

sternocleidomastoid muscle (each of a pair of long muscles which connect the sternum,
clavicle, and mastoid process of the temporal bone and serve to turn and nod the neck).
The condition is usually unilateral with little or no pain .
Other conditions : These include miliary tuberculosis (Miliary tuberculosis is

so named because the innumerable tiny spots that form in the lungs are the size of
millet, the small round seeds in bird food.) tuberculosis of the skin and tuberculosis of
the middle ear and ocular structure .

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Complications of tuberculosis :
Tuberculosis with HIV : HIV reactivates latent tuberculosis infection, makes

the disease more serious and renders treatment ineffective .
Laboratory diagnosis :
Specimen : Collection of the specimen depends on the nature of the infection

whether pulmonary or extrapulmonary .
Sputum , lung tissue , gastric lavage (washing out an organ a body cavity, or a
wound by flushing it with a fluid.) and bronchoalveolar lavage are the specimens
collected for the diagnosis of pulmonary tuberculosis.
Cerebrospinal fluid (CSF), pleural fluid, peritoneal fluid, urine, lymph node
tissue , bone marrow and blood are the other specimens frequently used in the
diagnosis of extra pulmonary tuberculosis.
1.Sputum is the specimen of choice for pulmonary tuberculosis . Sputum not the saliva is collected
in
the morning into a clean wide mouthed container, such as sputum cup. Collection of morning
sputum is
ideal .Gastric aspirate (Attach a syringe to the nasogastric tube. Gently insert the nasogastric tube
through the nose and advance it into the stomach. Withdraw (aspirate) gastric contents (2–5 ml)
using
the syringe attached to the nasogastric tube may be used in place of sputum , especially in young
children who cannot produce the sputum . In older children bronchial secretions may be collected
by
stimulation of the cough by using an aerosol solution of propylene glycol in 10 % sodium
chloride .

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2.Urine is the specimen of choice for diagnosis of genitourinary tuberculosis . This is collected
either as three consecutive early morning samples or as a single sample of completely voided urine
in 24 hours. The urine specimens are centrifuged at 300 rpm for 30 minute , and the sediments are
used for culture on selective media for M. tuberculosis .
3.CSF is collected for diagnosis of tubercular meningitis .The CSF is centrifuged and the sediment
after centrifugation is stained for smears and is inoculated on the media for culture.
4.Pleural fluid, peritoneal fluid and other exudates are collected in containers with
citrate to prevent coagulation . Bone marrow and liver tissue are usually collected from
patients with miliary tuberculosis and blood is collected from patients with HIV for
isolation of bacteria by culture .
5.It is essential to collect all these specimens before starting anti-tubercular

therapy.
Microscopy : Sputum microscopy is the most dependable and conventional method

of demonstration of Acid fast bacteria (AFB) by ZN (Ziehl- Neelsen ) method .
The sputum smears are made by using new slides every time .Prepare a smear of given bacterial
suspension .Allow smears to air dry and then heat fix it. Flood the smear with carbol fuchsin stain
and heat the slide from below till steam rises for 5 minutes.Do not boil the stain and ensure that
stain does not dry out. Allow the slide to cool for 5 minutes to prevent the breakage of slide in the
subsequent step. Wash with tap water. Decolourise the slide by using acid- alcohol or 20 %
sulphuric acid until carbol fuchsin fails to wash from smear. Wash with water and counter stain
with 1% aqueous solution of malachite green or methylene blue for 1 to 2 minutes. Wash smear
with tap water, dry and examine under oil- immersion objective.The slides should not be reused ,
because AFB may adhere to the surface of the slide and may not be removed from the slide during
the process of cleaning .
Ziehl –Neelsen method of M. tuberculosis
Auramine rhodamine stains are the fluorescent stains that are used as variation of
the traditional ZN stain for demonstration of Acid fast Bacteria.

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Culture : Culture is the definitive method to detect and identify M. tuberculosis .

The culture is also more sensitive diagnostic method than microscopy of the
smear, LJ medium is the definitive medium and the Middle brook 7H10 and 7H11
media are the agar based media and these are conventionally used for culture .
Since M. tuberculosis is a slow growing organism , a period of 6- 8 weeks is required

for colonies to appear on these conventional culture media after incubation at 37 0C.
Serodiagnosis : M. tuberculosis infection is associated with elevated levels of antibodies

in the serum . Various mycobacterial antigens have been used in these serological
tests , which include BCG, 5 and 6 kDa proteins of M. tuberculosis .
Detection of antibodies by serological test ,such as ELISA , is of limited value in the
diagnosis of pulmonary tuberculosis .
Latex agglutination test using latex particles coated with rabbit antibody against
M. tuberculosis has been used for demonstration of antigen in the CSF for diagnosis
of tuberculosis meningitis .
Tuberculin skin test : Tuberculin skin test (TST) is a widely used test for diagnosis of tuberculosis
.
Mantoux test is the recommended method of skin test .
The test is performed by intradermal injection of 0.1ml or 5 tuberculin units (TU)
PPD (Purified Protein Derivative) into the volar aspect (the volar surface of the
forearm is the portion of the forearm that is on the same side as the palm of the hand.)
of the forearm using a 27 –G needle .It is essential that PPD (Purified Protein
Derivative) is injected between the layers of the skin , but not subcutaneously .
Development of an induration (a hardened mass or formation) of 10 mm or more at
the site of injection after 48- 72 hours is considered a positive test . Induration less
than 5 mm is negative , while between 6 and 9mm is considered equivocal . Erythema
(superficial reddening of the skin, usually in patches, as a result of injury or irritation
causing dilatation of the blood capillaries). is not considered in reading of the test.
If the test is negative with the PPD(Purified Protein Derivative) of 5TU (tuberculin
units) , then the test may be repeated using PPD of 10 or 100 TU.
Purified protein derivatives of 1 TU is used when the recipient is considered to be
extremely hypersensitive to the antigen .

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Rapid and automated methods .
The conventional methods are very slow and time consuming and require 6 to 8 weeks
for isolation of M. tuberculosis. Hence recently more rapid and automated methods
are being increasingly used for diagnosis of tuberculosis . These recent methods
include automated radiometric culture methods .( eg. BACTEC), SEPTICHEK,
mycobacterial growth indicator tubes (MGITs).
Automated radiometric culture methods , such as BACTEC employ a liquid Middle

brook 7H12 medium containing radiometric palmitic acid labeled with radioactive
carbon -14 (14C).
The medium also contains several anti microbial agents to prevent the growth of other
non mycobacterial microbes .
The result of the test is noted by demonstration of radiolabeled 14CO2 produced during
the growth of mycobacteria . Growth of mycobacteria is usually detected within 9 to
16 days.
SEPTICHEK is another rapid method for isolation of mycobacteria .This is a non radiometric
method which is based on a biphasic broth based system that decreases the mean recovery time
versus conventional methods .
A new method employs MGITs (mycobacterial growth indicator tubes) which show
microbial growth and provide a quantitative index of M. tuberculosis growth .
Round bottom tubes with oxygen sensitive sensors at the bottom are used in the test

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Treatment :
Anti tuberculous drugs are classified as first line and second line drugs .
First line anti tubercular drugs :

First line anti tubercular drugs include Rifampicin, INH (Isoniazid, also known as
isonicotinic acid hydrazide (INH) , ethambutol , streptomycin and pyrazinamide.
All first line drugs with the exception of ethambutol are bactericidal .These drugs have less
toxicity and show greater efficacy than second line drugs.
Combination of four drugs ( INH, rifampicin, pyrazinamide and ethambutol is given for
a period of 6 - 7 months for treatment of smear positive cases of tuberculosis .
These are given three times a week for first 2 months , followed by only two drugs
(INH, rifampicin ) three times a week.
Emergence of natural drug resistance in M. tuberculosis is a major problem in
chemotherapy of tuberculosis .This occurs by mutation with a frequency of
approximately 106 cell divisions .
Multiple drug resistance :- If the cases of tuberculosis are treated with a single
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antitubercular drug, the subpopulation of tubercle bacilli susceptible to that drug are
killed , where as populations not susceptible to the drug continue to multiply .
Therefore the use of multiple antitubercular agents in the treatment of tuberculosis is
useful. The emergence of multi –resistant tuberculosis (MDR-TB) is a very serious
problem and is defined as resistance to rifampicin and INH with or with out resistance
to one or more other drugs.
This is because rifampicin and INH form the mainstay of short term chemotherapy ,
and M. tuberculosis strains resistant to both these drugs are unlikely to respond to
treatment .
Multiple drug resistance (MDR) is of two types – primary and secondary .
Primary MDR is defined as development of resistance to antitubercular treatment in

an individual who has no history of anti tubercular treatment .
It usually occurs : In patients residing in areas with a high prevalence ( common) of drug resistant
M. tuberculosis . In those exposed to drug resistant contagious tuberculosis and In those with HIV
infections and in the individuals using intravenous drugs.
Secondary MDR(Multiple drug resistance) is defined as emergence of antitubercular

resistance during the course of infection and antitubercular treatment . It develops
usually In patients treated with inappropriate drug regimen and In those not taking
antitubercular drugs regularly .
Second line antitubercular Drugs :
Second line antitubercular Drugs are used for the cases of tuberculosis where first line
drugs become ineffective. These include a large number of old and new drugs, such as
ciprofloxacin, cycloserine, ethionamide , kanamycin , ofloxacin, levofloxacin,
capreomycin and others. Directly observed therapy (DOT) is a method being recently
followed for treatment of cases of tuberculosis .
Prevention and Control.
These include chemoprophylaxis , vaccination and general health measures .
Chemoprophylaxis or preventive chemotherapy is carried out by use of antitubercular drugs
such as INH. INH is usually used for treatment of persons with latent tuberculosis. Patients with
HIV infection. Recent contact of patients with contagious tuberculosis in past 3 months.
Unvaccinated children and Elderly person with radiological evidence of tubercular disease . These
cases are treated by INH ,given in a dose of 5mg/kg daily for 6-12 months . Results of the study
have shown that chemoprophylaxis by INH has considerably reduced the risk of acquiring the
disease nearly by 90 %.
Vaccination : Vaccination against tuberculosis is carried out by administration of

the vaccine . BCG is a live attenuated vaccine prepared from attenuated strain of M.
bovis
MALARIA
Life Cycle

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The malaria parasite life cycle involves two hosts.
During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites

into the human host .
Sporozoites infect liver cells and mature into schizonts , which rupture and release
merozoites .
(Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can persist in the liver (if
untreated) and cause relapses by invading the bloodstream weeks, or even years later.) After
this initial replication in the liver (exo-erythrocytic schizogony ), the parasites undergo
asexual multiplication in the erythrocytes (erythrocytic schizogony ). Merozoites infect red
blood cells .
The ring stage trophozoites mature into schizonts, which rupture releasing merozoites 6.
Some parasites differentiate into sexual erythrocytic stages (gametocytes) .
Blood stage parasites are responsible for the clinical manifestations of the disease.
The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested

by an Anopheles mosquito during a blood meal .
The parasites’ multiplication in the mosquito is known as the sporogonic cycle .
While in the mosquito’s stomach, the microgametes penetrate the macrogametes generating
zygotes .
The zygotes in turn become motile and elongated (ookinetes) which invade the midgut
wall of the mosquito where they develop into oocysts .
The oocysts grow, rupture, and release sporozoites , which make their way to the mosquito’s
salivary glands.
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Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle.
Plasmodia are protozoa. Only the species Plasmodium falciparum, P vivax, P malariae, and P
ovale are usually infectious for humans. Of these, P falciparum is the most dangerous.
Plasmodium enters the human body as sporozoites ( infectious form) through the bite of
infected female Anopheles mosquito. The parasite initially multiply within the liver cells and
then attack the red blood cells resulting in their rupture .
The rupture of RBCs is associated with release of a toxic substance, haemozoin, which is
responsible for the chill and high fever recurring every three to four days .
When a female Anopheles mosquito bites an infected person , these parasites enters the
mosquito’s body and undergo further development .
The parasites multiply within them to form sporozoites that are stored in their salivary
glands .
When these mosquitoes bite a human , the sporozoites are introduced into his/ her body , there
by initiating the events mentioned above.
It is interesting to note that the malarial parasite requires two hosts – human and mosquitoes-
to complete its life cycle . The female Anopheles mosquito is the vector (transmitting agent )
too.
Pathogenesis.
The fever and chills of malaria are associated with the rupture of erythrocytic-stage schizonts.
In severe falciparum malaria, parasitized red cells may obstruct capillaries and
postcapillary venules, leading to local hypoxia(deficiency in the amount of oxygen reaching the
tissues) and the release of toxic cellular products. Obstruction of the microcirculation in the brain
(cerebral malaria) and in other vital organs is thought to be responsible for severe complications.
Epidemiology.
Malaria is distributed worldwide throughout the tropics and subtropics.
Diagnosis.
Diagnosis depends primarily on the identification of plasmodium in thick and thin blood
smears.
Control.
Treatment: The widespread resistance of P falciparum to chloroquine complicates treatment of

falciparum malaria.
Alternative drugs such as mefloquine, pyrimethamine/ sulfadoxine (FansidarR), quinine,

quinidine, halofantrine and artemisinin derivatives are used.
Chloroquine remains highly effective against P malariae and P ovale malaria, and against P
vivax everywhere except Papua New Guinea and parts of Indonesia, where significant resistance
has developed.
Disease caused by P vivax and P ovale requires primaquine to eradicate latent liver forms of the

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parasite.
Prevention: Malaria may be prevented by chemoprophylaxis (the use of drugs to prevent

disease) and personal protective measures against the mosquito vector and by community-wide
measures to control the vector.
Exposure to night-feeding Anopheles mosquitoes is reduced by using protective clothing, insect

repellents, insecticides, insecticide-impregnated bed nets, etc.
Mosquitoes may be reduced by destroying breeding places and by application of insecticides.

Vaccines are being developed.
The most characteristic symptom of malaria is fever. Other common symptoms include chills,
headache, nausea, and vomiting. Diarrhea, abdominal pain, and cough are occasionally seen.
As the disease progresses, some patients may develop the classic malaria paroxysm with bouts
(of an unpleasant feeling, you have it for a short period) of illness alternating with symptom-free
periods.
The malaria paroxysm comprises three successive stages. The first is a 15-to-60 minute cold
stage characterized by shivering and a feeling of cold.
Next comes the 2-to-6 hour hot stage, in which there is fever, sometimes reaching 41°C,
flushed, dry skin, and often headache, nausea, and vomiting.
Finally, there is the 2-to-4 hour sweating stage during which the fever drops rapidly and the
patient sweats.
Malaria is transmitted primarily by the bite of infected Anopheles mosquitoes.It can also be
transmitted by inoculation of infected blood .Anopheles feed at night and their breeding sites are
primarily in rural areas.
The greatest risk of malaria is therefore from dusk to dawn in rural areas. In many malaria-
endemic areas, there is little or no risk in urban areas.
Definitive diagnosis of malaria generally requires direct observation of malaria parasites in

Giemsa-stained thick and thin blood smears.
Thick blood smears are more difficult to interpret than thin blood smears but they are much
more sensitive, as more blood is examined.
Thin blood smears, in which parasites are seen within erythrocytes, are used to determine the
species of the infecting parasite.The presence of diagnostic forms can vary markedly with the
stage of the life cycle, especially early in disease.
In falciparum malaria, most organisms are not present in the peripheral blood because they are
sequestered (bind) in the microvascular tissue of internal organs.
If malaria is suspected, blood smears should be examined every 6 to 12 hr for at least 2 days.
New diagnostic methods include a rapid antigen-capture dipstick test and a technique for
detecting parasites with a fluorescent stain. Both of these tests are fast, easy to perform and are
highly sensitive and specific
Other diagnostic methods include assays to detect malaria antibodies and antigens, and
polymerase chain reaction/DNA and RNA probe techniques. These techniques are used
primarily in epidemiologic studies and immunization trials and rarely in the diagnosis of
individual patients.
QBC test.
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Quantitative Buffy Coat (QBC) is a direct and rapid test for diagnosis of malaria. It is based
on acridine orange staining of centrifuged peripheral blood samples in a microhematocrit tube (QBC)
and examination under UV light source (fluorescence microscopy).
QBC Test System.
The acridine orange stains all nucleic acid containing cells and the associated
fluorescence is observable under blue-violet light through a microscope.
QBC Malaria Test can detect as little as 1 parasite per μL of blood and establish diagnosis
earlier than thick film . QBC is established as an effective tool for diagnosing blood parasites
that cause malaria,and filariasis.
Principle:
Acridine orange binds deoxyribonucleic acids and ribonucleic acids. The malaria parasite binds
acridine orange in the nucleus and the cytoplasm and emits green and red fluorescence when excited
by blue light (at 460 nm) allowing the detection and examination of parasite morphology by
fluorescent microscopy.
The nuclei of the parasites emit yellowish green fluorescence whereas the cytoplasm exhibits
bright red fluorescence. RBCs are not stained by the dye, hence remain inconspicuous (not
clearly visible) under fluorescent light (dark background) while the brightly fluorescent parasites
are easily seen. The outlines of stained parasites are well observed .
Sample collection: Blood sample can be collected in either capillary finger-prick in a

ethylenediamine tetra acetate (EDTA) containing vials.
About the QBC tube:
The QBC glass capillary tube (Becton Dickinson) is 75 mm in length and 1.677 mm in
diameter. The tubes are internally coated with EDTA and heparin at the fill end and with
acridine orange stain and potassium oxalate at the other end.
Procedure:
1. Draw samples of blood ( 55 µl) in to the QBC tube by capillary action.

2. Rotate the tubes for 10 seconds to dissolve the contained residues in the blood.
3. Insert a close fitting cylindrical insert or plastic float { having a specific gravity (1.055)
i.e midway between that of plasma (1.028) and red blood cells (1.090)} inside
acridine orange-coated capillary tube.
4. Centrifuge the tubes at 12,000 rpm (revolution per minute) for 5 minutes. After
centrifugation blood components and malaria parasites separate based on density, and
concentrate in distinct layers.
Note: The float by virtue of its density settles on top of the centrifuged packed red cells. It
occupies 90% cross-sectional area of the tube which aids in the expansion of the
centrifugally separated cell layers. It is surrounded by three observable and now
measurable layers of the buffy coat.

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5. Insert the centrifuged QBC Malaria test into the Paraviewer (This gives the technician a 3
dimensional view when compared to the other tests ). Position the tube so the closure end
extends over the depressed area of the holder.
6. The area surrounding the float just beneath the buffy coat was examined under oil
immersion. Individual cells within this layer were easily seen by microscopy; the
malaria parasites staining green (DNA) and orange (RNA) under blue-violet light.
7. The entire circumference of the tube was examined systematically while moving away
from the buffy coat through the erythrocyte layer.
8. Each tube was examined until parasites were detected or for a maximum of 5 minutes.
Results/Analysis Falciparum malaria in QBC

Test
If sample contains P. falciparum malaria
parasites:
1.Crescent (a phase of a planet or a moon, when it appears to have one concave edge and one
convex edge; esp., of the moon just after new moon) shaped gametocytes (1) will appear
near the interface of the lymphocyte/monocyte and platelet layers.
A small number of (2) schizonts (an infected mosquito injects immature forms of the
parasite, called sporozoites, into the person’s bloodstream.
The sporozoites are carried by the blood to the liver, where they mature into forms known as
schizonts. and (3) mature trophozoites (A trophozoite is the activated, feeding stage in the life cycle of
certain protozoa such as malaria-causing Plasmodium falciparum )may appear in the granulocyte
layer.
Ring-shaped (4) immature trophozoites will appear throughout the red blood cell layer,
with a concentration near the interface with the granulocyte layer.

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Thick and Thin Blood smears.
The direct microscopic visualization of the malarial parasite on the thick and/or thin blood
smears has been the “gold standard” for malaria diagnosis.Thick blood film samples a relatively
large volume of blood thus allowing more efficient detection of parasites (increased sensitivity).
Thick smears consist of a thick layer of dehemoglobinized (lysed) red blood cells (RBCs) which
provides better opportunity to detect parasitic forms against a more transparent background.
However, they do not permit an optimal review of parasite morphology.
Making Thick Blood Smear.
1. Using the corner of a clean slide, spread the drop of blood in a circle the size of a
(diameter 1-2 cm). Do not make the smear too thick or it will fall off the slide.
(you should be able to read newsprint through it.)
2. Allow the smear to dry thoroughly. Insufficiently dried smears (and/or smears that are too
thick) can detach from the slides during staining. You can accelerate the drying by using
a fan or hairdryer. Do not fix thick smears with methanol or heat.
3. If there will be a delay in staining smears, dip the thick smear briefly in water to
hemolyse the RBCs.
Quality Control
Visually, the smear should appear as a round to oval smear of blood about 2 cm in
diameter. It should be of such thickness that newsprint can barely be seen through the wet
or dry smear.
Limitation of Thick Smear
Making a species identification of malarial parasites may be difficult to impossible,

even for experienced technicians.
A thin film should always be examined if a definitive identification based on

morphology is required. Smears must be prepared from anticoagulated
blood within one hour after
Venipuncture ((is the process of obtaining intravenous access for the purpose of venous
blood sampling). The morphology of parasitic forms and the erythrocytes become atypical
after that time from direct action of the anticoagulant.
Thin Blood Smear.
Thin smears consist of blood spread in a layer such that the thickness decreases
progressively toward monolayer. It allows optimal assessment of the morphology of any
parasitic forms that may be present. Thin blood film is prepared similarly to that of the
differential white-cell count.
Making Thin Blood Smear
1. Bring a clean spreader slide, held at a 45° angle, toward the drop of blood on the
specimen slide.
2. Wait until the blood spreads along the entire width of the spreader slide.
3.While holding the spreader slide at the same angle, push it forward rapidly and
smoothly.

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4.Wait until the thin films are completely dry before staining.
5.Fix the thin film with methanol (100% or absolute alcohol ) for 15-30 second and let it dry
completely before staining.
Note: fixation time for ethanol is 20 minutes
Limitation of a thin smear.
Parasitic forms may be missed in light infections. In such instances, a thick film must
be examined. Smears must be prepared from anticoagulated blood within 1 hour after
venipuncture (is the process of obtaining intravenous access for the purpose of venous
blood sampling).The morphology of parasitic forms and the RBC become atypical
after that time from the direct action of the anticoagulant. Staining of the thick/thin
smear with Giemsa Stain.
Place slides into the working Giemsa stain (2.5%) for 45-60 minutes.( named after
German chemist and bacteriologist Gustav Giemsa, is a nucleic acid stain used in
cytogenetics and for the histopathological diagnosis of malaria and other parasites.)
Remove thin smear slides and rinse by dipping 3-4 times in the Giemsa buffer. Thick
smears should be left in a buffer for 5 minutes. For perfect malaria staining, the pH of
the buffer should be 7.2. Dry the slides upright in a rack.
Note: As alternates to this 45-60 minutes in 2.5% Giemsa stain, the smears could be stained for
shorter times in more concentrated stains. One alternate is 10 minutes in 10% Giemsa; the
shorter stains yield faster results, but use more stain and might be of less predictable
quality.Microscopic examination. First screen the thick/thin smear at low magnification (10× or
20× objective lens), to detect large parasites(microfilaria) then examine the smear using oil
immersion objective.
Peripheral smear. Study of malarial parasite
Cholera
Vibrio cholerae causes Cholera.Vibrio cholera are Gram negative bacilli with rounded or slightly
pointed ends . They measure 1 to 3 µm in length and 0.5- 0.8 µm in diameter. Vibrio cholera is

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strongly aerobic and grows best under aerobic conditions .
Mode of Transmission:
Vibrio cholerae usually enters the body orally through contaminated water and
food.The incubation period is short and varies from 2 to 3 days after ingestion of the
bacteria .
Symptoms:
The condition shows an abrupt onset of watery diarrhea and vomiting . Severe abdominal cramp,
possibly caused by distention of the small intestine due to excretion of large volumes of intestinal
fluid .
Vomiting is another important manifestation of cholera and occurs in early stage of the
disease. This is caused by decreased gastric and intestinal motility.
Chlorea if left untreated can lead to severe loss of fluid and electrolytes due to diarrhea and
vomiting . In patients with severe disease , diarrhea leads to dehydration and patients may die
of cardiac arrhythmia (refers to a group of conditions that cause the heart to beat irregular, too
slowly, or too quickly) and renal failure.
Laboratory Diagnosis:
Fresh stool specimen collected before administration of antibiotics is the specimen
of choice .The stool may be collected by introducing a sterile soft rubber catheter or the
liquid stool may be collected directly in a screw capped container . If delay is anticipated , the
specimens may be preserved at 40C in a refrigerator or in transport media used for V. cholera.
Microscopy : Dark field microscopy is a useful method for demonstrating characteristic
motility of the bacilli and its inhibition by antisera . This is a rapid method of
examination of stool collected from cases or after enrichment
for 6 hrs . Direct immunofluorescence is another rapid method used for demonstration of
vibrios in the stool.
Treatment :-
Treatment of cholera includes
a) replacement of fluid and electrolytes .
b) Antibiotic therapy.
Replacement of fluid and electrolytes . Replacement of fluid by oral administration of fluid

containing glucose and electrolytes is the most successful and highly effective method for
treatment of cholera.The oral rehydration therapy (ORT) solution consisting of glucose,
sodium chloride, potassium chloride and sodium citrate is widely used. The glucose facilitates
absorption of sodium in the small intestine and salts present in the ORT restore the
electrolytes balance and therapy reverse acidosis .
Antibiotic Therapy :- Antibiotic therapy is a secondary importance and is a supplement to fluid

therapy . Tetracycline or doxycycline is the drug of choice for adults and Trimethoprim-
sulphamethoxazole for children. Pyrazolidone is the usually recommended treatment for
pregnant females suffering from cholera .
HEPATITIS
Hepatitis refers to an inflammatory condition of the liver. It’s commonly caused by a viral infection,
but there are other possible causes of hepatitis. These include autoimmune hepatitis and hepatitis that
occurs as a secondary result of medications, drugs, toxins, and alcohol. liver is located in the right
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upper area of your abdomen.It performs many critical functions that affect metabolism throughout
your body, including: bile production, which is essential to digestion.filtering of toxins from your
body.excretion of bilirubin (a product of broken-down red blood cells), cholesterol, hormones, and
drugs. breakdown of carbohydrates, fats, and proteins.activation of enzymes, which are specialized
proteins essential to body functions. storage of glycogen (a form of sugar), minerals, and vitamins (A,
D, E, and K) synthesis of blood proteins, such as albumin. synthesis of clotting factors.
Hepatitis is a clinical syndrome caused by many pathogens including viruses. There are six
medicinally important viruses that are called hepatitis viruses because their main site of
infection is liver. These viruses are hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C
virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV) and newly described G virus (HGV).
These viruses infect the liver and cause distinct clinical pathology by producing characteristic
symptoms of jaundice (Jaundice is a condition that causes skin and the whites of the eyes to turn
yellow) and production and release of liver enzymes in the serum.
Hepatitis A Virus : Hepatitis A Virus (HAV) is a picorna virus (Picornaviruses are a group of
related non-enveloped RNA viruses which infect vertebrates including mammals and birds) that is
most commonly transmitted by fecal –oral route. It has a relatively short incubation period of 3-4
weeks after which jaundice starts suddenly.
Symptoms :
Fatigue (extreme tiredness resulting from mental or physical exertion or illness.), nausea, vomiting,
fever, jaundice, anorexia (lack or loss of appetite for food) and rash are the most common signs and
symptoms of the disease. The condition is also associated with passing of dark coloured urine , pale
feces and elevated serum transmittance levels (The increase in circulating cobalamin levels ).
Transmission of infection- contaminated food or water is the main source of infection. Wide
outbreak can occur from a single contaminated source, such as uncooked vegetables, infected shell
fish and contaminated food and water.
Laboratory Diagnosis .
Specimens – These include (a) serum for antibody detection and (b) liver, bile , stool and
blood for HAB antigen and genome.
Direct antigen detection – Hepatitis A virus is present in stools during 2 weeks prior to
the onset of jaundice and up to 2 weeks after the onset of jaundice .
The virus can be detected in the stool during this period by using immunoelectron
microscopy.
Serodiagnosis – Enzyme linked immune sorbent assay (ELISA) is the method of choice
for IgM and IgG antibodies in the serum.
Other tests:- liver function tests are highly useful for supplementingthe
diagnosis of HAV infection .
Hepatitis A virus infection is associated with a consistent increase in serum alanine

aminotransferase (ALT) and aspartate aminotransferase (AST). Increase in ALT and AST levels
is nearly 4 -100 times more than the normal levels . Increase in serum levels of ALT and AST
are usually seen 1 week before. The ALT and AST remain at peak level within 3 to 10 days
after the onset of clinical illness .Serum bilirubin level is also increased , and it increases with
the appearance of jaundice.

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Treatment – No antiviral therapy is available against HAV infection. Treatment of the condition is
always supportive.The hepatitis A vaccine is available to prevent this infection. Most children begin
vaccination between ages 12 and 18 months.It’s a series of two vaccines. Vaccination for hepatitis A
is also available for adults and can be combined with the hepatitis B vaccine.
Prevention and control : Prevention of HAV infection depends on a) Vaccines

b)Prophylaxis (treatment given or action taken to prevent disease) with immune serum globulin and
c) measures to prevent feco –oral spread of infection .
Hepatitis B :-
Hepatitis B virus is a major cause of infectious hepatitis worldwide . Individuals with chronic,
HBV infection are the major reservoir of HBV infections . Those people with HBeAg in their
serum tend to have high viral titers and thus greater infectivity . Hepatitis B virus is present at a
high level in the serum.
The Hepatitis B virus can be transmitted in the following ways .
Perinatal transmission : This is a major route of transmission of the virus world wide .The
transmission occurs from infected mother to child due to contact with mother’s infected blood
during the time of delivery.
Parenteral transmission: This transmission occurs due to transfusion of HBV infected blood and
blood products .Patients with hemophilia (Hemophilia is usually an inherited bleeding disorder in
which the blood does not clot properly. This can lead to spontaneous bleeding as well as
bleeding following injuries or surgery. ), renal dialysis and those receiving organ transplantation and
intravenous drug users remain at increased risk of infection.
The risk of acquiring HBV among health workers after needle stick injury from infected
individuals is estimated to be as high as 5%. Sexual transmission of HBV.
Laboratory Diagnosis .
Serodiagnosis (a diagnosis involving tests on blood serum or other serous (any body fluid)
fluid of the body)– Diagnosis of acute infection is made by demonstration of HBsAg as
well as HBeAg in the serum. (Hepatitis B e antigen (HBeAg) is a small polypeptide that exists
in a free form in the serum of individuals during the early phase of hepatitis B infection, soon
after hepatitis B surface antigen (HBsAg) becomes detectable. Serum levels of both
HBeAg and HBsAg rise rapidly during the period of viral replication.)Both HBsAg and
HBeAg are the important serum markers of acute HBV.
They indicate viral replication. When viral replication slows, HBeAg disappears and anti-HBeAg
is detected.Hepatitis B surface antibody (HBSAb) produced may persist for many years . This is
followed by demonstration of IgM antibodies against hepatitis B core antigen (HBcAb).
Other Tests:
These tests include elevation of ALT and AST .(alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) . High levels are found in acute hepatitis (1000- 2000
IU/ml).
Treatment :- No specific antiviral treatment is available for patients with acute HBV
infection. Supportive and symptomatic care continue to be the mainstay of therapy for most
of the patients. Therapy is recommended for patients with chronic hepatitis B infection.
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Interferon and nucleoside analogs , such as lamivudine , adefovir and telbivudine are the
antiviral drugs used widely.
Hepatitis C virus :- Hepatitis C virus is a flavivirus (have single-stranded RNA as their genetic
material) with an RNA genome and is the most important cause of parenteral non-A ,non-B
hepatitis world wide.
Hepatitis C is exclusively a human disease .Patients who are infected with the virus are
the important reservoir of infection . Blood or blood products and also organs of infected
patients are the major sources of infection .
Hepatitis C can be transmitted by following methods .
Blood transfusion . Blood transfusion is the most important route of transmission of HCV.
The current risk of transfusion derived HCV is estimated to be one case in every 100,000
units transfused .
Parenteral transmission. HCV is transmitted parenterally

a) through transfusion of infected blood or blood products ,
b) transplantation of organs from infected donors and
c) also by sharing of contaminated needles among intravenous drug users. The use of
intravenous drugs is most important risk factor responsible for around 50% of both acute
and chronic infections .
Sexual transmission : Sexual transmission is believed to be responsible for approximately

20 % of cases of hepatitis C .
Perinatal transmission:- Perinatal transmission is possible and is observed in fewer than

5% of children born to HCV infected mothers.
Laboratory Diagnosis :
ELISAs including , Hepatitis C virus genotyping .
Treatment : A combination therapy of pegylated interferon (Pegylation of the interferon

increases the amount of time the interferon remains in the body by increasing the size of the
interferon molecule.) and antiviral agent ribavirin.
Interferons are a group of signaling proteins made and released by host cells in response to the
presence of several viruses. In a typical scenario, a virus-infected cell will release interferons
causing nearby cells to heighten their anti-viral defenses.
Hepatitis D Virus :- Hepatitis D virus is the smallest of known human pathogens that
causes infections in humans.
It is an RNA virus , which is structurally unrelated to hepatitis A, B or C virus. Hepatitis D
virus is unique in being an incomplete virus and requires the presence of HBV to
replicate and infect other hepatocytes (Hepatocytes are the chief functional cells of the liver
and perform an astonishing number of metabolic, endocrine and secretory functions.).
Hence ,HDV infection occurs in those patients who suffer from HBV infection.
Infection appears to be more commonly transmitted through contaminated blood and blood
products . Sharing of contaminated needles in intravenous drug users is believed to be the
most common method of transmitting HDV. The sexual and perinatal transmission of HDV
is also described.

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Reverse transcriptase PCR (Reverse transcription polymerase chain reaction is a laboratory

technique combining reverse transcription of RNA into DNA and amplification of specific
DNA targets using polymerase chain reaction)is the most sensitive method for detection
of HDV RNA in blood in the stage of co-infection.
Treatment : No specific therapy is available for treatment of HDV infection of liver.

Lamivudine and ribavirin appear to be ineffective against HBV and HDV co- infection.
Antiviral therapy with interferon is also ineffective in patients with chronic infections .
Vaccination with HBV vaccine protects against subsequent HDV infection . HDV virus is
prevented best in patients already infected with HBV by avoiding the use of HDV
contaminated blood or blood products.
Hepatitis E virus : -
Hepatitis E virus (HEV) is the primary cause of enterically transmitted non-A non –B hepatitis
virus(NANBH) ,most commonly seen in developing countries including India. The virus has many
similarities with HAV. The virus was first observed during the electron microscopy of feces
contaminated with enteric NANHB.
Hepatitis E virus is currently classified in the family of Calciviridae (They are positive-sense, single-
stranded RNA which is not segmented).
Hepatitis E virus is transmitted primarily by fecal oral route due to fecal contamination of water in
endemic areas.
Tropical climate , poor sanitization and poor personal hygiene all contribute to the epidemic of
the disease in developing countries.
Hepatitis E virus usually causes an acute , self limiting disease similar to HAV. Earlier it was
mistaken for HAV due to clinical and epidemiological similarity.
The incubation period of HEV infection varies from 2 to 9 weeks with an average of 35 days .
Hepatitis E virus causes a serious infection in pregnant women.
It causes fulminant disease ( a rare syndrome of massive necrosis of liver parenchyma and a
decrease in liver size (acute yellow atrophy) that usually occurs after infection with certain hepatitis
viruses, exposure to toxic agents, or drug- induced injury.) in pregnant women, especially in last
trimester of pregnancy and has a high fatality rate of 15 to 20%.
Diagnosis :-
The serodiagnosis of HEV infection is carried out by Western blot and ELISAs .
HEV IgM and IgG helps to differentiateacute and chronic infections.
PCR is also used to detect HEV RNA in serum and stool specimen of infected patients.
Hepatitis G virus.
Hepatitis G virus (HGV) is similar to viruses of Flaviviridae family(of positive, single-stranded,

enveloped RNA viruses), which includes HCV. Hepatitis G virus is an RNA virus and its genome
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codes for 2900 amino acids . Hepatitis G virus is a blood borne virus , which is transmitted by
transfusion of contaminated blood or blood products . HGV co-infection is observed in 6% of
chronic HBV infection and in 10% of chronic HCV infection.
Although HGV RNA has been demonstrated in patients with acute , chronic and fulminant
hepatitis , patients with multiple transfusions and hemodialysis , blood donors and intravenous
drug addicts , its role in the pathogenesis of hepatitis is yet to be elucidated .
Meningitis
Neisseria meningitidis causes meningitis .
Meningitis is an inflammation of the membranes (meninges) surrounding the brain and

spinal cord. The swelling from meningitis typically triggers symptoms such as headache,
fever and a stiff neck
Morphology : N. meningitidis are Gram negative , spherical or oval cocci arranged in

pairs with the adjacent sides flattened . The cocci are generally intracellular in PMN in
smears (A PMN is a type of white blood cell. Also called granular leukocyte, granulocyte,
and polymorphonuclear leukocyte ) from pus cells and other specimens .They measure
0.6- 0.8 µm in diameter.Freshly isolated bacteria are usually capsulated .
They are non motile and nonsporing.
Culture : Meningococci are strict aerobes. They grow optimally at a temperature between 360C and
390C and optimum pH of 7.4 – 7.6 .Their growth is enhanced by incubation in a moist
atmosphere in the presence of 5% CO2.
Meningococci are fastidious bacteria (A fastidious organism is any organism that has
complex or particular nutritional requirements) with complex nutritional requirements .
They do not grow on ordinary media , but grow well on the medium enriched with blood
or serum, such as blood agar , chocolate agar and Mueller –Hinton agar.
Identifying features of N. meningitidis.
Gram -ve diplococci arranged in pairs. On blood agar ,produces convex gray , and translucent
colonies.
Oxidase test positive. Catalase test positive .

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N. meningitidis on Blood agar
Ferments glucose and maltose with production of acid .Does not ferment sucrose or lactose.
Oxidase Test : The test can be performed in two ways : In the first method , 1% solution of
oxidase reagent ( tetramethyl paraphylene -diamine dihydrochloride ) is poured on the culture
media – The Neisseria colonies turn deep purple .
In the second method , a few colonies of Neisseria are rubbed with a glass rod on a
strip of filter paper moistened with oxidase reagent . A deep purple colour develops
immediately.
Cell wall components and Antigenic structure :
The cell wall of pathogenic meningococci contains a toxic LPS(

lipo polysaccharide) of endotoxin .
Antigenic structures :
Depending on group specific capsular polysaccharide antigens , meningococci are
subdivided into 13 serogroups (A, B, C, D, X, Y, Z, W 135, 29E, H, I, K and L).
Meningococci belonging to group A,B and C are responsible for most of the
epidemics and out breaks of meningitis.
Group Y and group W 135 meningococci cause disease more commonly than
groups X and Z.
Pathogensis and Immunity :
N. meningitidis colonizes the human nasopharynax , and under specific conditions

, invades the blood stream and then reaches the brain, causing meningitis .

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Virulence factors :
N. meningitidis has three important virulent factors , which are responsible for causing
diseases . These are (a) capsular polysaccharides b) LOS endotoxin and c) IgA
protease.
Capsular polysaccharides : N. meningitidis is surrounded by a prominent

polysaccharide capsule that is antiphagocytic.The capsule is an important virulence
factor, which contributes to the virulence by inhibiting phagocytosis . The capsule
protects meningococci from destruction by the leukocytes inside the phagocytic vesicle
of the leukocyte, they survive intracellular death , multiply and then migrate to sub-
epithelial spaces.
LOS Endotoxin : LOS Endotoxin is present in the outer membrane of N.

meningitidis. It is responsible for damage of blood vessels associated with
meningococcal infections. The endotoxin comprises two antigenic
determinant components
a) a protein component and
b) a carbohydrate component .
The continuous production and release of endotoxin by N. meningitidis cause

severe endotoxin reaction , seen in patients with meningo coccal disease .
IgA protease : IgA protease is the other important virulence factor .The enzyme acts
by clearing the secretory IgA, thus helping the bacteria to attach to the epithelial
cells of the upper respiratory tract .
Pathogensis of meningitis : Initially N. meningitidis causes a localized infection by

colonizing the nasopharynx. From this site , the meningococci invade the submucosa by
circumventing the host defense mechanisms and gain access to the central nervous systems
(CNS) , meningo cocci reach CNS by the following ways .
1. Invasion of blood stream : This is the most common mode of spread of

meningococci. Once inside the blood stream, the meningococci escape the immune
surveillance ( eg. antibodies, complement mediated bacterial killing, neutrophil
phagocytosis ) of the host and subsequently reach distant sites including the CNS.

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2. Direct contiguous spread : Meningococci can also reach the CNS by direct
contiguous spread from nasopharynx. Inside CNS , the bacteria multiply and survive
because host defense mechanisms ( such as immunoglobulins, neutrophils and
complement) appear to have limited role in controlling multiplication of bacteria
continues in the CSF which subsequently causes a cascade of meningeal
inflammation.
Source and Transmission of infection .
Human is the only reservoir of meningococcal infection . Nasopharyngeal secretion is

the most common source of infection . Meningococci are transmitted by airborne
droplets of infected nasopharyngeal secretions ( the most common source of infection ).
Family members living in crowded conditions and older people are more
susceptible to infections.
Clinical Syndromes :
Meningo coccal meningitis caused by N. meningitidis is most common in children

and young adults . It is a febrile illness ( a sudden fever or elevation in body
temperature) of short duration characterized by headache and stiff neck .
Lethargy or drowsiness is frequent . Confusion , agitated delirium and stupor (delirium
is used to describe an acute confusional state; stupor, a state in which vigorous stimuli
are needed to elicit a response) are rarer .
Mental obtundation (refers to less than full alertness/ dulled or less sharp), stupor and
coma due to increased intracranial pressure are some of the noted complications at
the end stage of the disease .
Specimens . Cerebrospinal fluid and blood are the specimens of choice for
demonstration of meningococci in the early stage of meningitis. Nasopharyngeal
swabs are useful to detect carriers .
The CSF is collected by lumbar puncture (the procedure of taking fluid from the spine
in the lower back through a hollow needle) and blood by venipuncture in strict
aseptic conditions. (venipuncture -the puncture of a vein as part of a medical
procedure, typically to withdraw a blood sample or for an intravenous injection).

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Microscopy :
Gram staining of the CSF is a very useful method for determination of

meningococci.
Culture : Isolation of N.meningitidis from the CSF , blood confirms the diagnosis
of meningococcal infection. The CSF is inoculated immediately on a nonselective
media , such as blood agar or chocolate agar , and incubated at 35 -36 0C under 5%
CO2 for 18-24 hours . The colonies of meningo cocci are small, round, translucent,
and convex with a smooth glistening (shining with a sparkling light) surface .
Blood is inoculated immediately into blood culture bottles containing either glucose
broth or sodium taurocholate broth and incubated at 35 -36 0C in the presence of
CO2 .
Antigen detection : Detection of soluble polysaccharide antigen in the CSF is a
useful method for diagnosis of meningococcal meningitis , counter current
immunoelectrophoresis , latex agglutination test and bacterial coaggluation test using
specific antibodies are the rapid tests frequently used to detect the soluble antigen in the
CSF.
Serodiagnosis : Indirect hemagglutination test and ELISA are useful for the demonstration of
antibodies against specific polysaccharide antigen in the serum.
Treatment :
Intravenous penicillin G is the recommended drug for treatment of meningo coccal
disease. The MIC of penicillin usually ranges from 0.01 to 0.05µgm/ml.
Chloramphenicol , rifampicin, erythromycin, tetracycline and cephalosporins
(ceftriaxone, cefotaxime,cefuroxime) are useful in the treatment of bacterial
meningitis . Ceftriaxone has an additional advantage of eradicating the
nasopharyngeal carriage of meningococci. Chloramphenicol is useful for
patients who are allergic to penicillin.
Prevention and control :
This include chemoprophylaxis and vaccines .
Chemoprophylaxis : Antimicrobial chemoprophylaxis of close contacts is the key factor for

preventing secondary cases of sporadic meningococcal disease (occurring at irregular intervals or
only in a few places; scattered or isolated) .Person to person transmission can be interrupted by
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administration of antibiotics , which eradicate the asymptomatic nasopharyngeal carrier state .

Sulfonamide , rifampicin , ciprofloxacin and ceftriaxone are the drugs frequently used eradicate
meningococci from the nasopharynx. However ciprofloxacin is not recommended for children ,
because it has been found to cause cartilage damage in immature experimental animals .
Immunoprophylaxis : Immunoprophylaxis by vaccination with group specific

meningococcal capsular polysaccharides of groups A,C,Y and W135 meningococci is
very useful for prevention of meningococcal disease.
Vaccines :
Quadrivalent meningococcal polysaccharide vaccine ( MPSV4) : It has been
shown to be highly effective in preventing disease caused by A,C,Y and W135
serogroups of meningo cocci. The vaccine is given intra muscularly. Use of this
vaccine is indicated for population at risk during outbreak of infection caused by one
of these serogroups of meningococci. These vaccines developed against group .C.Y
and W135 are poorly immunogenic under 2 years of age . These vaccines , however
produce good antibody response in children above 2 years of age.
Tetravalent meningococcal polysaccharide -protein conjugate vaccine (MCV4) :
MCV4 is being used for the persons aged 11 – 55 years for vaccination against
meningococci in the United States . This is recommended for groups of population
at risk , which include a) military recruits b ) travelers to areas epidemic to
meningo coccal disease.
Syphilis
Treponema pallidum is the causative agent of syphilis, the most common sexually
transmitted disease.
Morphology.
T. pallidum shows the following morphological features . T. pallidum is a thin, coiled spirochete. It
measures 0.1µm in breadth and 5-15 µm in length. It has six to ten sharp and angular coils ,
which are present at regular interval of 1 µm. It is actively motile. T. pallidum is too thin to be seen
by microscopy in specimens stained by simple Gram staining.
Culture : T. pallidum does not grow in artificial culture media . T. pallidum had been
maintained for a long time by subculture in animals .
Source and transmission of infection.
T. pallidum is a strict human pathogen and does not naturally occur in any animal
species . Humans are the only natural hosts .Infected human hosts secreting T. pallidum in
serious transudates (Exudate is fluid that leaks around the cells of the capillaries caused by
inflammation) from moist lesions such as primary chancre ( a non painful ulcer), condyloma
latum (Any knob-like or warty growth on the genitals ), mucous patch (a broad, flat,
erosive lesion of secondary syphilis that occurs on moist skin or mucous membranes and is

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often marked by a yellowish discharge During the secondary stage of syphilis, mucous
patches) etc. are the sources of infection .
Transmission of syphilis occurs Primary through sexual contact by inoculation of the

spirochetes through mucosal membranes and abrasions on epithelial surfaces. By vertical
transmission transplacentaly . Vertical transmission of early syphilis during pregnancy
results in a congenital infection in at least 50-80 % of exposed neonates. By transfusion of
T. pallidum contaminated blood .
Pathogenesis of syphilis :
T. pallidum causes disease by invasion and multiplication at the site of infection,
then spreading via circulation and producing disseminated disease ( infectious)
.Immune response of the host is believed to be responsible primarily for tissue
destruction and for causing pathogenic lesions observed in patients with syphilis.
On sexual contact T. pallidum from infected person is passed to another through
intact mucous membrane or through minor skin abrasions.The organism invade the
skin at these lesions and multiply at the site of infection.Chancre is the primary
lesion, which develop at the site of infection .Subsequently , the treponemes get
transmitted in the blood stream and produce disseminated lesions ( papular skin
rashes, mucous patches in the oropharynx etc.)
Specimens : Specimens for microscopy includes serous transudates from moist lesions ,
such as primary chancre ( a non painful ulcer), condyloma latum (Any knob-like or warty
growth on the genitals ) , mucous patch (a broad, flat, erosive lesion of secondary syphilis
that occurs on moist skin or mucous membranes and is often marked by a yellowish
discharge During the secondary stage of syphilis, mucous patches) etc. Serum is used for
serodignosis and cerebrospinal fluid (CSF) is used for diagnosis of neurosyphilis.
Microscopy : Dark field microscopy is useful for diagnosis of primary , secondary or

congenital syphilis by demonstration of treponemes in the clinical specimen .Dark field
microscopy is particularly useful for diagnosis early in the disease before appearance of
serum antibodies .Dark field microscopy although useful has many limitations . First it is
reliable only when examined by an experienced analyst. Specimen from oral cavity cannot
be used because saprophytic nonpathogenic treponemes are present as normal flora of the
oropharynx.
Direct antigen detection : Direct fluorescent antibody T. pallidum (DFA- TP) is a sensitive
and better method for direct detection of treponemal antigen in the exudates for diagnosis of
syphilis. The test using fluorescent tagged T. pallidum antibodies is used to detect
treponemal antigen directly in the acetone fixed smears of the exudates . The test is 85 to
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92% sensitive.
Serodiagnosis :- Serology is the mainstay in the diagnosis of syphilis . The serological test
depending on the nature of the antigen used can be classified as
a) nontreponemal tests and b) treponema specific tests .
Nontreponemal tests- Nontreponemal tests are non specific serological tests used for the
diagnosis of syphilis .
These tests are also called standard tests of syphilis .This group of tests use nontreponemal
antigen ( known as cardiolipin) and detect reagin antibodies (Most reaginic antibodies are
the immunoglobulin E (IgE) fraction in the blood. It works by detecting the nonspecific
antibodies that your body produces while fighting the infection. ).
VDRL Test : VDRL test is a slide flocculation test used widely for the diagnosis of
syphilis. The test is so named because it was developed first in the Venereal Disease
Research Laboratory , New York.
This is a simple and more rapid test, which uses cardiolipin antigen with added lecithin and
cholesterol. In this test the serum is inactivated at 56 0C for 30 minutes and a measured
volume of serum is placed on a special cavity slide .
The cardiolipin antigen after preparation is added to serum sample on a slide and is
rotated on a VDRL rotator for a specified period of 4 minutes.The reaction is read
under a low power objective of microscope .
In a positive test , the cardiolipin antigen reacts with reagin antibodies present in the
infected serum and forms the visible clumps.
In a negative test, cardiolipin continues to remain as uniform crystals in the
serum.
The result of the test is reported as reactive, weak reactive or non reactive
depending on the extend of the formation of the clumps.

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VDRL test can also be performed on CSF samples .
Unlike serum samples, CSF samples are not heated prior to the test. The VDRL test becomes
positive 4-5 weeks after exposure to T. pallidum and 1-2 weeks after appearance of chancre. The
VDRL test is a highly sensitive test. Sensitivity of the test depends on the stage of the disease.
Treponema specific tests : The Treponema specific tests measures antibodies specific for T.
pallidum. These tests use
a) live T. pallidum strains (T. pallidum immobilization test),
b) killed T. pallidum (T. pallidum agglutination test)
c) T. pallidum extracts as antigens Enzyme immunoassay (EIA) .
Treatment : Penicillin is the drug of choice for treatment of all the stages of syphilis .A
single intramuscular dose of benzathine penicillin G ( 50000 units /kg , not to exceed 2.4
million units) is effective for treatment of primary , secondary and early latent syphilis .
Doxycycline or tetracycline may be used for non pregnant patients allergic to penicillin .
HIV
Human immunodeficiency virus (HIV) is a retro virus that causes acquired
immunodeficiency syndrome ( AIDS).HIV is a Lentivirus , a sub family of Lentiviridae in
the family of retrovirus.
Morphology : HIV is a spherical enveloped virus , which measures up to 120 nm in

diameter.
It has a unique three layered structure.
i) the innermost genome layer .
ii) middle cone shaped nucleocapsid and
iii)an outer membrane of glycoprotein surrounded by lipoprotein envelope .

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Schematic diagram of HIV
Viral genome :
HIV genome is most complex of human retroviruses .
The genome is diploid and consists of two identical copies of single stranded
positive sense RNA genome .The HIV genome is most complex of human
retroviruses .
It contains three major genes gag, pol and env characteristic of all retroviruses .All these genes
encode for the structural proteins .
Source and transmission of infection .
HIV is primarily a human infection .
Humans infected with HIV and AIDs are the reservoir of infection .
The high titer of HIV is found in the blood, semen and vaginal secretions of the
infected people; hence these are important sources of infection .The virus is also
present in the breast milk of an infected mother.
Transmission of HIV infection .
Sexual transmission : HIV is transmitted primarily through sexual contact and

constitutes more than 70% of the HIV transmission .Sexual transmission is more
common in heterosexual women and men than in homosexual men world wide.Varied
sexual behaviors such as a) more number of sexual partners b) Sex with commercial
sex workers and homosexuals.
Transmission by blood transfusion . HIV is also transmitted by transfusion of

infectious blood or blood products , such as serum , plasma and cells from HIV
positive individuals .
It can also be transferred by the organs donated from the HIV positive individuals .

2
Parenteral transmission : Parenteral transmission occurs largely among drug users

.Injection users of illicit drugs are commonly infected through the use of contaminated
needles .
Mother to child transmission : Mother to infant transmission can occur by vertical

transmission or by perinatal transmission .
Vertical infection occurs in the following ways .
1. The fetus in uterus can be infected by vertical transmission of virus through the
placenta or through the amniotic membrane if the membrane are infected or inflamed .
Vertical transmission is most common during delivery of the baby of the infected
mother. The risk of vertical transmission is greatly increased with an increase in
duration of contact with the maternal blood and cervical vaginal secretions.
2. Perinatal infection in HIV occurs during the birth process or through breast
feeding.
Transmission of HIV during breast feeding usually occurs by 6 months.
Clinical Syndromes:
The course of untreated HIV infection is usually 10 years or longer . The disease
progress through the stages of
a) Primary infection
b) Dissemination of virus to lymphoid organs
c) Clinical latency and
d) A late stage of profound immunosuppression known as full blown AIDS .
HIV is associated with the following syndromes .
Acute HIV infection .
Acute HIV infection is characterized by rapid rise in plasma viremia (the presence
of viruses in the blood) with concomitant drop in CD4 count (CD4 (cluster of
differentiation 4) is a glycoprotein found on the surface of immune cells such as T
helper cells, monocytes, macrophages, and dendritic cells.) (A normal range for CD4
cells is about 500-1,500) after an incubation period of 3 to 6 weeks .
The symptoms of HIV are non specific and include low grade fever , fatigue ,
malaise , rash , head ache and lymphadenopathy (of an inflammatory type , producing
swollen or enlarged lymph nodes), spontaneous resolution may occur within weeks .
HIV antibodies are usually absent in the serum at the onset of the illness, but begin
to appear after 3 to 4 weeks of the infection .
Asymptomatic HIV infection : This period is followed by an asymptomatic or

clinically latent stage during which the patient continues to remain asymptomatic

2
for several months to years .This stage is characterized by a low level of viral
replication and gradual fall in CD4 count.The serum is positive for HIV antibodies in
these patients.Another characteristic of the stage of latency is persistent generalized
lymphadenopathy (of an inflammatory type , producing swollen or enlarged lymph
nodes), which may last for several years .During this stage , virus continuous to
replicate in the lymph nodes . This is a benign condition but may progress to AIDS
related complex or AIDS.
AIDS related complex. (ARC) : Aids related complex is characterized by

lymphadenopathy (of an inflammatory type , producing swollen or enlarged lymph
nodes), and fever. This has an insidious onset and may be associated with malaise (a
general feeling of discomfort, illness,) and weight loss. Diarrhea , night sweats ,
fatigue and opportunistic infections are the presenting symptoms . The patients with
ARC may progress to AIDS in a few months.
AIDS : AIDS is the end stage disease of the HIV infection .
It denotes the irreversible breakdown of immune system of the host making the
infected host highly susceptible to a wide range of progressive opportunistic infections
or unusual malignancies.
AIDS is characterized by deterioration of immune response as evidenced by CD4 cell

decrease response .The onset of clinical manifestation correlates with A reduction in
number of CD4 T cells to less than 450/µL. Increased level of virus in the blood .
Presence of p24 antigen in the blood . When CD4 count falls less than 200/µL, the
patient develops full blown AIDS. This stage is characterized by development of HIV
wasting syndrome with weight loss and diarrhea for 1 month.
This is also associated with many opportunistic infections such as TB, pneumonia,
meningitis and other diseases.
Serodiagnosis .
Serodiagnosis includes demonstration of antibodies and viral antigens .
Demonstration of antibodies . Detection of specific antibodies to HIV in

serum is the most commonly used method for serodiagnosis of patients with HIV
and AIDS .
Detectable level of antibodies is demonstrated in most individuals with in 6 – 12 weeks

after infection and in all the individuals within 6 months of infection .
The antibody based serological testing in HIV is of two types.
a) Screening test b)supplementary or confirmatory tests .
Screening tests :
ELISA : ELISA is the most frequently used test for detection of both HIV -1 and HIV
– 2 specific antibodies in the serum .

2
This test is highly sensitive and specific and commercial ELISA kits are available ,
which detect both HIV -1 and HIV -2 antibodies in the serum .
ELISA can also be used for demonstration of antibodies in the saliva . This is very
useful for testing injectable drugs users from which it may be difficult to collect blood
due to collapsed blood vessels .
Rapid tests : Rapid tests include dot blot assay , latex agglutination , gelatin
agglutination , HIV spot and comb test etc.
These tests are simple tests , which can be performed in any laboratory with out
requiring any expensive instrument or skilled man power .
Moreover , test results can be read rapidly within 30 minutes of receipt of the specimen
.
Supplementary or confirmatory tests : Western blot , line immunoassay and indirect

immunofluorescence assay are the most commonly used serologic confirmatory tests .
Western blot : It is the most common confirmatory test used in HIV serology .
In this test , HIV viral antigens are separated as gp 160, gp 120 , p66, p55, pp51, gp41,
p31, p24, p17 and p15 depending on their electrophoretic mobility by polyacrylamide
gel electrophoresis .
These antigens are then blotted on to strips of nitrocellulose paper.
These strips are treated with test serum .
Antibodies to these HIV proteins , if present in test serum , combine with different
fragments of HIV and then react with enzyme conjugated antihuman globulin .
These strips are washed , followed by addition of a suitable substrate , which produces
coloured bands.The position of the coloured band on the strip indicates the antigen
with which the antibody has reacted .The demonstration of multiple bands indicates a
positive test .
The test is considered positive if it shows bands against at least two of the three viral
proteins , namely p24, gp 41 and gp 120 and gp 160 .The test is considered positive if
multiple bands are seen with multiple proteins, which are encoded by three genes (gag,
pol and env).
This represents p24 of gag gene core protein , p31 of pol gene reverse transcriptase
,gp41 ,gp120 or gp 160 of env gene surface antigens .
Treatment :
Antiretro viral treatment is the mainstay in HIV treatment .
The goals of antiretroviral therapy are to inhibit replication of HIV

and to reduce morbidity and death .

2
Anti HIV drugs : The anti HIV drugs can be broadly classified
as a) nucleoside analog reverse transcriptase inhibitors (NRTIs)
b)non -nucleoside analog reverse transcriptase inhibitors (NNRTIs) or
c) protease inhibitors .
Antiretro viral drugs against HIV.
Nucleoside reverse transcriptase inhibitors(NRTIs).
Zidovudine (AZT), Didanosine, Lamivudine,Stavudine.
Non nucleoside reverse transcriptase inhibitors (NNRTIs).
Nevirapine, Delaviridine.
Protease inhibitors (PI).
Ritonavir, Indinavir , Nelfinavir.
Nucleoside analog reverse transcriptase inhibitors (NRTIs) – these inhibit the enzyme
reverse transcriptase and alter their incorporation into DNA to cause chain
termination .
These agents prevent the spread of the virus to uninfected cells .
Zidovudine is recommended for treatment of asymptomatic or mildly symptomatic

people with CD4 count of less than 500/µL.
This is also recommended for treatment of pregnant women to reduce the possibility
of transmission of virus to the fetus. The toxicity associated with high doses of
AZT (Zidovudine) and the emergence of resistance to AZT is the main disadvantage
of monotherapy with AZT. Zidovudine is also used effectively to reduce significant
transmission of HIV from mother to infant . The treatment decreases vertical
transmission at all levels of maternal viral load.
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) inhibit the enzyme by

blocking the morphogenesis of the virion by inhibiting the cleavage of the Gag and
Gag core polyproteins .This in turn prevents activation of the virion.
Protease Inhibitors : Protease Inhibitors prevent the maturation of viral particle during
the late stage of viral replication.
Gonorrhea .
The genus Neisseria consists of Gram –ve , aerobic , non sporing , non motile cocci
typically arranged in pairs ( diplio cocci) with adjacent sides flattened together .The bacteria
belongs to the genus are oxidase positive and mostly catalase positive . They ferment sugars
with production of acid but not gas.
The genus Neisseria consists of 10 species . Neisseria gonorrhoeae and Neisseria meningitidis

2
are the two important species that cause human infections .Neisseria gonorrhoeae is a strict
human pathogen . It is the causative agent of gonorrhea , one of the most common sexually
transmitted disease world wide.
Morphology – N. gonorrhoeae are Gram –ve , aerobic , diplio cocci. They are mostly
intracellular found within the polymorphonuclear ( PMN) leukocytes (A type of immune cell
that has granules (small particles) with enzymes that are released during infections, allergic
reactions, and asthma. ... A polymorphonuclear leukocyte is a type of white blood cell. Also
called granular leukocyte, granulocyte )– and some cells contains as hundred cocci.
Culture : N. gonorrhoeae is a fastidious (requires complex media ) coccus .
It requires complex media for growth . The cocci grow on enriched media , such as blood or
chocolate agar. These cannot grow on ordinary media , such as nutrient agar .
They are aerobes , but can also grow anaerobically . They grow optimally at a temperature
range of 35 –36 0C. They fail to grow at temperature less than 250C or greater than 37 0C.
Transmission of disease :
Only humans especially asymptomatic infected men and women are reservoirs of infections.
Purulent urethra (discharging pus) or cervical discharge is the most common sources of
infection
The infection is transmitted primarily by sexual contact.
Clinical syndromes :
Gonorrhea is sexually transmitted disease . It is primarily a genital infection restricted to the
urethra in men and cervix in women . The incubation period varies from 2 to 8 days.
Gonorrhea in men - A symptomatic acute infection is seen in approximately 95% of all
infected men. Urethritis) (inflammation of the urethra) is the major clinical manifestation , with
burning micturition (the action of urinating) and serious urethral discharge as the initial
manifestation . Subsequently the discharge becomes more profuse (abundant) , purulent
(discharging pus ) and even blood tinged .
Gonorrhea in women - In women, endocervix is the primary site ( 80-90 %) of infection

because gonococci invade only the endocervical columnar epithelial cells . The presence of
vaginal discharge , dysuria (painful or difficult urination) and mild lower abdominal pain are
the common symptoms in women.

2
Laboratory diagnosis :
Specimens : The genital ( urethral discharge , cervical discharge etc) , rectal and pharyngeal
specimens are collected for the isolation and identification of gonococci.
The specimens are inoculated on a non selective medium ( blood agar or chocolate agar) and on
a selective medium ( eg. modified Thayer Martin medium).
Neisseria gonorrhoeae on chocolate agar.

The colonies of gonococci on chocolate agar after 48 hours of incubation at 35 - 36 0C in
the presence of 5 -10 % CO2 are small, round , translucent and convex with finely granular
surface.
On Thayer Martin medium , the colonies show similar morphology as that on chocolate agar .
The mixed microbial flora present in the clinical specimens is suppressed by the selective
media.
However the vancomycin present in the selective media inhibits some strains of gonococci.
Detection of gonococcal antigen : The gonococcal antigen can be detected by both direct
fluorescent antibody test and direct enzyme immune assay (EIA ) in urethral discharge and
endocervical discharge and other clinical specimens .
Treatment :
Penicillin is the drug of choice for penicillin - sensitive strains of N. gonorrhoeae.
Alternative drugs in case of penicillin resistance or in penicillin allergic individuals
.Ceftriaxone , Cefixime, Ciprofloxacin or Ofloxacin .A single dose of any of these antibiotics
is given as an initial therapy in uncomplicated urethritis ((inflammation of the urethra),
cervicitis or rectal or pharyngeal infections in adults.
A single dose of Ceftriaxone 125 mg intramuscularly or cefixime ( 400 mg), Ciprofloxacin

(500mg) or Ofloxacin( 400 mg) as a single dose orally is also effective.

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1

Chapter 1. Introduction to the Science of microbiology. Major divisions

EPCP Department of Pharmacognosy Page 1

EPCP Department of Pharmacognosy Page 2

Pasteur made another valuable contribution to microbiology by developing an attenuated

EPCP Department of Pharmacognosy Page 3

1) The organism is regularly found in the lesions of the disease.

EPCP Department of Pharmacognosy Page 4

All biological systems have the following characteristics in common.

There is no field of human endeavour ( attempt) , whether it be in Industry or agriculture , or in

EPCP Department of Pharmacognosy Page 7

Features distinguishing Prokaryotic from Eukaryotic cells.

EPCP Department of Pharmacognosy Page 8

(Ribosome- The site of protein

Cytoplasmic nature and Absent Absent

Ribosomes 70S’ distributed in the 80 S’ arrayed on membrane as in

S’ refers to the sedimentation coefficient of a particle in the ultra centrifuge.

Bacteria are prokaryotic microorganisms . The Eucaryotic microorganisms include the

EPCP Department of Pharmacognosy Page 9

system of classification is based on three levels of cellular organization which evolved to

1.Contribution of Louis Pasteur.2 marks.?Repeated.

EPCP Department of Pharmacognosy Page 10

2.Koch’s postulates?. 2 marks.Repeated.

EPCP Department of Pharmacognosy Page 11

Chapter 2. Different methods of Classification of microbes and study of

Procryotes with a cell wall structure characteristic of Gram + ve bacteria.

Prokaryotes that lack a cell wall .

Bergey’s manual is the international standard for bacterial taxonomy.

Morphologically three different bacteria are recognised .

Micrococcus :- when they occur singly eg. Micro coccus .

Diplococci:- when predominantly in pairs. A coccus is divided in one plane in to two

Strepto cocci:- ( Greek – streptos, twisted). When they live in chains.

EPCP Department of Pharmacognosy Page 12

Staphylo cocci:- When they are found in a group .

2. Cylindrical or rod shaped :- Bacillus, singular – bacilli, Bacillus meaning – stick.

Diplo bacillus :- When they occur in pairs.

Strepto bacillus:- These are found in

Vibrio or comma:- They are comma (,) shaped which cause

chlorea. Spirilla:- These are longer rigid rods with several

Spirochetes:- Unlike the spirilla, which use flagella to move,

spirochetes move by means of an axial filament , which is

contained under an external flexible sheath.

EPCP Department of Pharmacognosy Page 13

Structure of Bacterial cell:-

EPCP Department of Pharmacognosy Page 14

Pharmaceutically important principal bacterial groups according to the organisation of

4. Facultative anaerobic Gram- ve rods:-

EPCP Department of Pharmacognosy Page 15

5. Anaerobic , Gram(-ve) , straight , curved & helical rods.

6. Dissimulatory Sulpahte/ Sulphur reducing Bacteria- These bacteria are found in

7. Anaerobic gram-ve Cocci:- They are non-motile, non-endospore forming anaerobic

EPCP Department of Pharmacognosy Page 16

responsible for a number of diseases known as the spotted fever group.

Endemic murine typhus :-(Endemic occurring frequently in a particular region or population

8. Mycoplasmas:- Most species of mycoplasma are aerobes or facultative anaerobes , don’t

EPCP Department of Pharmacognosy Page 17

Actinomycosis- a non-contagious disease caused by the bacterium Actinomyces Israelii , which

EPCP Department of Pharmacognosy Page 18

16. Gliding sheathed & budding and or appendaged bacteria:-

21. Actinomycetes :- Actinomycetes are common inhabitants of soil. Being filamentous

1.Spirochaetes?. 2 marks.Repeated in 4 question papers.

EPCP Department of Pharmacognosy Page 19

2.Discuss classification of Microorganisms. 5 marks.?