DMLT Microbiology Book

Morphology and Classification of Bacteria MODULE
Microbiology
1
Notes
MORPHOLOGY AND
CLASSIFICATION OF BACTERIA
1.1 INTRODUCTION
Microorganisms are a heterogeneous group of several distinct classes of living
beings. Based on the difference in cellular organization and biochemistry, the
kingdom protista has been divided into two groups namely prokaryotes and
eukaryotes. Bacteria and blue-green algae are prokaryotes, while fungi, other
algae, slime moulds and protozoa are eukaryotes. Bacteria are prokaryotic
microorganisms that do not contain chlorophyll. They are unicellular and do not
show true branching, except in higher bacteria like actinomycetales.
OBJECTIVES
After reading this lesson, you will be able to:
z describe the structure of Prokaryotic and Eukaryotic cell
z explain the size of bacteria
z classify bacteria based on the shape and arrangements
z describe the structure of bacterial cell wall
z describe the phases of Growth curve
z explain the factors affecting the growth of bacteria
1.2 PROKARYOTES
The prokaryotic cells have the following characteristics such as
z No organelles, all the action takes place in the cytosol or cytoplasmic
membrane
MICROBIOLOGY 1
MODULE Morphology and Classification of Bacteria
Microbiology z Most bacteria possess peptidoglycan, a unique polymer that makes its
synthesis a good target for antibiotics
z Protein synthesis takes place in the cytosol with structurally different
ribosome’s
Notes
Fig. 1.1: Prokaryote Cell
Fig. 1.2: Eukaryote Cell
Difference between Prokaryotic and Eukaryotic Cells
Character Prokaryotes Eukaryotes
Nucleus Absent. No nuclear Present with nuclear

envelope envelope and nucleolus
Membrane-bound Absent Present. Includes

organelles mitochondria, chloroplasts
(plants), lysosomes
Chromosome (DNA) Single coiled chromosome Multiple linear

in cytoplasm ‘nucleoid’ chromosomes with histone
region in association with proteins
‘histone-like’ proteins
2 MICROBIOLOGY
Microbiology
Cell wall Eubacteria have a cell wall No cell wall in animal
of peptidoglycan Archaea cellsPlant cell walls =
have cell walls of celluloseFungal cell walls =
pseudomurein chitin
Mitotic division Absent Present
Ribosomes 70S. Free in cytoplasm 80S. Both free in cytoplasm

and attached to rough
E.R.70S in mitochondria Notes
and chloroplasts
Flagella when present consist of consist of 9+2 arrangement

protein flagellin of microtubules
Cytoplasmic membrane Eubacteria= Fatty acids Fatty acids joined to

lipids joined to glycerol by ester glycerol by ester linkage
linkageArchaea=
Hydrocarbons joined to
glycerol by ether linkage
Mitochondria Absent Present
Lysosomes Absent Present
Golgi apparatus Absent Present
Endoplasmic Reticulum Absent Present
1.3 BACTERIA
The major characteristics of Bacteria are based on their size, shape and
arrangements
1.3.1 Size
The unit of measurement used in bacteriology is the micron (micrometer)
1 micron (μ) or micrometer (μm) – one thousandth of a

millimeter
1 millimicron (mμ) or nanometer (nm) – one thousandth of a micron
or one millionth of a
millimeter
1 Angstrom unit (Å) – one tenth of a nanometer
The limit of resolution with the unaided eye is about 200 microns. Bacteria are
smaller which can be visualized only under magnification. Bacteria of medical
importance generally measure 0.2 – 1.5 μm in diameter and about 3-5 μm in
length.
MICROBIOLOGY 3
Microbiology 1.3.2 Microscopy

The morphological study of bacteria requires the use of microscopes. Microscopy
has come a long way since Leeuwenhoek first observed bacteria using hand-
ground lenses.
The types of microscope are
Notes (i) Light or optical microscope

(ii) Phase contrast microscope
(iii) Dark field/ Dark ground microscope
(iv) Electron microscope
Light or optical microscope

They are of two types namely Simple and Compund Microscope
z Simple Microscope consists of a single lens. A hand lens is an example of
a simple Microscope.
z Compound Microscope consists of two or more lenses in series. The image
formed by the first lens is further magnified by another lens.
Bacteria may be examined under the compound microscope, either in the living
state or after fixation and staining. Examination of wet films or hanging drops
indicates the shape, arrangements, motility and approximately size of the cells.
But due to lack of contrast details cannot be appreciated.
Phase contrast microscope

This imposes the contrast and makes evident the structure within the cells that
differ in thickness or refractive index. The difference in the refractive index
between bacteria cells and the surrounding medium makes them clearly visible.
Retardation, by a fraction of a wavelength, of the rays of light that pass through
the object, compared to the rays passing through the surrounding medium,
produces phase difference between the two types of rays.
Dark field / Dark ground microscope

Another method of improving the contrast is the dark field microscope in which
reflected light is used instead of the transmitted light used in the ordinal
microscope. The contrast gives an illusion of increased resolution, so that very
slender organisms such as spirochete, not visible under ordinary illumination,
can be clearly seen under the dark field microscope.
4 MICROBIOLOGY
Electron Microscope Microbiology
Beams of electron are used instead of beam of light, used in light microscope.
The object which is held in the path of beam scatters the electrons and produces
an image which is focused on a fluorescent viewing screen. Gas molecules
scatter electron, therefore it is necessary to examine the object in a vacuum.
Notes
INTEXT QUESTIONS 1.1
Match the following
Microscopes Properties:
1. Light microscope (a) reflected light
2. Phase contrast microscope (b) electron beam
3. Dark field microscope (c) light beam
4. Electron microscope (d) refractive index
1.3.3 Stained Preparations

Live bacteria do not show the structural detail under the light microscope due
to lack of contrast. Hence staining techniques are used to produce colour
contrast. Routine methods of staining of bacteria involve dying and fixing
smears – procedures that kill them. Bacteria have an affinity to basic dyes due
to acidic nature of their protoplasm. The commonly used staining techniques are
Simple Stains
Dyes such as methylene blue or basic fuchsin are used for simple staining. They
provide colour contrast, but impart the same colour to all bacteria.
Negative Staining
Bacteria are mixed with dyes such as Indian ink or nigrosin that provide a
uniformly coloured background against which the unstained bacteria stand out
in contrast. Very slender bacteria like spirochetes that cannot be demonstrated
by simple staining methods can be viewed by negative staining.
Impregnation Methods
Cells and structures too thin to be seen under ordinary microscope may be
rendered visible if they are impregnated with silver on the surface. These are
used for demonstration of spirochetes and bacterial flagella.
MICROBIOLOGY 5
Microbiology Differential Stains

These stains impart different colours to different bacteria or bacterial structures,
the two most widely used differential stains are the Gram stain and Acid fast
stain. The gram stain was devised by histologist Christian Gram as a method of
staining bacteria in tissues.
Gram positive cells are simpler chemical structure with a acidic protoplasm. It
Notes has a thick peptidoglycan layer. Teichoic acids are intertwined among the
peptidoglycan and the teichoic acids are the major surface antigen determinants
Gram negative cells are more complex, they are rich in lipids. The membrane
is bilayered as phospholipids, proteins and lipopolysaccharide. Lipopoly-
saccharides (LPS) are also known as endotoxin. Gram negative cells have a
peptidoglycan layer which is thin and formed by just one or two molecules. No
Teichoic acids are found in the cell wall of Gram negative bacteria. The Outer
membrane has Lipopolysaccharide channels with porins which transfer the
solutes across. Lipoprotein cross link outer membrane and peptidoglycan layer
Gram reaction may be related to the permeability of the bacterial cell wall and
cytoplasmic membrane to the dye-iodine complex, the Gram-negative, but not
the Gram-positive cells, permitting the outflow of the complex during
decolourisation. Gram staining is an essential procedure used in the identification
of bacteria and is frequently the only method required for studying their
morphology.
The acid fast stain was discovered by Ehrlich, who found that after staining with
aniline dyes, tubercle bacilli resist decolourisation with acids. The method as
modified by Ziehl and Neelsen, is in common use now.

Match the following:
1. Simple stain (a) Silver
2. Negative stain (b) acids
3. Impregnation method (c) iodine complex
4. Acid fast stain (d) Methylene blue
5. Gram stain (e) Indian ink
1.4 SHAPE OF THE BACTERIA

Depending on their shape, bacteria are classified into several varieties
1. Cocci (from kokkos meaning berry) are spherical or oval cells
6 MICROBIOLOGY
2. Bacilli (from baculus meaning rod) are rod shaped cells Microbiology
3. Vibrios are comma shaped curved rods and derive their name from their
characteristics vibratory motility.
4. Spirilla are rigid spiral forms.
5. Spirochetes (from speira meaning coil and chaite meaning hair) are flexuous
spiral forms
Notes
6. Actinomycetes are branching filamentous bacteria, so called because of a
fancied resemblance to the radiating rays of the sun when seen in tissue
lesions (from actis meaning ray and mykes meaning fungus)
7. Mycoplasmas are bacteria that are cell wall deficient and hence do not
possess a stable morphology. They occur as round or oval bodies and as
interlacing filaments.
Fig. 1.3: Shapes of bacteria.
MICROBIOLOGY 7
Microbiology
INTEXT QUESTION 1.3

1. Bacilli (a) coma
2. Cocci (b) flexous spiral form
Notes 3. Vibrio (c) rigid spiral form
4. Sprillum (d) rod shaped
5. Spirochetes (e) spherical shaped
Bacteria sometime show characteristic cellular arrangement or grouping.
According to the plane of cellular division, cocci may be arranged in pairs
(diplococci), chains (streptococci), groups of four (tetrads) or eight (sarcina),
or grape like clusters (staphylococci).
Fig. 1.4: Arrangement of Cocci.

1. Diplococci (a) groups of four
2. Streptococci (b) groups of eight
3. Tetrads (c) occurs in pairs
4. Sarcina (d) grape like clusters
5. Staphylococci (e) occurs in chains
8 MICROBIOLOGY
Microbiology
1.5 BACTERIAL STRUCTURE
The outer layer or cell envelope consists of two components, a rigid cell wall
and beneath it a cytoplasmic or plasma membrane. The cell envelope encloses
the protoplasm, comprising the cytoplasm, cytoplasmic inclusions such as
ribosomes and mesosomes, granules, vacuoles and the nuclear body.
Cell wall Notes

Beneath the external structures is the cell wall. It is very rigid & gives shape to
the cell. Its main function is to prevent the cell from expanding & eventually
bursting due to water uptake. Cell Wall constitutes a significant portion of the
dry weight of the cell and it is essential for bacterial growth & division. The cell
wall cannot be seen by direct light microscopy and does not stain with simple
Fig. 1.5
MICROBIOLOGY 9
Microbiology stains. It may be demonstrated by microdissection, reaction with specific

antibodies, mechanical rupture of the cell, differential staining procedures or by
electron microscopy.
Chemically the cell wall is composed of peptidoglycan. Mucopeptide
(peptidoglycan or murien) formed by N acetyl glucosamine & N acetyl muramic
acid alternating in chains, cross linked by peptide chains. Embedded in it are
Notes polyalcohol called Teichoic acids. Some are linked to Lipids & called
Lipoteichoic acid. Lipotechoic acid link peptidoglycan to cytoplasmic membrane
and the peptidoglycan gives rigidity.
The functions of Teichoic acid are
z gives negative charge
z major antigenic determinant
z transport ions
z anchoring
z external permeability barrier
Characteristics Gram Positive Gram Negative

Thickness Thicker Thinner
Variety of amino acids Few Several
Lipids Absent Present
Teichoic acid Present absent
Outer Membrane
Outer membrane is found only in Gram-negative bacteria, it functions as an
initial barrier to the environment and is composed of lipopolysaccharide (LPS)
and phospholipids
Lipopolysaccharide (LPS)
The LPS present on the cell walls of Gram-negative bacteria account for their
endotoxic activity and antigen specificity.
A bacterium is referred as a protoplast when it is without cell wall. Cell wall
may be lost due to the action of lysozyme enzyme, which destroys peptidoglycan.
This cell is easily lysed and it is metabolically active but unable to reproduce.
A bacterium with a damaged cell wall is referred as spheroplasts. It is caused
by the action of toxic chemical or an antibiotic, they show a variety of forms
and they are able to change into their normal form when the toxic agent is
removed, i.e. when grown on a culture media
10 MICROBIOLOGY
Cytoplasmic membrane Microbiology
Cytoplasmic membrane is present immediately beneath the cell wall, found in

both Gram positive & negative bacteria and it is a thin layer lining the inner
surface of cell wall and separating it from cytoplasm. It acts as a semipermeable
membrane controlling the flow of metabolites to and from the protoplasm.
Cytoplasm Notes
The cytoplasm is a Colloidal system containing a variety of organic and
inorganic solutes containing 80% Water and 20% Salts, Proteins. They are rich
in ribosomes, DNA & fluid. DNA is circular and haploid. They are highly coiled
with intermixed polyamines & support proteins. Plasmids are extra circular
DNA.
1 μm
Fig. 1.6
Ribosomes
They are the centers of protein synthesis. They are slightly smaller than the
ribosomes of eukaryotic cells
Mesosomes
They are vesicular, convoluted tubules formed by invagination of plasma
membrane into the cytoplasm. They are principal sites of respiratory enzymes
and help with cell reproduction
Cytoplasmic Inclusions
The Inclusion bodies are aggregates of polymers produced when there is excess
of nutrients in the environment and they are the storage reserve for granules,
MICROBIOLOGY 11
Microbiology phosphates and other substances. Volutin granules are polymetaphosphates

which are reserves of energy and phosphate for cell metabolism and they are also
known as metachromatic granules.
Nucleus
The Nucleus is not distinct and has no nuclear membrane or nucleolus and the
genetic material consist of DNA. The cytoplasmic carriers of genetic information
Notes
are termed plasmids or episomes.
Capsule
Capsule is the outer most layer of the bacteria (extra cellular). It is a condensed
well defined layer closely surrounding the cell. They are usually polysaccharide
and if polysaccharide envelops the whole bacterium it is capsule and their
production depends on growth conditions. They are secreted by the cell into the
external environment and are highly impermeable. When it forms a loose mesh
work of fibrils extending outward from the cell they are described as glycocalyx
and when masses of polymer that formed appear to be totally detached from the
cell and if the cells are seen entrapped in it are described as slime layer.
The Capsule protects against complement and is antiphagocytic. The Slime layer
& glycocalyx helps in adherence of bacteria either to themselves forming
colonial masses or to surfaces in their environment and they resists phagocytosis
and desiccation of bacteria.
Flagella
Flagella are long hair like helical filaments extending from cytoplasmic
membrane to exterior of the cell. Flagellin is highly antigenic and functions in
cell motility. The location of the flagella depends on bacterial species as polar
situated at one or both ends which swims in back and forth fashion and lateral
at along the sides.
The parts of flagella are the filament, hook and the basal body. Filament is
external to cell wall and is connected to the hook at cell surface, the hook & basal
body are embedded in the cell envelope. Hook & filament is composed of protein
subunits called as flagellin. Flagellin is synthesized within the cell and passes
through the hollow centre of flagella. The arrangement of flagella may be
described as
(i) Monotrichous – single flagella on one side
(ii) Lophotrichous – tuft of flagella on one side
(iii) Amphitrichous – single or tuft on both sides
(iv) Peritrichous – surrounded by lateral flagella
12 MICROBIOLOGY
Microbiology
Structure Flagella Type Example
Monotrichous Vibrio cholerae
Lophotrichous Bartonella
bacillifornis
Amphitrichous Spirillum serpens
Notes
Peritrichous Escherichia coli
Fig. 1.7: Flagella.
Various types of mobility is observed because of the presence of the flagella as

Serpentine motility is seen with Salmonella, Darting motility with Vibrio and
Tumbling motility with Listeria monocytogenes
Pili / Fimbriae
Hair-like proteinaceous structures that extend from the cell membrane to
external environment are pili which are otherwise known as fimbriae. They are
thinner, shorter and more numerous than flagella and they do not function in
motility. The fimbriae is composed of a subunit called pilin.
There are two types pili namely Non-sex pili (Common pili) eg. fimbriae or type
IV and the sex pili. The fimbriae are antigenic and mediate their adhesion which
inhibits phagocytosis. The sex pili help in conjugation.

1. Monotrichous (a) single or tuft on both sides
2. Lophotrichous (b) surrounded by lateral flagella
3. Amphitrichous (c) single flagella on one side
4. Peritrichous (d) tuft of flagella on one side
Spore
Some bacteria have the ability to form highly resistant resting stage called
spores, which helps them to overcome adverse environmental conditions that are
unfavorable for vegetative growth of cell. They are not a reproductive form and
MICROBIOLOGY 13
Microbiology Characteristics of Bacteria Cell Structures
Structure Functions(s) Predominant chemical

composition
Flagella Swimming movement Protein
Pili
Notes Sex pilus Stabilizes mating bacteria Protein
during DNA transfer by
conjugation
Common pili or fimbriae Attachment to surfaces; Protein

protection against
phagotrophic engulfment
Capsules (includes “slime Attachment to surfaces; Usually polysaccharide;

layers” and glycocalyx) protection against phagocytic occasionally polypeptide
engulfment, occasionally
killing or digestion;
protection against
desiccation
Cell wall
Gram-positive bacteria confers rigidity and shape on Peptidoglycan (murein)

cells complexed with teichoic
acids
Gram-negative bacteria confers rigidity and shape; Peptidoglycan (murein)

outer membrane is surrounded by phospholipid
permeability barrier; protein-lipopolysaccharide
associated LPS and proteins “outer membrane”
have various functions
Plasma membrane Permeability barrier; Phospholipid and protein

transport of solutes; energy
generation; location of
numerous enzyme systems
Ribosomes Sites of translation (protein RNA and protein

synthesis)
Inclusions Often reserves of nutrients; Highly variable;

additional specialized carbohydrate, lipid, protein
functions or inorganic
Chromosome Genetic material of cell DNA
Plasmid Extrachromosomal genetic DNA

material
14 MICROBIOLOGY
not a storage granule. These spores are resistant to bactericidal agents and Microbiology
adverse physical conditions. Each spore can give rise to only one endospore
which play a role in heat resistance. Spores consists of three layers namely core,
cortex and spore coat
Notes
Fig. 1.8: Spere
1.6 GROWTH AND MULTIPLICATION OF BACTERIA

Bacteria divide by binary fission and cell divides to form two daughter cells.
Nuclear division precedes cell division and therefore, in a growing population,
many cells having two nuclear bodies can be seen. Bacterial growth may be
considered as two levels, increase in the size of individual cells and increase in
number of cells. Growth in numbers can be studied by bacterial counts that of
total and viable counts. The total count gives the number of cells either living
or not and the viable count measures the number of living cells that are capable
of multiplication.
1.6.1 Bacterial Growth Curve

When bacteria is grown in a suitable liquid medium and incubated its growth
follows a definite process. If bacterial counts are carried out at intervals after
innoculation and plotted in relation to time, a growth curve is obtained. The
curve shows the following phase
(i) Lag phase

Immediately following innoculation there is no appreciable increase in number,
though there may be an increase in the size of the cells. This initial period is the
time required for adaptation to the new environment and this lag phase varies
with species, nature of culture medium and temperature.
MICROBIOLOGY 15
Microbiology (ii) Log or exponential phase

Following the lag phase, the cell starts dividing and their numbers increase
exponentially with time.
(iii) Stationary phase

After a period of exponential growth, cell division stops due to depletion of
nutrient and accumulation of toxic products. The viable count remains stationary
Notes as an equilibrium exists between the dying cells and the newly formed cells.
(iv) Phase of decline

This is the phase when the population decreased due to cell death.
Stationary
phase
Log Decline
Number of phase phase
bacteria Lag
phase
Time
Fig. 1.8: The growth curve of bacteria showing different phases
The various stages of bacterial growth curve are associated with morphological
and physiological alterations of the cells. The maximum cell size is obtained
towards the end of the lag phase. In the log phase, cells are smaller and stained
uniformily. In the stationary phase, cells are frequently gram variable and show
irregular staining due to the presence of intracellular storage granules. Sporulation
occurs at this stage. Also, many bacteria produce secondary metabolic products
such as exotoxins and antibiotics. Involution forms are common in the phase of
decline.
1.7 FACTORS THAT AFFECT THE GROWTH OF

BACTERIA
Many factors affect the generation time of the organism like temperature,
oxygen, carbon dioxide, light, pH, moisture, salt concentration.
Nutrition
The principal constituents of the cells are water, proteins, polysaccharides,
lipids, nucleic acid and mucopeptides. For growth and multiplication of bacteria,
the minimum nutritional requirement is water, a source of carbon, nitrogen and
some inorganic salts.
16 MICROBIOLOGY
Bacteria can be classified nutritionally, based on their energy requirement and Microbiology
on their ability to synthesise essential metabolites. Bacteria which derive their
energy from sunlight are called phototrophs, those who obtain energy from
chemical reactions are called chemotrophs. Bacteria which can synthesise all
their organic compounds are called autotrophs and those that are unable to
synthesise their own metabolites are heterotrophs.
Some bacteria require certain organic compounds in minute quantities. These Notes
are know as growth factors or bacterial vitamins. Growth factors are called
essential when growth does not occur in their absence, or they are necessary for
it.
Oxygen
Depending on the influence of oxygen on growth and viability, bacteria are
divided into aerobes and anaerobes.
Aerobic bacteria require oxygen for growth. They may be obligate aerobes like
cholera, vibrio, which will grow only in the presence of oxygen or facultative
anaerobes which are ordinarily aerobic but can grow in the absence of oxygen.
Most bacterial of medical importance are facultative anaerobes. Anaerobic

bacteria, such as clostridia, grow in the absence of oxygen and the obligate
anaerobes may even die on exposure to oxygen. Microaerophilic bacteria are
those that grow best in the presence of low oxygen tension.
Carbon Dioxide
All bacteria require small amounts of carbon dioxide for growth. This
requirement is usually met by the carbon dioxide present in the atmosphere.
Some bacteria like Brucella abortus require much higher levels of carbon
dioxide.
Temperature
Bacteria vary in their requirement of temperature for growth. The temperature
at which growth occurs best is known as the optimum temperature. Bacteria
which grow best at temperatures of 25-40°C are called mesophilic. Psychrophilic
bacteria are those that grow best at temperatures below 20°C. Another group of
non pathogenic bacteria, thermophiles, grow best at high temperatures, 55-80°C.
The lowest temperature that kills a bacterium under standard conditions in a

given time is known as thermal death point.
MICROBIOLOGY 17
Microbiology Moisture and Drying

Water is an essential ingredient of bacterial protoplasm and hence drying is lethal
to cells. The effect of drying varies in different species.
Light
Bacteria except phototrophic species grow well in the dark. They are sensitive
Notes to ultraviolet light and other radiations. Cultures die if exposed to light.
H-ion concentration
Bacteria are sensitive to variations in pH. Each species has a pH range, above
or below which it cannot survive and an optimum pH at which it grows best.
Majority of pathogenic bacteria grow best at neutral or slightly alkaline pH (7.2
– 7.6)
Osmotic Effect
Bacteria are more tolerant to osmotic variation than most other cells due to the
mechanical strength of their cell wall. Sudden exposure to hypertonic solutions
may cause osmotic withdrawal of water and shrinkage of protoplasm called
plasmolysis.
WHAT YOU HAVE LEARNT

z Bacteria are prokaryotic microorganism that do not contain chlorophyll
z They are unicellular and do not exhibit true branching.
z The morphological study of bacteria requires the use of microscope like
optical or light microscope, phase control microscope, dark/field microscope,
electron microscope
z Staining techniques like simple stain, negative stain, impregnation stain,
differential stains are used to exhibit structure of bacteria
z Bacteria are classified based on the shape as cocci, bacilli, vibrio, Spirilla.
And based on arrangements they are classified as diplococci, streptococci,
tetrads, sarcina, staphylococci
z Bacterial cell has cell wall, inner protoplasm and other components
z Bacterial growth phase has a lag phase, log phase, stationary phase and a
decline phase
18 MICROBIOLOGY
Microbiology
TERMINAL QUESTIONS
1. Describe the structure of cell wall
2. Classify bacteria based on shaped and arrangement with examples
3. Explain the factors affecting the growth of the bacteria
Notes
4. Describe growth curve
ANSWERS TO INTEXT QUESTIONS
1.1
1. (c) 2. (d) 3. (a) 4. (b)
1.2
1. (d) 2. (e) 3. (a) 4. (b) 5. (c)
1.3
1. (d) 2. (e) 3. (a) 4. (c) 5. (b)
1.4
1. (c) 2. (e) 3. (a) 4. (b) 5. (d)
1.5
1. (c) 2. (d) 3. (a) 4. (b)
MICROBIOLOGY 19
MODULE Common Staining Technique
Microbiology
2
Notes
COMMON STAINING
TECHNIQUE
2.1 INTRODUCTION
Staining is technique used in microscopy to enhance contrast in the microscopic
image. Stains and dyes are frequently used in biological tissues for viewing,
often with the aid of different microscopes. Stains may be used to define and
examine bulk tissues (highlighting, for example, muscle fibers or connective
tissue), cell populations (classifying different blood cells, for instance), or
organelles within individual cells.
Bacteria have nearly the same refractive index as water, therefore, when they
are observed under a microscope they are opaque or nearly invisible to the naked
eye. Different types of staining methods are used to make the cells and their
internal structures more visible under the light microscope.
Microscopes are of little use unless the specimens for viewing are prepared
properly. Microorganisms must be fixed & stained to increase visibility,
accentuate specific morphological features, and preserve them for future use
OBJECTIVES
z describe the need for staining techniques
z explain terms related to staining techniques
z discuss the substances used as stain
z enlist various staining techniques
z classify & explain various stains
20 MICROBIOLOGY
Common Staining Technique MODULE
Microbiology
2.2 TERMS RELATED TO STAINING
Stain
A stain is a substance that adheres to a cell, giving the cell color. The presence
of color gives the cells significant contrast so they are much more visible.
Different stains have different affinities for different organisms, or different parts
of organisms. They are used to differentiate different types of organisms or to
view specific parts of organisms Notes
Staining
Staining is an auxiliary technique used in microscopy to enhance contrast in the
microscopic image. Stains and dyes are frequently used in biology and medicine
to highlight structures in biological tissues for viewing, often with the aid of
different microscopes.
Fixation
Fixation by itself consists of several steps–aims to preserve the shape of the cells
or tissue involved as much as possible. Sometimes heat fixation is used to kill,
adhere, and makes them permeable so it will accept stains
What can be used as stain
The substance be used as a stain must be colored or it should react in the system
to give a colored product, because of which some portion of the system becomes
colored and the rest remains colorless. Staining renders the organism more
visible, it displays the structure and finer details of bacteria and it helps to
differentiate between organisms
Staining techniques
Direct staining - The organism is stained and background is left unstained
Negative staining - The background is stained and the organism is left unaltered

Fill in the blanks:
1. Staining is primarily used to enhance ................ in the image
2. Substances that adhere to cell giving colour to cell are called as ................
3. ................ aims to preserve the shape of the cells
4. The organism is stained and background is left unstained in ................
staining technique
5. Background is stained and the organism is left unchanged in ................
staining technique
MICROBIOLOGY 21
Microbiology
2.3 KINDS OF STAINS
Stains are classified as
z Simple stain
z Differential stain
z Structural or special stains
Notes
Simple Staining
The staining process involves immersing the sample (before or after fixation and
mounting) in dye solution, followed by rinsing and observation. Many dyes,
however, require the use of a mordant, a chemical compound that reacts with
the stain to form an insoluble, coloured precipitate. When excess dye solution
is washed away, the mordanted stain remains. Simple staining is one step method
using only one dye. Basic dyes are used in direct stain and acidic dye is used
in negative stain. Simple staining techniques is used to study the morphology
better, to show the nature of the cellular contents of the exudates and also to study
the intracellular location of the bacteria
Commonly used simple stains are
z Methylene blue
z Dilute carbol fuchsin
z Polychrome methylene blue
Loeffler’s Methylene Blue
Method of Staining
Flood the smear with methylene blue, allow for 2 minutes, pour off the stain and
allow the air to dry by keeping in a slanting position and by this the organism
will retain the methylene blue stain
Use
Methylene blue staining is used to make out clearly the morphology of the
organisms eg. H.influenzae in CSF, Gonococci in urethral pus
Polychrome Methylene Blue
Preparation
Allow Loeffler’s Methylene blue to ‘ripen’ slowly. Methylene blue stain is kept
in half filled bottles, aerate the content by shaking at intervals, Slow oxidation
of methylene blue forms a violet compound and Stain gets polychrome property.
The ripening nearly takes 12 months and this is hastened by addition of 1%
potassium carbonate
22 MICROBIOLOGY
Use Microbiology
Polychrome Methylene Blue is used to demonstrate Mc Fadyean reaction of

B.anthracis and in this the blue bacilli Is surrounded by purple capsular material
Dilute Carbol Fuchsin

Preparation
Prepare carbol fuchsin and dilute it to 1/15 using distilled water Notes
Method of staining
Flood the smear and let stand for 30 seconds, wash with tap water and blot gently
to dry
Use
To stain throat swab from patients of suspected Vincent’s angina, (Borrelia are
better stained), it is used as a counter stain in Gram stain and to demonstrate the
morphology of Vibrio cholerae (comma shaped)

Fill in the blanks:
1. Chemicals that reacts with stain to form precipitates are called as ..............
2. .............. dyes is used in direct stain
3. .............. dyes is used in negative stain
4. Commonly used simple stains are .............., .............. & ..............
2.4 DIFFERENTIAL STAINS

Gram Staining
Differential Stains use two or more stains and allow the cells to be categorized
into various groups or types. Both the techniques allow the observation of cell
morphology, or shape, but differential staining usually provides more information
about the characteristics of the cell wall (Thickness). Gram staining (or Gram’s
method) is an emprical method of differentiating bacterial species into two large
groups (Gram-positive and Gram-negative) based on the chemical and physical
properties of their cell wall. The Gram stain is almost always the first step in
the identification of a 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
MICROBIOLOGY 23
Microbiology indeterminate groups as well. The word Gram is always spelled with a capital,
referring to Hans Christian Gram, the inventor of Gram staining
Gram staining Principles

Gram staining is used to determine gram status to classify bacteria broadly. It
is based on the composition of their cell wall. Gram staining uses crystal violet
Notes to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain
to mark all bacteria. Gram status is important in medicine; the presence or
absence of a cell wall will change the bacterium’s susceptibility to some
antibiotics. Gram-positive bacteria stain dark blue or violet. Their cell wall is
typically rich with peptidoglycan and lacks the secondary membrane and
lipopolysaccharide layer found in Gram-negative bacteria
Gram Staining Technique

1. Crystal violet acts as the primary stain. Crystal violet may also be used as
a simple stain because it dyes the cell wall of any bacteria.
2. Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell
wall).
3. Decolorizer is used next to remove the primary stain (crystal violet) from
Gram Negative bacteria (those with LPS imbedded in their cell walls).
Decolorizer is composed of an organic solvent, such as, acetone or ethanol
or a combination of both.)
4. Finally, a counter stain (Safranin), is applied to stain those cells (Gram
Negative) that have lost the primary stain as a result of decolorization
Gram Reaction
Gram-positive bacteria are those that are stained dark blue or violet by Gram
staining. This is in contrast to Gram-negative bacteria, which cannot retain the
crystal violet stain, instead taking up the counter stain (safranin or fuchsine) and
appearing red or pink. Gram-positive organisms are able to retain the crystal
violet stain because of the high amount of peptidoglycan in the cell wall. Gram-
positive cell walls typically lack the outer membrane found in Gram-negative
bacteria.
Gram-negative bacteria are those bacteria that do not retain crystal violet dye
in the Gram staining protocol. In a Gram stain test, a counter stain (commonly
safranin) is added after the crystal violet, coloring all Gram-negative bacteria
with a red or pink color. The test itself is useful in classifying two distinct types
of bacteria based on the structural differences of their cell walls. On the other
hand, Gram-positive bacteria will retain the crystal violet dye when washed in
a decolorizing solution.
24 MICROBIOLOGY
Microbiology
Notes
Fig. 2.1: Gram Reaction.
MICROBIOLOGY 25
Microbiology

Fill in the blanks:
1. Gram staining uses ............... to stain cell walls, ............... as mordant &
............... as counter stain
Notes 2. Gram positive bacteria stains ............... colour
3. Gram negative bacteria stains ............... colour
4. Gram positive organism are able to retain crystal violet stain because of high
amount of ............... in the cell wall
2.5 ACID-FAST STAINING

The Ziehl–Neelsen stain, also known as the acid-fast stain, widely used
differential staining procedure. The Ziehl – Neelsen stain was first described by
two German doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich
Neelsen (1854 to 1894) a pathologist. In this type some bacteria resist
decolourization by both acid and alcohol and hence they are referred as acid-
fast organisms. This staining technique divides bacteria into two groups namely
acid-fast and non acid-fast. This procedure is extensively used in the diagnosis
of tuberculosis and leprosy. Mycobacterium tuberculosis is the most important
of this group, as it is responsible for the disease called tuberculosis (TB) along
with some others of this genus
Principle
Mycobacterial cell walls contain a waxy substance composed of mycolic acids.
These are β-hydroxy carboxylic acids with chain lengths of up to 90 carbon
atoms. The property of acid fastness is related to the carbon chain length of the
mycolic acid found in any particular species
Ziehl- Neelsen Procedure

1. Make a smear. Air Dry. Heat Fix.
2. Flood smear with Carbol Fuchsin stain
z Carbol Fuchsin is a lipid soluble, phenolic compound, which is able
to penetrate the cell wall
3. Cover flooded smear with filter paper
4. Steam for 10 minutes. Add more Carbol Fuchsin stain as needed
5. Cool slide
6. Rinse with DI water
26 MICROBIOLOGY
7. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol contains Microbiology
3% HCl and 95% ethanol, or you can declorase with 20% H2SO4
8. Tilt slide 45 degrees over the sink and add acid alcohol drop wise (drop
by drop) until the red color stops streaming from the smear
9. Rinse with DI water
10. Add Loeffler’s Methylene Blue stain (counter stain). This stain adds blue
color to non-acid fast cells. Leave Loeffler’s Blue stain on smear for 1 Notes
minute
11. Rinse slide. Blot dry.
12. Use oil immersion objective to view.
Fig. 2.2: Ziehi-Neelsen acid fast staining procedure
Fig. 2.3
MICROBIOLOGY 27
Microbiology

Fill in the blanks:
1. Organisms that resist decolourisation by acid and alcohol are called as
................
Notes 2. ................... staining is used in the diagnosis of Tuberculosis and Leprosy
3. Acid fastness is related to ................... length of mycolic acid
4. Acid fast staining groups bacteria into two groups namely ................... &
...................
2.6 SPECIAL STAINS

z Stains for Metachromatic granules
z Stain for spores
z Stain for capsules
z Stain for spirochetes
z Stain for flagella
Albert’s Staining for C. diphtheriae

In all cases of suspected Diphtheria, stain one of the smears with Gram stain.
If Gram stained smear shows morphology suggestive of C.diphtheriae, proceed
to do Albert staining which demonstrates the presence or absence of metachromatic
granules.
C.diphtheriae are thin Gram positive bacilli, straight or slightly curved and often
enlarged (clubbing) at one or both ends and are arranged at acute angles giving
shapes of Chinese letters or V shape which is characteristic of these organisms.
Present in the body of the bacillus are numerous metachromatic granules which
give the bacillus beaded or barred appearance. These granules are best
demonstrated by Albert’s stain.
Albert staining
Albert stain I
z Toluidine blue 0.15 gm
z Malachite green 0.20 gm
z Glacial acetic acid 1.0 ml
z Alcohol(95%) 2.0 ml
z Distilled water 100 ml
28 MICROBIOLOGY
Albert stain II Microbiology
z Iodine 2.0 gm
z Potassium iodide 3.0 gm
z Distilled water 300 ml
Albert staining Procedure

z Cover the heat-fixed smear with Albert stain I. Let it stand for two minutes.
Notes
z Wash with water.
z Cover the smear with Albert stain II. Let it stand for two minutes.
z Wash with water, blot dry and examine.
z To demonstrate metachromatic granules in C.diphtheriae. These granules
appear bluish black whereas the body of bacilli appear green or bluish green.
Capsule staining
The purpose of the capsule stain is to reveal the presence of the bacterial capsule,
the water-soluble capsule of some bacterial cells is often difficult to see by
standard simple staining procedures or after the Gram stain. The capsule staining
methods were developed to visualize capsules and yield consistent and reliable
results
Capsule may appear as clear halo when a fresh sample is stained by Grams or
Leishman stain, Negative staining- using - India ink, Nigrosin
India ink
Commercially available India ink is used undiluted
Procedure
z Place a loop full of India ink on the slide
z A small portion of the culture is emulsified in the drop of ink
z Place a clean cover slip over the preparation without bubbles. Press down
gently
z Examine under dry objective
Uses
India ink is used to demonstrate capsule which is seen as unstained halo around
the organisms distributed in a black background eg. Cryptococcus
Endospore Staining
Bacterial endospores are metabolically inactive, highly resistant structures
produced by some bacteria as a defensive strategy against unfavorable
MICROBIOLOGY 29
Microbiology environmental conditions. Primary stain - is malachite green, which stains

both vegetative cells and endospores and heat is applied to help the primary stain
penetrate the endospore. Decolorized with water, which removes the malachite
green from the vegetative cell but not the endospore, Safranin - counterstain
any cells which have been decolorized, At the end of the staining process,
vegetative cells will be pink, and endospores will be dark green
Notes Flagella stain

z Flagella are fragile appendages
z Cannot be seen under ordinary microscope
z Hence the surface is coated with a precipitate to form a colloidal substance
z This precipitate serves as a layer of stainable material
Components
1. 1% Osmic acid
2. Mordant
10% Tannic acid
Sat.potassium alum
10% Ferric chloride
3. Fontana’s silver solution
Use
This is used to demonstrate the flagella and the organisms stain black and
flagella appear light brown

Fill in the blanks:
1. Presence or absence of metachromatic granules is demonstrated by .............
staining
2. ............. chemical is used in capsule staining
3. ............. is used as primary stain in Endospore staining
4. ............. is the counter stain in endospore staining
30 MICROBIOLOGY
Microbiology

z Staining is technique used in microscopy to enhance contrast in the
microscopic image.
z Stain is a substance that adheres to a cell, giving the cell colour.
z Strains are classified as Simple stain, Differential stain and Special stains. Notes
z Gram staining is used to differentiate bacterial species as Gram-positive and
Gram-negative based on the chemical and physical properties of cell wall.
z Acid Fast staining technique or Ziehl Neelsen stain divides bacteria into
acid fast and non-acid-fast and this is used in diagnosis of tuberculosis and
Leprosy.
z Albert staining technique demonstrates the presence or absence of
metachromatic grannules which is used in identification of C. diphtheria
bacilli.
TERMINAL QUESTIONS
1. List staining techniques
2. Describe different kinds of stains
3. Explain gram staining
4. Explain Acid fast staining
2.1
1. Contrast
2. Stain
3. Fixation
4. Direct
5. Negative
MICROBIOLOGY 31
Microbiology 2.2
1. Mordant
2. Basic
3. Acidic
4. Methylene blue, Polychrome methylene blue & Dilute carbol fuchsin
Notes
2.3
1. Crystal violet, Iodine & Fuchsin
2. Dark blue or violet
3. Red or pink
4. Peptidoglycan
2.4
1. Acid fast organism
2. Acid fast staining
3. Carbon chain
4. Acid fast and Non acid fast
2.5
1. Albert’s
2. Indian ink
3. Malachite green
4. Safranin
32 MICROBIOLOGY
Nutrition and Growth of Bacteria MODULE
Microbiology
3
Notes
NUTRITION AND GROWTH OF
BACTERIA
3.1 INTRODUCTION
Bacteria are prokaryotic organisms that do not contain chlorophyll. They are
unicellular and do not show true branching. They differ from eukaryotes in not
having a nuclear membrane, a nucleolus, and cell organelles like mitochondria,
golgi apparatus and endoplasmic reticulum. They have a single circular
chromosome.
This chapter will deal with the growth and multiplication of bacteria and their
requirements for the same. It will deal with the energy requirements and their
ability to synthesise essential metabolites.
OBJECTIVES
z discuss salient aspects of nutrition, gaseous requirements, temperature
requirements and other physiological requirements for growth
z describe how bacteria grow and multiply
z list the salient features of the bacterial growth curve
3.2 GROWTH OF BACTERIA

3.2.1 Bacterial nutrition
The bacterial cell has the same general chemical pattern as the cells of other
organisms. The bacterial cell contains water (80% of total weight), proteins,
polysaccharides, lipids, nucleic acids, mucopeptides and low molecular weight
compounds.
MICROBIOLOGY 33
MODULE Nutrition and Growth of Bacteria
Microbiology For growth and nutrition of bacteria, the minimum nutritional requirements are
water, a source of carbon, a source of nitrogen and some inorganic salts. Water
is the vehicle of entry of all nutrients into the cell and for the elimination of waste
products.
Bacteria can be classified nutritionally based on their energy requirements and
on their ability to synthesise essential metabolites. Bacteria which derive energy
Notes from sunlight are called phototrophs. Those that obtain energy from chemical
reactions are called chemotrophs. Bacteria that can synthesise all their organic
compounds are called autotrophs. They are able to use atmospheric carbon
dioxide and nitrogen. They are capable of independent existence in water and
soil. They are of no medical importance. Some bacteria are unable to synthesise
their own metabolites. They depend on preformed organic compounds. They are
called heterotrophs. These bacteria are unable to grow with carbon dioxide as
the sole source of carbon. Their nutritional requirements vary widely. Some may
require only a single organic substance like glucose. Others may need a large
number of different compounds like amino acids, nucleotides, lipids, carbohydrates
and coenzymes.
Bacteria require a supply of inorganic salts. They require anions like phosphate
and sulphate anions and cations like sodium, potassium, magnesium, iron and
calcium. Some ions like cobalt may be required in trace amounts.
Some bacteria require certain organic compounds in minute quantities. These
are called growth factors or bacterial vitamins. Growth factors are called
essential when growth does not occur in their absence. Accessory growth factors
are those which enhance growth without being absolutely necessary for it. In
many cases, bacterial vitamins are same as vitamins necessary for nutrition of
mammals, for example, B group vitamins – thiamine, riboflavin, pyridoxine,
nicotinic acid, folic acid and vitamin B12.
3.2.2 Gaseous Requirements

Depending on the influence of oxygen on growth and survival, bacteria are
divided into aerobes and anaerobes. Aerobic bacteria require oxygen for
growth. They may be obligate aerobes or facultative anaerobes. Obligate
aerobes grow only in the presence of oxygen, for eg. Cholera bacillus.
Facultative anaerobes are ordinarily aerobic but can grow in the absence of
oxygen, though less abundantly. Most bacteria of medical importance are
facultative anaerobes. Anaerobic bacteria, such as clostridia grow in the
absence of oxygen. Obligate anaerobes may even die on exposure to oxygen.
Microaerophilic bacteria are those that grow best in the presence of low oxygen
tension.
34 MICROBIOLOGY
In case of aerobes, atmospheric oxygen is the final electron acceptor in the Microbiology
process of respiration (aerobic respiration). In this case, the carbon and energy
source may be completely oxidised to carbon dioxide and water. Energy is
provided by the production of energy-rich phosphate bonds and the conversion
of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). This process
is called oxidative phosphorylation.
Anaerobic bacteria use compounds like nitrates or sulphates instead of oxygen
as final electron acceptors in the process of respiration (anaerobic respiration). Notes
A more common process used by these bacteria in anaerobic metabolism is
fermentation. It is defined as the process by which complex organic compounds,
such as glucose, are broken down by the action of enzymes into simpler
compounds without the use of oxygen. This process leads to the formation of
several organic end products such as organic acids and alcohols, as well as of
gas (carbon dioxide and hydrogen). For example, Escherichia coli ferments
glucose with the production of acid and gas. It also ferments lactose. During the
process of fermentation, energy-rich phosphate bonds are produced by the
introduction of organic phosphate into intermediate metabolites. This process
is known as substrate-level phosphorylation. The energy-rich phosphate
groups so formed are used for conversion of ADP to ATP.
All bacteria require some amounts of carbon dioxide for growth. This is obtained
from the atmosphere or from the cellular metabolism of the bacterial cell. Some
bacteria like Brucella abortus require much higher levels of carbon dioxide (5-
10%) for growth. They are called capnophilic.
3.2.3 Temperature Requirements

Bacteria vary in their requirement of temperature for growth. For each species,
there is a “temperature range”, and growth does not occur above the maximum
or below the minimum of this range. The temperature at which growth occurs
best is known as the “optimum temperature”. In the case of most pathogenic
bacteria, the optimum temperature is 37ºC.
Bacteria which grow best at temperatures of 25-40ºC are called mesophilic, for
example Escherichia coli. Psychrophilic bacteria are those that grow best at
temperatures below 20ºC. They are soil and water saprophytes and may cause
spoilage of refrigerated food. Thermophilic bacteria are those which grow best
at high temperatures, 55-80ºC. They may cause spoilage of underprocessed
canned food. Some thermophiles, for example Geobacillus stearothermophilus,
form spores that are highly thermoresistant.
3.2.4 Other physiological Requirements

Moisture and drying, hydrogen ion concentration, light, osmotic effect and
mechanical and sonic stress may also influence the growth and multiplication
of bacteria.
MICROBIOLOGY 35
Microbiology 3.2.5 Growth and Multiplication of Bacteria

Bacteria divide by binary fission. When a bacterial cell reaches a certain size,
it divides to form two daughter cells. Nuclear division is followed by cell
division.
The interval of time between two cell divisions, or the time required for a
bacterium to give rise to two daughter cells under optimum conditions, is called
Notes the generation time or the population doubling time. In Escherichia coli and
many other medically important bacteria, the generation time is about 20
minutes. Some bacteria are slow-growing. The generation time in tubercle bacilli
is about 20 hours. In lepra bacilli, it is as long as about 20 days.
When bacteria are grown in a vessel of liquid medium, multiplication is arrested
after a few cell divisions due to depletion of nutrients or accumulation of toxic
products. This is a batch culture. By the use of special devices for replenishing
nutrients and removing bacterial cells (chemostat or turbidostat), it is possible
to maintain a continuous culture of bacteria for industrial or research purposes.
When bacteria multiply in host tissues, the situation may be intermediate
between a batch culture and a continuous culture. The source of nutrients may
be inexhaustible but the bacteria have to fight the defence mechanisms of the
host. Bacteria growing on solid media (for example blood agar, MacConkey
agar) form colonies. Each colony represents a cluster of cells derived from a
parent cell. In liquid media, growth is diffuse.
Bacterial growth may be considered at two levels: increase in the size of the
bacterial cell and increase in the number of cells. Growth in numbers can be
studied by bacterial counts. Two types of bacterial counts can be made: total
count and viable count. The total count gives the total number of cells in the
sample, irrespective of whether they are living or not. It can be done by various
methods, for example direct counting under the microscope using counting
chambers. The viable count measures the number of living cells, that is, cells
capable of multiplication. Viable counts are obtained by dilution or plating
methods. In the dilution method, the suspension, whose cell count is to be
determined, is serially diluted. The dilutions are made to the point beyond which
unit quantities do not yield growth when inoculated into suitable liquid media.
Each dilution is inoculated into the respective tubes containing liquid media. The
viable count is statistically evaluated from the number of tubes showing growth.
This method is not accurate but is used for the estimation of “presumptive
coliform count” in drinking water. The presumptive coliform count is a method
of estimating the level of pollution of drinking water. In the plating method,
appropriate dilutions are inoculated on solid media, either on the surface of
plates or as pour plates. The number of colonies that develop after incubation
gives an estimate of the viable count. The method commonly employed is that
described by Miles and Misra (1938) in which serial dilutions are dropped on
the surface of dried plates and colony counts obtained.
36 MICROBIOLOGY
3.2.6 Bacterial growth curve Microbiology
When a bacterium is seeded into a suitable liquid medium and incubated, its
growth follows a definite course. If bacterial counts are made at intervals after
inoculation and plotted in relation to time, a growth curve is obtained. The curve
shows the following phases:
Lag phase: Immediately following the seeding of a culture medium, there is no
appreciable increase in number, though there may be an increase in the size of Notes
the cells. This initial period is the time required for adaptation to the new
environment. The necessary enzymes and metabolic intermediates are built up
in adequate quantities for multiplication to proceed. The maximum cell size is
obtained towards the end of lag phase. The duration of the lag phase varies with
the species, size of the inoculum, nature of the culture medium and environmental
factors such as temperature.
Log (logarithmic) or exponential phase: Following the lag phase, the cells
start dividing and their numbers increase exponentially or by geometric
progression with time. If the logarithm of the viable count is plotted against time,
a straight line will be obtained. In this phase, cells are smaller and stain
uniformly.
Stationary phase: After a varying period of exponential growth, cell division
stops due to depletion of nutrients and accumulation of toxic products. The
number of new cells formed is just enough to replace the number of cells that
die. Equilibrium exists between the dying cells and the newly formed cells. So,
the viable count remains stationary. In this phase, cells are frequently gram
variable and show irregular staining. Sporulation occurs at this stage.
Phase of decline: This is the phase when the population decreases due to cell
death. Besides nutritional exhaustion and toxic accumulation, cel death may also
be caused by autolytic enzymes.

1. Bacteria that can synthesise all their organic compounds are called:
A. Phototrophs B. Chemotrophs
C. Autotrophs D. heterotrophs
2. Organisms that are ordinarily aerobic but can grow in the absence of oxygen
are called:
A. Aerobes B. Facultative anaerobes
C. Anaerobes D. Obligate anaerobes
MICROBIOLOGY 37
Microbiology 3. The process by which complex organic compounds, such as glucose, are
broken down by the action of enzymes into simpler compounds without the
use of oxygen is called:
A. Fermentation B. Substrate-level phosphorylation
C. Oxidative phosphorylation D. Photosynthesis
4. Organisms which are mesophilic grow at:
Notes
A. Temperature of 0-20ºC B. Temperature of 50-60ºC.
C. Temperature of 25-40ºC D. Concentration of 5-10% CO2
5. In the bacterial growth curve, the phase in which there is an exponential
increase in the number of cells is:
A. Lag phase B. Log phase
C. Stationary phase D. Phase of decline

z Bacteria are unicellular organisms (prokaryotes) which lack nuclear
membrane, nucleolus and other cell organelles like mitochondria, endoplasmic
reticulum and golgi apparatus.
z Bacterial cell contains water (80% of total weight), proteins, polysaccharides,
lipids, nucleic acids, mucopeptides and low molecular weight compounds.
z For growth and multiplication of bacteria, the mimimal nutritional
requirements are water, a source of carbon, a source of nitrogen and some
inorganic salts.
z Phototrophs are bacteria that derive energy from sunlight while chemotrophs
derive theirs from chemical reactions.
z Autotrophs are bacteria that synthesise all their organic compounds while
heterotrophs are unable to do so.
z Heterotrophs depend on preformed organic compounds.
z Aerobic bacteria require oxygen for growth and may be obligate aerobes
or facultative anaerobes.
z Obligate aerobes grow only in the presence of oxygen.
z Facultative anaerobes grow in the presence or absence of oxygen.
z Anaerobic bacteria grow in the absence of oxygen.
z Obligate anaerobes may even die on exposure to oxygen.
z Microaerophilic bacteria grow best in the presence of low oxygen tension.
38 MICROBIOLOGY
z Depending on requirements of temperature for growth, bacteria can be Microbiology
classified as mesophilic (25-40ºC), psychrophilic (below 20ºC) and
thermophilic (55-80ºC).
z Moisture and drying, hydrogen ion concentration, light, osmotic effect and
mechanical and sonic stress may also influence the growth and multiplication
of bacteria.
z Bacteria divide by binary fission.
Notes
z When the bacterial cell reaches a certain size, it divides to form two daughter
cells.
z Nuclear division is followed by cell division.
z The time interval between two cell divisions is the generation time or the
population doubling time. It may vary from 20 minutes (coliform bacilli)
to 20 hours (tubercle bacilli) to 20 days (lepra bacilli).
z The bacterial growth curve consists of a lag phase, a log phase, a stationary
phase and a decline phase. This is seen in a liquid medium.
z In the lag phase, the bacteria adapt to the environment. There is no
appreciable increase in cell number.
z In the log phase, there is exponential increase in the number of bacterial
cells.
z In the stationary phase, there is no increase or decrease in the number of
bacterial cells.
z In the decline phase, there is a decrease in the bacterial population due to
cell death.
TERMINAL QUESTIONS
1. Describe the classification of bacteria based on their nutritional requirements.
2. Explain the temperature requirements of bacteria with suitable examples.
3. Describe the gaseous requirements of bacteria.
4. Define fermentation and give an example of fermentation.
4. Describe the bacterial growth curve with suitable diagrams.

1. C 2. B 3. A 4. C 5. B
MICROBIOLOGY 39
MODULE Sterilisation and Disinfection
Microbiology
4
Notes
STERILISATION AND
DISINFECTION
4.1 INTRODUCTION
Disinfection and sterilization are essential for ensuring that medical and surgical
instruments do not transmit infectious pathogens to patients. Because sterilization
of all patient-care items is not necessary, health-care policies must identify,
primarily on the basis of the items’ intended use, whether cleaning, disinfection,
or sterilization is indicated.
OBJECTIVES
z define terms related to Sterilization and Disinfection
z classify items to be sterilised or disinfected
z discuss different Methods of sterilisation
z describe Evaluation and in Process Monitoring of Sterilization Procedures
z discuss Methods of disinfection
z describe the Testing of disinfectants
4.2 DEFINITIONS OF TERMS

Sterilization: Sterilization describes a process that destroys or eliminates all
forms of microbial life and is carried out in health-care facilities by physical or
chemical methods.
40 MICROBIOLOGY
Sterilisation and Disinfection MODULE
Disinfection: Disinfection describes a process that eliminates many or all Microbiology
pathogenic microorganisms, except bacterial spores, on inanimate objects.
Cleaning: Cleaning is removal of visible soil (e.g., organic and inorganic
material) from objects and surfaces. It is normally accomplished manually or
mechanically using water with detergents or enzymatic products.
Decontamination: Decontamination removes pathogenic microorganisms from
objects so they are safe to handle, use, or discard.
Notes
Classification of Materials to be Sterilised / Disinfected
Earle H. Spaulding devised a rational approach to disinfection and sterilization
of patient-care items and equipment. This has three categories
Critical Items
Critical items confer a high risk for infection if they are contaminated with any
microorganism. Thus, objects that enter sterile tissue or the vascular system must
be sterile because any microbial contamination could transmit disease. This
category includes surgical instruments, cardiac and urinary catheters, implants,
and ultrasound probes used in sterile body cavities etc.
Semi-critical Items
Semi-critical items contact mucous membranes or non-intact skin. This category
includes respiratory therapy and anaesthesia equipment, some endoscopes,
laryngoscope blades, esophageal manometry probes, cystoscopes, anorectal
manometry catheters, and diaphragm fitting rings etc.
Noncritical Items
Noncritical items are those that come in contact with intact skin but not mucous
membranes. Intact skin acts as an effective barrier to most microorganisms;
therefore, the sterility of items coming in contact with intact skin is “not critical.”
They can be
Non-critical patient care items: bedpans, blood pressure cuffs, crutches and
computers
Non-critical environmental surfaces

1. Sterilization (a) Removal of visible soil
2. Disinfection (b) Removal of Pathogenic Microorganisms
3. Cleaning (c) Destroys all forms of Microbes
4. Decontamination (d) Removal of Pathogenic Microorganism
except bacteria spores
MICROBIOLOGY 41
Microbiology
4.3 METHODS OF STERILIZATION
The various methods of sterilization are:
1. Physical Method
(a) Thermal (Heat) methods
(b) Radiation method
Notes (c) Filtration method
2. Chemical Method
3. Gaseous method
Methods of sterilization/disinfection
Physical Chemical Physio-

chemical
Liquid
Sunlight Heat Vibration Radiation Filtration
Alcohols
Non-ionizing Earthenware Aldehydes
Dry heat Moist heat Ionizing Phenolics
Asbestos
Red heat Below 100°C Halogens
Flaming Electomagnetic Sintered glass
At 100°C Heavy metals
Incineration Above 100°C Particulate Membrane Surface active agents
Hot air oven Dyes
Infra red Gaseous
Formaldehyde
Ethylene oxide
Plasma
4.3.1 Heat Sterilization

Heat sterilization is the most widely used and reliable method of sterilization,
involving destruction of enzymes and other essential cell constituents. The
process is more effective in hydrated state where under conditions of high
humidity, hydrolysis and denaturation occur, thus lower heat input is required.
Under dry state, oxidative changes take place, and higher heat input is required.
This method of sterilization can be applied only to the thermostable products,
but it can be used for moisture-sensitive materials for which dry heat (160-
180°C) sterilization, and for moisture-resistant materials for which moist heat
(121-134°C) sterilization is used.
The efficiency with which heat is able to inactivate microorganisms is dependent
upon the degree of heat, the exposure time and the presence of water. The action
of heat will be due to induction of lethal chemical events mediated through the
action of water and oxygen. In the presence of water much lower temperature
42 MICROBIOLOGY
time exposures are required to kill microbe than in the absence of water. In this Microbiology
processes both dry and moist heat are used for sterilization.
Dry Heat Sterilization: Examples of Dry heat sterilization are:
1. Incineration
2. Red heat
3. Flaming Notes
4. Hot air oven
It employs higher temperatures in the range of 160-180°C and requires
exposures time up to 2 hours, depending upon the temperature employed. The
benefit of dry heat includes good penetrability and non-corrosive nature which
makes it applicable for sterilizing glass-wares and metal surgical instruments.
It is also used for sterilizing non-aqueous thermo-stable liquids and thermo-
stable powders. Dry heat destroys bacterial endotoxins (or pyrogens) which are
difficult to eliminate by other means and this property makes it applicable for
sterilizing glass bottles which are to be filled aseptically.
Hot-air oven
Dry heat sterilization is usually carried out in a hot air oven, which consists of
the following:
(i) An insulated chamber surrounded by an outer case containing electric
heaters.
(ii) A fan
(iii) Shelves
(iv) Thermocouples
(v) Temperature sensor
(vi) Door locking controls.
Operation
(i) Articles to be sterilized are first wrapped or enclosed in containers of
cardboard, paper or aluminium.
(ii) Then, the materials are arranged to ensure uninterrupted air flow.
(iii) Oven may be pre-heated for materials with poor heat conductivity.
(iv) The temperature is allowed to fall to 40°C, prior to removal of sterilized
material.
Moist Heat Sterilization: Moist heat may be used in three forms to achieve
microbial inactivation
MICROBIOLOGY 43
Microbiology 1. Dry saturated steam – Autoclaving

2. Boiling water/ steam at atmospheric pressure
3. Hot water below boiling point
Moist heat sterilization involves the use of steam in the range of 121-134°C.
Steam under pressure is used to generate high temperature needed for
sterilization. Saturated steam acts as an effective sterilizing agent. Steam for
Notes sterilization can be either wet saturated steam (containing entrained water
droplets) or dry saturated steam (no entrained water droplets).
Fig. 4.1: An Autoclave
Autoclaves use pressurized steam to destroy microorganisms, and are the most
dependable systems available for the decontamination of laboratory waste and
the sterilization of laboratory glassware, media, and reagents. For efficient heat
transfer, steam must flush the air out of the autoclave chamber. Before using the
autoclave, check the drain screen at the bottom of the chamber and clean if
blocked. If the sieve is blocked with debris, a layer of air may form at the bottom
of the autoclave, preventing efficient operation. Autoclaves should be tested
periodically with biological indicators like spores of Bacillus stearothermophilus
to ensure proper function. This method of sterilization works well for many
metal and glass items but is not acceptable for rubber, plastics, and equipment
that would be damaged by high temperatures (Figure 4.1).
Autoclaves, or steam sterilizers essentially consist of following:
1. A cylindrical or rectangular chamber, with capacities ranging from 400 to
800 litres.
44 MICROBIOLOGY
2. Water heating system or steam generating system Microbiology
3. Steam outlet and inlet valves

4. Single or double doors with locking mechanism.
5. Thermometer or temperature gauge
6. Pressure gauges
Operation Notes
For porous loads (dressings) sterilizers are generally operated at a minimum
temperature of 134°C for one hour, and for bottled fluid, sterilizers employing
a minimum temperature of 121°C are used. Ensure that there should be sufficient
water in the autoclave to produce the steam. The stages of operation of
autoclaves include air removal, steam admission and sterilization cycle (includes
heating up, holding/exposure, and cooling stages).
Gaseous Sterilization
The chemically reactive gases such as formaldehyde, (methanol, H.CHO) and
ethylene oxide (CH2)2O possess biocidal activity. Ethylene oxide is a colorless,
odorless, and flammable gas.
The mechanism of antimicrobial action of the two gases is assumed to be through
alkylations of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and
amino groups of nucleic acids. The concentration ranges (weight of gas per unit
chamber volume) are usually in range of 800-1200 mg/L for ethylene oxide and
15-100 mg/L for formaldehyde with operating temperatures of 45-63°C and 70-
75°C respectively.
Both of these gases being alkylating agents are potentially mutagenic and
carcinogenic. They also produce acute toxicity including irritation of the skin,
conjunctiva and nasal mucosa.
(a) Ethylene oxide sterilizer: An ethylene oxide sterilizer consists of a
chamber of 100-300-Litre capacity and surrounded by a water jacket. Air
is removed from sterilizer by evacuation, humidification and conditioning
of the load is done by passing sub-atmospheric pressure steam, then
evacuation is done again and preheated vaporized ethylene oxide is passed.
After treatment, the gases are evacuated either directly to the outside
atmosphere or through a special exhaust system.
Ethylene oxide gas has been used widely to process heat-sensitive devices,
but the aeration times needed at the end of the cycle to eliminate the gas
made this method slow.
MICROBIOLOGY 45
Microbiology (b) Low temperature steam formaldehyde (LTSF) sterilizer: An LTSF

sterilizer operates with sub atmospheric pressure steam. At first, air is
removed by evacuation and steam is admitted to the chamber.
Liquid Sterilization
(a) Peracetic Acid liquid sterilization: Peracetic acid was found to be
sporicidal at low concentrations. It was also found to be water soluble, and
Notes
left no residue after rinsing. It was also shown to have no harmful health
or environmental effects. It disrupts bonds in proteins and enzymes and may
also interfere with cell membrane transportation through the rupture of cell
walls and may oxidize essential enzymes and impair vital biochemical
pathways.
In a low-temperature liquid chemical sterile processing system, several
steps must be followed for effective sterilization:
1. Pre-cleaning of the devices is necessary because many devices have
small connected lumens.
2. Leak testing is done to ensure there are no leaks that could allow fluid
to enter/leak the ampoules/vials and cause damage.
3. The appropriate tray/container must then be selected, and if the device
has lumens, the appropriate connector attached.
4. The sterilant concentrate is provided in a sealed single- use cup and
requires no pre-mixing or dilution.
The disadvantages of this method of sterilization are that the devices must
be immersible, must fit in the appropriate tray, and must be able to withstand
the 55°C temperature the process uses.
(b) Hydrogen Peroxide Sterilization: This method disperses a hydrogen
peroxide solution in a vacuum chamber, creating a plasma cloud. This agent
sterilizes by oxidizing key cellular components, which inactivates the
microorganisms. The plasma cloud exists only while the energy source is
turned on. When the energy source is turned off, water vapor and oxygen
are formed, resulting in no toxic residues and harmful emissions. The
temperature of this sterilization method is maintained in the 40-50°C range,
which makes it particularly well-suited for use with heat-sensitive and
moisture-sensitive medical devices. The instruments are wrapped prior to
sterilization, and can either be stored or used immediately.
There are five phases of the hydrogen peroxide processing cycle:
1. A vacuum phase creates a vacuum in the chamber and the pressure
drops to less than one pound per square inch. This phase lasts about
20 minutes.
46 MICROBIOLOGY
2. In the injection phase, the aqueous hydrogen peroxide is introduced Microbiology
into the vacuum chamber and is vaporized into a gas, which creates
a rise in pressure due to the increase of molecules.
3. During the diffusion phase the hydrogen peroxide vapor spreads
throughout the chamber and the increased pressure drives the sterilant
into the packs, exposing the instrument surfaces to the sterilant and
killing the microorganisms.
Notes
4. During the plasma phase the radio frequency energy is applied,
stripping the electrons from some of the molecules and producing a
low-temperature plasma cloud. Following this reaction, the activated
compounds lose their high energy and recombine to form oxygen and
water.
5. The purpose of the venting phase is to introduce filtered air into the
chamber and return the chamber to atmospheric pressure so that the
door can be opened. It lasts about one minute.

Match the following
1. Dry heat Sterilisation (a) Hydrogen peroxide Sterilizer
2. Moist heat (b) Formaldehyde Sterilizer
3. Gas Sterilization (c) Autoclave
4. Liquid Sterilisation (d) Hot air Oven
4.3 RADIATION STERILIZATION

Many types of radiation are used for sterilization like electromagnetic radiation
(e.g. gamma rays and UV light), particulate radiation (e.g. accelerated
electrons).The major target for these radiation is microbial DNA. Gamma rays
and electrons cause ionization and free radical production while UV light causes
excitation.
Radiation sterilization with high energy gamma rays or accelerated electrons has
proven to be a useful method for the industrial sterilization of heat sensitive
products. But some undesirable changes occur in irradiated products, an
example is aqueous solution where radiolysis of water occurs.
Radiation sterilization is generally applied to articles in the dry state; including

surgical instruments, sutures, prostheses, unit dose ointments, plastic syringes
MICROBIOLOGY 47
Microbiology and dry pharmaceutical products. UV light, with its much lower energy, and poor
penetrability finds uses in the sterilization of air, for surface sterilization of
aseptic work areas, for treatment of manufacturing grade water, but is not
suitable for sterilization of pharmaceutical dosage forms.
Gamma ray Sterilizer: Gamma rays for sterilization are usually derived from
cobalt-60 source, the isotope is held as pellets packed in metal rods, each rod
Notes carefully arranged within the source and containing 20 KCi of activity. This
source is housed within a reinforced concrete building with 2 m thick walls.
Articles being sterilized are passed through the irradiation chamber on a
conveyor belt and move around the raised source.
Ultraviolet Irradiation: The optimum wavelength for UV sterilization is 260

nm. A mercury lamp giving peak emission at 254 nm is the suitable source of
UV light in this region.
Electron Accelerator
There are two types of electron accelerator machines, the electrostatic accelerator
which produces electrons with maximum energies of 5 MeV, and the microwave
linear accelerator which produces electrons with maximum energies of 10 MeV.
Higher energies cause better penetration into the product but there is a risk of
induced radiation.
A high energy electron beam is generated by accelerating electrons from a hot

filament down an evacuated tube under high potential difference, and then
additional energy is imparted to this beam in a pulsed manner by a synchronized
traveling microwave. Articles to be sterilized are arranged on a horizontal
conveyor belt and are irradiated from one or both sides.
Filtration Sterilization
Filtration process does not destroy but removes the microorganisms. It is used
for both the clarification and sterilization of liquids and gases as it is capable
of preventing the passage of both viable and non viable particles.
The major mechanisms of filtration are sieving, adsorption and trapping within
the matrix of the filter material. Sterilizing grade filters are used in the treatment
of heat sensitive injections and ophthalmic solutions, biological products and air
and other gases for supply to aseptic areas. They are also used in industry as part
of the venting systems on fermentors, centrifuges, autoclaves and freeze driers.
Membrane filters are used for sterility testing.
48 MICROBIOLOGY
Application of filtration for sterilization of gases: HEPA (High efficiency Microbiology
particulate air) filters can remove up to 99.97% of particles >0.3 micrometer in
diameter. Air is first passed through prefilters to remove larger particles and then
passed through HEPA filters. The performance of HEPA filter is monitored by
pressure differential and airflow rate measurements.
There are two types of filters used in filtration sterilization

Notes
(a) Depth filters: Consist of fibrous or granular materials so packed as to form
twisted channels of minute dimensions. They are made of diatomaceous
earth, unglazed porcelain filter, sintered glass or asbestos.
(b) Membrane filters: These are porous membrane about 0.1 mm thick, made
of cellulose acetate, cellulose nitrate, polycarbonate, and polyvinylidene
fluoride, or some other synthetic material.The membranes are supported on
a frame and held in special holders. Fluids are made to transverse
membranes by positive or negative pressure or by centrifugation.
Application of filtration for sterilization of liquids: Membrane filters of 0.22

micrometer nominal pore diameter are generally used, but sintered filters are
used for corrosive liquids, viscous fluids and organic solvents. The factors which
affects the performance of filter is the titre reduction value, which is the ratio
of the number of organism challenging the filter under defined conditions to the
number of organism penetrating it. The other factors are the depth of the
membrane, its charge and the tortuosity of the channels.
Evaluation and In Process Monitoring of Sterilization Procedures

Dry Heat Sterilization
Physical indicator: In this process temperature record chart is made of each

sterilization cycle with dry heat sterilization. This chart forms the batch
documentation and is compared against a master temperature records. The
temperature should be taken as the coolest part of the loaded sterilizer, further
information on heat distribution and penetration within sterilizer can be gained
by the use of thermocouple place at selected site in the chamber or injected into
test packs or bottles.
Chemical indicator: It is based on the ability of heat to alter the chemical or

physical characteristics of variety of chemical substances. This change should
take place only when satisfactory condition for sterilization prevails. Thus
conforming that sterilization cycle has been successfully completed. Chemical
indicators generally undergo melting or colour change.
MICROBIOLOGY 49
Microbiology Biological indicator: The biological indicators are the standardized bacterial
spore preparations which are usually in the form of suspension in water or culture
medium or of spore dried on paper or plastic carriers, they are placed in sterilizer.
After the sterilization process the aqueous suspension /spores are on carriers are
aseptically transferred to an appropriate nutrient medium, which is then
incubated and occasionally seen for the growth. Clostridium species is generally
Notes used for dry heat sterilization indicator.
Indicators Sterilization Principle Device Parameter
Methods monitored
Physical Dry heat Temperature Temperature Temperature

recording charts recording charts
Chemical Dry heat Temperature Browne’s tube Temperature,

sensitive Time
coloured
solution
Temperature A temperature Temperature

sensitive sensitive white
chemical wax concealing
a black marked
Biological Dry heat Temperature Bacillus D value

sensitive subtilis
microbes
Moist Heat Sterilization

Physical Indicator: In this process temperature record chart is made of each
sterilization cycle with dry heat sterilization. This chart of the batch documentation
is compared against a master temperature records. The temperature should be
taken as the coolest part of the loaded sterilizer, further information on heat
distribution and penetration within sterilizer can be gained by the use of
thermocouple place at selected site in the chamber or injected into test packs or
bottles.
Chemical Indicator: It is based on the ability of heat to alter the chemical or

physical characteristics of variety of chemical substances. This change should
take place only when satisfactory condition for sterilization prevails. Thus
conforming that sterilization cycle has been successfully completed chemical
indicator generally undergoes melting or colour change.
Biological Indicator: Spores of G. steareothermophylus in sealed ampoules of

culture medium are used for moist heat sterilization monitoring and these may
50 MICROBIOLOGY
be incubated directly at 55°C, thus may eliminate the need of aseptic transfer Microbiology
(Table 3).
Aseptic transfer is also avoided by use of self-contained units where the spores
strip and the nutrient medium are present in the same device ready for mixing
after use.
The bacterial spores should have following qualities
(i) It should be non-pathogenic Notes
(ii) Should possess above average resistant to the particular sterilization
process.
Indicator Sterilization Principle Device Parameter

monitored
Physical Moist heat Temperature Temperature Temperature

recording recording
charts charts
Chemical Moist heat Temperature Browne’s tube Temperature,

sensitive Time
coloured
solution
Steam A device which Saturated steam

sensitive is impregnated
chemical into a carrier
material.
Biological Moist heat Temperature Geobacillus D value

sensitive stearother-
microbes mophilus
Gaseous Sterilization
Physical Indicator: Gas concentration is measured independently of pressure
rise, often by reference to weight of gas used.
Chemical Indicator: The chemical indicator used here are Royach Sacket, the
indicator paper impregnated with reactive chemical which undergoes a distinct
colour change on reaction. Chemical indicators are valuable monitors of the
condition prevailing at the coolest of most in accessible part of a sterilizer.
Biological Indicator: As with chemical indicator they are usually packed in

dummy packs located at strategic sites in the sterilizer. Alternatively for gaseous
sterilization, these may also be placed in tubular helix device. The species of
bacteria generally used for gaseous sterilization are B.subtilis var.niger and
B.subtilis var.golbigii
MICROBIOLOGY 51
Microbiology Radiation Sterilization

Physical Indicator: In radiation sterilization a plastic or perspex dosimeter
which gradually darkens in proportion to the radiation it absorbs give an accurate
measure of the radiation dose and is considered to be the best technique currently
available for the radiation sterilization process.
Chemical Indicator: Chemical dosimeter acidified with cerric ammonium

Notes sulphate or cerric sulphate solution .These responds to irradiation by dose
change in the applied density. Those are considered best and accurately measure
relation dose.
Biological Indicator: These consist of standardized bacterial spore preparation
which are usually in the form of suspension in water or culture medium or of
spore dried on paper or plastic carriers, they are placed in sterilizer.
After the sterilization process the aqueous suspension /spores are on carriers are
aseptically transferred to an appropriate nutrient medium, which is then
incubated and periodically observed for the growth. Clostridium species is
generally used for dry heat sterilization indicator
Filtration Sterilization
Physical Indicator: Sterilizing filters are subjected to a bubble point pressure
test. This is a technique for determining the pore size of a filter, and may also
be used to check the integrity of certain types of filters. The principle of the test
is that the wetted filter in its assembled unit is subjected to an increasing air or
nitrogen gas pressure difference. The pressure difference recorded when the first
bubble of gas breaks away from the filter is related to maximum pore size. When
the gas pressure is further increased slowly there is general eruption of bubble
over the entire surface. The pressure difference here is related to the mean pore
size. Pressure difference below the expected value would signify a damage or
faulty filter.
Biological Indicator: Filtration sterilization requires a different approach from
biological monitoring, the test effectively measure in the ability of a filter to
produce a sterile filtrate from a culture of suitable organism S. marcesence, a
small gram negative rod shape bacterium. B. diminuta used as a biological
indicator having a dimension 0.5 micrometres and 0.3 micrometre respectively
has been used for filters of 0.45 micrometre and 0.22 micrometre. The extent
of the passage of this organism through membrane filter is enhanced by
increasing the filtration pressure. Thus successful sterile filtration depends
markedly on the challenge condition. Such tests are used as the part of filter
manufacture characterization and quality assurance process, and user’s initial
validation procedure.
52 MICROBIOLOGY
Microbiology
4.4 CHEMICAL METHODS OF DISINFECTION
Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate
surfaces. Some chemicals when used at apropriate concentration for appropriate
duration can be used for sterilization and are called sterilant liquids. Those
chemicals that can be safely applied over skin and mucus membranes are called
antiseptics.
An ideal antiseptic or disinfectant should have following properties: Notes
1. Should have wide spectrum of activity
2. Should be able to destroy microbes within practical period of time
3. Should be active in the presence of organic matter
4. Should make effective contact and be wettable
5. Should be active in any pH
6. Should be stable
7. Should have long shelf life
8. Should be speedy
9. Should have high penetrating power
10. Should be non-toxic, non-allergenic, non-irritative or non-corrosive
11. Should not have bad odour
12. Should not leave non-volatile residue or stain
13. Efficacy should not be lost on reasonable dilution
14. Should not be expensive and must be available easily
Such an ideal disinfectant is not yet available. The level of disinfection achieved
depends on contact time, temperature, type and concentration of the active
ingredient, the presence of organic matter, the type and quantum of microbial
load. The chemical disinfectants at working concentrations rapidly lose their
strength on standing.
Classification of disinfectants:
1. Based on consistency
(a) Liquid (E.g., Alcohols, Phenols)
(b) Gaseous (Formaldehyde vapour)
2. Based on spectrum of activity
(a) High level
(b) Intermediate level
(c) Low level
MICROBIOLOGY 53
Microbiology 3. Based on mechanism of action

(a) Action on membrane (E.g., Alcohol, detergent)
(b) Denaturation of cellular proteins (E.g., Alcohol, Phenol)
(c) Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2,
Halogens)
Notes (d) Alkylation of amino-, carboxyl- and hydroxyl group (E.g.,

Formaldehyde)
(e) Damage to nucleic acids (Formaldehyde)
Alcohols
Mode of action: Alcohols dehydrate cells, disrupt membranes and cause
coagulation of protein.
Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
Application: A 70% aqueous solution is more effective at killing microbes than

absolute alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin.
Isopropyl alcohol is preferred to ethanol. It can also be used to disinfect surfaces.
It is used to disinfect clinical thermometers. Methyl alcohol kills fungal spores,
hence is useful in disinfecting inoculation hoods.
Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable
Aldehydes
Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group,
and probably damages nucleicacids. It kills all microorganisms, including
spores.
Examples: Formaldehyde, Gluteraldehyde
Application: 40% Formaldehyde (formalin) is used for surface disinfection and

fumigation of rooms, chambers, operation theatres, biological safety cabinets,
wards, sick rooms etc. Fumigation is achieved by boiling formalin, heating
paraformaldehyde or treating formalin with potassium permanganate. It also
sterilizes bedding, furniture and books. 10% formalin with 0.5% tetraborate
sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize
thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments
etc. An exposure of at least 3 hours at alkaline pH is required for action by
gluteraldehyde. 2% formaldehyde at 40°C for 20 minutes is used to disinfect
wool and 0.25% at 60oC for six hours to disinfect animal hair and bristles.
54 MICROBIOLOGY
Disadvantages: Vapors are irritating (must be neutralized by ammonia), has Microbiology
poor penetration, leaves non-volatile residue, activity is reduced in the presence
of protein. Gluteraldehyde requires alkaline pH and only those articles that are
wettable can be sterilized.
Phenol
Mode of action: Act by disruption of membranes, precipitation of proteins and Notes
inactivation of enzymes.
Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol),
hexachlorophene, chlorhexidine, chloroxylenol (Dettol)
Applications: Joseph Lister used it to prevent infection of surgical wounds.
Phenols are coal-tar derivatives. They act as disinfectants at high concentration
and as antiseptics at low concentrations. They are bactericidal, fungicidal,
mycobactericidal but are inactive against spores and most viruses. They are not
readily inactivated by organic matter. The corrosive phenolics are used for
disinfection of ward floors, in discarding jars in laboratories and disinfection of
bedpans. Chlorhexidine can be used in an isopropanol solution for skin
disinfection, or as an aqueous solution for wound irrigation. It is often used as
an antiseptic hand wash. 20% Chlorhexidine gluconate solution is used for pre-
operative hand and skin preparation and for general skin disinfection.
Chlorhexidine gluconate is also mixed with quaternary ammonium compounds
such as cetrimide to get stronger and broader antimicrobial effects (eg. Savlon).
Chloroxylenols are less irritant and can be used for topical purposes and are more
effective against gram positive bacteria than gram negative bacteria.
Hexachlorophene is chlorinated diphenyl and is much less irritant. It has marked
effect over gram positive bacteria but poor effect over gram negative bacteria,
mycobacteria, fungi and viruses. Triclosan is an organic phenyl ether with good
activity against gram positive bacteria and effective to some extent against many
gram negative bacteria including Pseudomonas. It also has fair activity on fungi
and viruses.
Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is
inactivated by anionic soaps. Chloroxylenol is inactivated by hard water.
Halogens
Mode of action: They are oxidizing agents and cause damage by oxidation of
essential sulfydryl groups of enzymes. Chlorine reacts with water to form
hypochlorous acid, which is microbicidal.
Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine
compounds (tincture iodine, iodophores)
MICROBIOLOGY 55
Microbiology Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic.

Iodine can be combined with neutral carrier polymers such as polyvinylpyrrolidone
to prepare iodophores such as povidone-iodine. Iodophores permit slow release
and reduce the irritation of the antiseptic. For hand washing iodophores are
diluted in 50% alcohol. 10% Povidone Iodine is used undiluted in pre and
postoperative skin disinfection. Chlorine gas is used to bleach water. Household
bleach can be used to disinfect floors. Household bleach used in a stock dilution
Notes of 1:10. In higher concentrations chlorine is used to disinfect swimming pools.
0.5% sodium hypochlorite is used in serology and virology. Used at a dilution
of 1:10 in decontamination of spillage of infectious material. Mercuric chloride
is used as a disinfectant.
Disadvantages: They are rapidly inactivated in the presence of organic matter.
Iodine is corrosive and staining. Bleach solution is corrosive and will corrode
stainless steel surfaces.
Heavy Metals
Mode of action: Act by precipitation of proteins and oxidation of sulfydryl
groups. They are bacteriostatic.
Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury
salts (e.g., mercurochrome, merthiolate)
Applications: 1% silver nitrate solution can be applied on eyes as treatment for
opthalmia neonatorum (Crede’s method). This procedure is no longer followed.
Silver sulphadiazine is used topically to help to prevent colonization and
infection of burn tissues. Mercurials are active against viruses at dilution of
1:500 to 1:1000. Merthiolate at a concentration of 1:10000 is used in
preservation of serum. Copper salts are used as a fungicide.
Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by
organic matter.
Surface Active Agents

Mode of actions: They have the property of concentrating at interfaces between
lipid containing membrane of bacterial cell and surrounding aqueous medium.
These compounds have long chain hydrocarbons that are fat soluble and charged
ions that are water-soluble. Since they contain both of these, they concentrate
on the surface of membranes. They disrupt membrane resulting in leakage of cell
constituents.
Examples: These are soaps or detergents. Detergents can be anionic or cationic.
Detergents containing negatively charged long chain hydrocarbon are called
anionic detergents. These include soaps and bile salts. If the fat-soluble part is
56 MICROBIOLOGY
made to have a positive charge by combining with a quaternary nitrogen atom, Microbiology
it is called cationic detergents. Cationic detergents are known as quaternary
ammonium compounds (or quat). Cetrimide and benzalkonium chloride act as
cationic detergents.
Application: They are active against vegetative cells, Mycobacteria and
enveloped viruses. They are widely used as disinfectants at dilution of 1-2% for
domestic use and in hospitals.
Notes
Disadvantages: Their activity is reduced by hard water, anionic detergents and
organic matter. Pseudomonas can metabolise cetrimide, using them as a carbon,
nitrogen and energy source.
Dyes
Mode of action: Acridine dyes are bactericidal because of their interaction with
bacterial nucleic acids.
Examples: Aniline dyes such as crystal violet, malachite green and brilliant
green. Acridine dyes such as acriflavin and aminacrine. Acriflavine is a mixture
of proflavine and euflavine. Only euflavine has effective antimicrobial properties.
They are more effective against gram positive bacteria than gram negative
bacteria and are more bacteriostatic in action.
Applications: They may be used topically as antiseptics to treat mild burns.
They are used as paint on the skin to treat bacterial skin infections. Melachite
green is used in LJ medium for growth of Mycobacterium tuberculosis.
Hydrogen Peroxide
Mode of action: It acts on the microorganisms through its release of nascent
oxygen. Hydrogen peroxide produces hydroxyl-free radical that damages
proteins and DNA.
Application: It is used at 6% concentration to decontaminate the instruments,
equipments such as ventilators. 3% Hydrogen Peroxide Solution is used for skin
disinfection and deodorising wounds and ulcers. Strong solutions are sporicidal.
Disadvantages: Decomposes in light, broken down by catalase, proteinaceous
organic matter drastically reduces its activity.
Beta-propiolactone (BPL)
Mode of action: It is an alkylating agent and acts through alkylation of carboxyl-
and hydroxyl-groups.
Properties: It is a colorless liquid with pungent to slightly sweetish smell. It is
a condensation product of ketane with formaldehyde.
MICROBIOLOGY 57
Microbiology Application: It is an effective sporicidal agent, and has broad-spectrum activity.

0.2% is used to sterilize biological products. It is more efficient in fumigation
that formaldehyde. It is used to sterilize vaccines, tissue grafts, surgical
instruments and enzymes
Disadvantages: It has poor penetrating power and is a carcinogen.
Notes Testing of Disinfectants

A disinfectant must be tested to know the required effective dilution, the time
taken to effect disinfection and to periodically monitor its activity. As disinfectants
are known to lose their activity on standing as well as in the presence of organic
matter, their activity must be periodically tested.
Different methods are:

1. Koch’s method
2. Rideal Walker Method
3. Chick Martin test
4. Capacity use dilution test (Kelsey-Sykes test)
5. In-use test
Koch’s method: Spores of Bacillus anthracis were dried on silk thread and were
subjected to action of disinfectants. Later, it was washed and transferred to solid
medium.
Rideal Walker method: This method relies on the estimation of phenol
coefficient. Phenol coefficient of a disinfectant is calculated by dividing the
dilution of test disinfectant by the dilution of phenol that disinfects under
predetermined conditions. Disadvantages of the Rideal-Walker test are: No
organic matter is included; the microorganism Salmonella typhi may not be
appropriate; the time allowed for disinfection is short; it should be used to
evaluate phenolic type disinfectants only.
Chick Martin test: This test also determines the phenol coefficient of the test
disinfectant. Unlike in Rideal Walker method where the test is carried out in
water, the disinfectants are made to act in the presence of yeast suspension (or
3% dried human feces). Time for subculture is fixed at 30 minutes and the
organism used to test efficacy is S.typhi as well as S.aureus. The phenol
coefficient is lower than that given by Rideal Walker method.
Capacity use dilution test (Kelsey-Sykes test)

The capacity test (Kelsey-Sykes) determine the appropriate use dilution of the
disinfectants. The stability test (Maurer) determines the stability and long term
58 MICROBIOLOGY
effectiveness of disinfectant dilution. The capacity and stability test help to Microbiology
determine the choice of a disinfectant.
In-use test:
The routine monitoring of disinfectant in use can be done by the ‘in use’ test
(Kelsey & Maurer). This test is intended to estimate the number of living
organism in a vessel of disinfectant in actual use. The disinfectant that is already
in use is diluted 1 in 10 by mixing 1 ml of the disinfectant with 9 ml of sterile Notes
nutrient broth. Ten drops of the diluted disinfectant (each 0.02 ml) is placed on
two nutrient agar plates. One plate is incubated at 37°C for 3 days while the other
is held at room temperature for 7 days. The number of drops that yielded growth
is counted after incubation. If there growth in more than five drops on either
plate, it represents failure of disinfectant.

1. ................. species is used as indicator in dry heat sterilization
2. Spores of ..................... is used in moist heat sterilisation
3. Chemicals that can be safely applied over skin is .................
4. Spores of ................. used for testing of disinfectants

The various methods of sterilization are:
z Physical Method
(a) Thermal (Heat) methods
(b) Radiation method
(c) Filtration method
z Chemical Method
z Gaseous method
Classification of disinfectants:
z Based on consistency
(a) Liquid (E.g., Alcohols, Phenols)
(b) Gaseous (Formaldehyde vapour)
MICROBIOLOGY 59
Microbiology z Based on spectrum of activity

(a) High level
(b) Intermediate level
(c) Low level
z Based on mechanism of action
(a) Action on membrane (E.g., Alcohol, detergent)
Notes (b) Denaturation of cellular proteins (E.g., Alcohol, Phenol)
(c) Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2,
Halogens)
(d) Alkylation of amino-, carboxyl- and hydroxyl group (E.g.,
Formaldehyde)
(e) Damage to nucleic acids (Formaldehyde)
TERMINAL QUESTIONS
1. Define sterilisation and disinfection?
2. Describe the working of an Autoclave in a flowchart?
3. Classify disinfectant based on their mechanism of action.
4. What are various physical methods of sterilisation describe any one of them.
5. How to test for efficacy of disinfectents?

4.1
1. (c) 2. (d) 3. (a) 4. (b)
4.2
1. (d) 2. (c) 3. (b) 4. (a)
4.3
1. Clostridium
2. G. Steareothermophilus
3. Antiseptics
4. Bacillus Anthracisis
60 MICROBIOLOGY
Bio Medical Waste Management MODULE
Microbiology
5
Notes
BIO MEDICAL WASTE
MANAGEMENT
5.1 INTRODUCTION
Bio medical waste (BMW) may be defined as any solid, fluid or liquid waste
material including its container and any other intermediate products which is
generated during short term and long term care consisting of observational,
diagnostic, therapeutic and rehabilitative services for a person suffering or
suspected to be suffering from disease or injury or during research pertaining
to production & testing of biologicals during immunization of human beings.
From total quantity of waste generated by health care activities almost 80-90%
is general waste comparable to domestic waste. This comes from the administrative
and housekeeping functions of Hospital and laboratories. The balance 10-20%
of waste is considered hazardous and / or infectious. This lesson discusses about
biomedical waste management.
OBJECTIVES
z describe the concept of bio medical waste management

z explain steps of bio medical waste management
z segregate and dispose waste materials appropriately
MICROBIOLOGY 61
MODULE Bio Medical Waste Management
Microbiology Steps of biomedical waste management:
Notes
Segregation should be done as per categories.

Category Type of Container Colour coding
1. Human anatomical waste Plastic bag yellow
2. Animal waste Plastic bag yellow
3. Microbiology and Plastic bag yellow/red
Biotechnology waste
4. Waste sharp Puncture proof Blue/white
container
5. Discarded Medicines Plastic bag Black
and cytotoxic waste
6. Solid soiled waste plastic bag yellow/red
7. Solid waste plastic bag blue(all disposable
plastics)
8. Liquid waste – –
9. Incineration ash plastic bag black
10. Chemical waste (solid) plastic bag black

Match the following
Waste Colour Coding
1. Human Anatomical waste a. Blue/white
2. Vials of cytotoxic drugs b. Red
3. Plastic IV Bottles c. Yellow
4. Needles d. Black
62 MICROBIOLOGY
Location of containers: All containers having different coloured plastic bags Microbiology
should be located at the point of generation of waste i.e. near diagnostic services
areas.
Bags: It should be ensured that waste bags are filled upto only three fourth
capacity, tied securely and removed from the site of the generation regularly and
timely.
Certain categories of waste, which may need pre-treatment (decontamination /
Notes
disinfection) at the site of generation such as plastic and sharp materials, etc,
should be removed from the site of generation only after treatment.
Storage of Waste
z No untreated BMW should be stored beyond 48 hours.
z If necessary to store beyond 48 hours, the authorized person must take
permission of the prescribed authority.
z The authorized person should ensure that waste does not adversely affect
human health and environment.
Transportation
Within the hospital

z Waste routes should be designated and separate time should be earmarked
for BMW to reduce chances of its mixing with general waste.
z Dedicated wheeled trolleys are used and they should be cleaned and
disinfected in case of any spillage.
z Trolleys should not have any sharp edges and should be easy to clean.
Outside the hospital
z BMW shall be transported only in vehicles authorized by competent authority
as specified by government.
Treatment and Disposal of Waste

General waste
z Most of the waste (80-90% generated in the hospital is general waste). This
waste is non toxic and non infectious and comprises of paper, left over food
articles, peels of fruits, disposable and paper container for tea / coffee etc,
card board boxes, outer cover or wrappings.
z These general wastes should be put into black coloured polyethylene bags
and deposited at the municipal dumps- It is subsequently collected by local
civic authorities.
z Safe disposal by local municipal authority.
MICROBIOLOGY 63
Microbiology Bio medical waste

z Always remember to disinfect and mutilate the waste before its final
disposal
z Remember the following while treating the bio medical waste
Anatomical waste to be deep buried
Syringes to be cut (with hub cutters) and chemically disinfected with
Notes 1% bleaching powder solution at source of generation before final
disposal into sharps pit
Infected plastics to be chemically disinfected or autoclaved, shredded
and recycled and sent for final disposal into municipal dumps
Incineration: The specific requirements regarding norms of combustion
efficiency and emission level have been defined in BMW rules 1998.
z Suitably designed pollution control devices should be installed.
z Incinerator should be certified from pollution control board.
z In case of small hospitals, joint facilities for incineration can be developed.
z The chlorinated plastic bags should not be incinerated.
z Waste to be incenerated shall not be chemically treated with any chlorinated
disinfectants.
z The functioning of the incinerator and the number of cycles operated per
day should be documented in a log book.
z Regular monitoring of the process should be done as per CPCB norms.
z The ash produced by incinerator should be sent for secure land filling and
should also be periodically checked for toxic metals.
Deep Burial: BMW rule 1998 mentions waste under category 1and 2 can be
accorded deep burial and this shall be an option available only in towns with
population less than five lakhs and in rural areas.The location of the deep burial
site will be authorized by the prescribed authority and it should be distant from
residential areas and it should be ensured that no contamination occurs of any
surface waste or ground water. The area should not be prone to flooding or
erosion.
Autoclave and Microwave: Category 3, 4, 6 & 7 can be treated by these
techniques.
Shredding: Plastic (IV bottles, syringes, catheter etc.), sharps (needles, blade,
glass) should be shredded after chemical treatment/ microwaving/ autoclaving.
Needle Destroyers: They can be used for disposal of needles directly without
chemical treatment.
Secured landfill: Incinerator ash, discarded medicines, cytoxic substances and
solid chemical waste should be treated by this option.
64 MICROBIOLOGY
Sharp pit: Sharp waste can be disposed in a circular or rectangular pit, after Microbiology
disinfection. Pit can be dug and lined with bricks, masonry or concrete rings. The
pit should be covered with heavy concrete slab which is penetrated by galvanized
steel pipe projecting about 1.5m above the slab with an internal diameter of upto
20mm. Pit should be 2-5m deep and 1-2m wide (WHO) When pit is full it can be
sealed completely, after another has been prepared. It should be half filled with
waste, and then covered with lime within 50 cm of surface before filling the rest
Notes
of the pit with soil.
Safety Measures
Health Care Workers (HCW) require following Personal Protection Equipments

(PPE):
z Gloves
z Masks
z Protective glasses
z Plastic aprons
z Gum boots for waste handlers
z Hepatitis B and Tetanus immunization
All accidents while doing therapeutic, diagnostic and handling waste should be
recorded. All waste handlers should be made aware of risks involved in handling
BMW.
Training
Entire HCW should be made aware of BMW Rule 1998 through training
programmes

Match the following
Lab speciemen PPE
1. Blood collection (a) Gloves and Mask
2. Sputum (b) Gloves, Mask, Apron and Goggles
3. Blood products (c) Gloves
4. Urine specimens (d) Gloves, Mask
MICROBIOLOGY 65
Microbiology

z The biomedical waste is the waste that is generated during the diagnosis,
treatment or immunization of human beings or animals or in research
activities pertaining thereto, or in the production or testing of biological
components.
Notes
z Steps of biomedical waste management are a) segregation b) storage c)
transport d) disposal.
z The biomedical wastes are categorized into ten according to its characteristics
taking into account treatment and disposal.
z The treatment options for biomedical waste as per the schedule I of the
Rules are incineration, deep burial, autoclave, microwave, chemical treatment,
destruction and shredding, and disposal in secured land fills.
TERMINAL QUESTIONS
1. Define and classify biomedical waste.
2. Describe the steps of biomedical waste management.
ANSWERS OF INTEXT QUESTIONS
5.1
1. (c) 2. (b) 3. (d) 4. (a)
5.2
1. (b) 2. (d) 3. (a) 4. (c)
66 MICROBIOLOGY
Laboratory Safety and Standards Precautions MODULE
Microbiology
6
Notes
LABORATORY SAFETY AND
STANDARDS PRECAUTIONS
6.1 INTRODUCTION
Working in a laboratory usually involves working with various chemical,
physical, and biological hazards. Because the hazards vary from laboratory to
laboratory, employers must address the hazards specific to their laboratories.
Standard precautions are meant to reduce the risk of transmission of blood borne
and other pathogens from both recognized and unrecognized sources. They are
the basic level of infection control precautions which are to be used, as a
minimum, in the health care settings.
OBJECTIVES
After reading this lesson, you will be able to
z describe preparing the laboratory
z explain about common symbols used in laboratories
z explain fire safety and prevent fire accidents in the laboratories
z describe and follow equipment safety
z explain and follow universal standard precautions
6.2 PREPARING FOR LABORATORY WORK

Before starting to work in a laboratory, you must familiarize with the following:
z The hazards of the materials in the lab, as well as appropriate safe handling,
storage and emergency protocols. Read labels and material safety data
MICROBIOLOGY 67
MODULE Laboratory Safety and Standards Precautions
Microbiology sheets (MSDSs) before moving, handling or opening chemicals. Never use
a product from an unlabeled container, and report missing labels to your
supervisor.
z The agents, processes and equipment in the laboratory. If you are unsure
of any aspect of a procedure, check with your supervisor before proceeding.
z The location and operation of safety and emergency equipment such as fire
Notes extinguishers, eye wash and shower, first aid and spill response kits, fire
alarm pull stations, telephone and emergency exits
z Emergency spill response procedures for the materials you will handle
During laboratory work

z Restrict laboratory access to authorized persons only. Children are not
permitted in labs.
z Smoking; eating; drinking; storing food, beverages or tobacco; handling
contact lenses are not permitted in laboratories.
z Wear lab coats (knee length) and safety glasses in laboratories employing
chemicals, biohazards or radioisotopes. Open shoes, such as sandals, should
never be worn in the lab.
z Keep work places clean and free of unwanted chemicals, biological
specimens, Avoid leaving reagent bottles, empty or full, on the floor.
z Work only with materials once you know their safe handling and storage.
z Never pipette by mouth; use mechanical transfer devices.
z Keep exits and passageways clear at all times.
z Ensure that access to personal protective equipments are not blocked.
z Report accidents and dangerous incidents (“near-misses”) promptly to your
supervisor
z Wash your hands thoroughly before leaving the laboratory.
z Perform procedures that liberate infectious bio-aerosols in a biological
safety cabinet
z Handle all human blood and body fluids as if potentially infectious.
Cleaning up before leaving

Perform a safety check at the end of each experiment and before leaving the lab.
make sure to:
z Turn off gas, water, electricity, vacuum and compression lines and heating
apparatus
68 MICROBIOLOGY
z Return unused materials, equipment and apparatus to their proper storage Microbiology
locations
z Dispose of all waste material.
z Remove defective or damaged equipment immediately, and arrange to have
it repaired or replaced
z Decontaminate any equipment or work areas that may have been in contact
with hazardous materials. Notes
z Leave behind protective clothing (lab coats, gloves, etc.) when leaving the
laboratory.
6.3 SYMBOLS TO BE IDENTIFIED BY ALL

LABORATORY TECHNICIANS
MICROBIOLOGY 69
Microbiology
6.4 FIRE SAFETY
Laboratory fires can by caused by bunsen burners, runaway chemical reactions,
electrical heating units, failure of unattended or defective equipment, or
overloaded electrical circuits. Familiarize yourself with the operation of the fire
extinguishers and the location of pull stations, emergency exits and evacuation
routes where you work. In the event that the general alarm is sounded use the
Notes evacuation routes established for your area and follow the instructions of the
Evacuation Monitors. Once outside of the building, move away from the doors
to enable others to exit.
The fire triangle

Fire cannot occur without an ignition source, fuel and an oxidizing atmosphere
(usually air), the three elements that comprise what is called the “fire triangle”:
Fire will not be initiated if any one of these elements is absent, and will not be
sustained if one of these elements is removed. This concept is useful in
understanding prevention and control of fires. For example, the coexistence of
flammable vapours and ignition sources should be avoided, but when flammable
vapours cannot be controlled elimination of ignition sources is essential.
Learn how to use the extinguisher in your lab, as there will be no time to read
instructions during an emergency. Attempt to fight small fires only, and only if
there is an escape route behind you. Remember to have the extinguisher
recharged after every use.
z P: Pull and twist the locking pin to break the seal.
z A: Aim low, and point the nozzle at the base of the fire.
z S: Squeeze the handle to release the extinguishing agent.
z S: Sweep from side to side until the fire is out.
z Be prepared to repeat the process if the fire breaks out again
70 MICROBIOLOGY
Microbiology
6.5 GLASSWARE SAFETY
Use a dustpan and brush, not your hands, to pick up broken glass. Discard broken
glass in a rigid container separate from regular garbage and label it appropriately.
Equipment Safety
Every effort should be made to prevent equipment from becoming contaminated.
To reduce the likelihood of equipment malfunction that could result in leakage, Notes
spill or unnecessary generation of aerosolized pathogens:
z Review the manufacturer’s documentation. Keep for future reference.
z Use and service equipment according to the manufacturer’s instructions.
z Ensure that anyone who uses a specific instrument or piece of equipment
is properly trained in setup, use and cleaning of the item.
Ensure that equipment leaving the laboratory for servicing or disposal is
appropriately decontaminated. Complete a Certificate of Equipment
Decontamination.
Centrifuges
z Check glass and plastic centrifuge tubes for stresslines, hairline cracks and
chipped rims before use. Use unbreakable tubes whenever possible.
z Avoid filling tubes to the rim.
z Use caps or stoppers on centrifuge tubes. Avoid using lightweight materials
such as aluminum foil as caps.
z Use sealed centrifuge buckets (safety cups) or rotors that can be loaded and
unloaded in a biological safety cabinet. Decontaminate the outside of the
cups or buckets before and after centrifugation. Inspect o-rings regularly and
replace if cracked or dry.
z Ensure that the centrifuge is properly balanced.
z Do not open the lid during or immediately after operation, attempt to stop
a spinning rotor by hand or with an object, or interfere with the interlock
safety device.
z Decant supernatants carefully and avoid vigorous shaking when re-
suspending.
Heating baths, water baths

z Never use laboratory ovens for preparation of food for human consumption
z Glassware that has been rinsed with an organic solvent should be rinsed
with distilled water before it is placed in a drying oven.
MICROBIOLOGY 71
Microbiology
6.6 UNIVERSAL / STANDARD PRECAUTIONS
Universal Precautions
These guidelines refer to the precautions, consistently used for all patients
regardless of their infection status and diagnosis. The main objective is to
prevent exposure of staff and patients to blood and body fluids.
Notes z Don’t eat, drink, smoke or apply cosmetics (including lip balm).
z Don’t insert or remove contact lenses.
z Don’t bite nails or chew on pens.
z Don’t mouth pipette.
z Limit access to the laboratory to trained personnel only.
z Assume all patients are infectious for HIV or other blood borne pathogens.
z Use appropriate barrier precautions to prevent skin an mucous membrane
exposure, including wearing gloves at all times and masks, goggles, gowns
or aprons if there is a risk of splashes or droplet formation.
z Wash hands thoroughly and other skin surfaces after gloves are removed
and immediately after any contamination.
z Avoid injuries to sharps such as needles and scalpels.
Standard Precautions
In 1996, CDC developed a new system of standard precaution synthesizing the
features of universal precautions and body substance isolation. Standard
precautions are used in the care of all patients and apply to blood, all body fluids,
secretion and excretion except sweat regardless of whether they contain visible
blood.
Standard precautions are guidelines and procedures designed to reduce the risk
of transmission of microorganisms from both recognized and unrecognized
sources of infection in healthcare settings.
Standard Precautions Include

z Hand washing
z Barrier protection
z Safe handling of sharp items
z Safe handling of specimen (blood etc)
z Safe handling of spillage of blood/body fluid
z Use of disposable/ sterile items
72 MICROBIOLOGY
Hand Washing Microbiology
Single most important method to limit cross transmission of nosocomial

pathogens
Multiple opportunities exist for HCW hand contamination by Direct patient care
and Inanimate environment
This is an ideal safety precaution and gloves should not be regarded as a Notes
substitute for hand washing.
For General Patient Care (Hand Contamination)

z Wash hands thoroughly in running water with soap without missing any
area. For effective hand washing first wash palms with fingers followed by
back of hands, knuckles, thumbs, fingertips and wrists,. Rinse and dry
thoroughly.
z Wash hand immediately after accidental contamination with blood/body
fluid, before eating and drinking and after removing gowns/coats and
gloves.
z Leave soap bars in dry containers to prevent contamination with
microorganism.
The 6 stages of effective hand hygiene
MICROBIOLOGY 73
Microbiology For Hand Disinfection (Hygienic Hand Wash)

Required in high risk health care settings (ICU, neonatal units, nursery)
z Use 2-4% chlorhexidine gluconate/ detergent solution, povidone iodine/
detergent solution containing 0.75% available Iodine.
z Do not use alcoholic hand rubs as substitute for hand washing except for
rapid hand decontamination between patient contacts.
Notes
Barrier Protection
Gloves
z Wear while collecting/ handling blood specimens and blood soiled items.
z Wear while disposing waste.
z Remove before handling door knobs, telephone, pens performing office
work.
z Discard if cracked, discoloured or punctured.
z Discard if blood spills on them.
z Don’t reuse disposable gloves.
z Wash hands when gloves are removed or changed.
Masks
z Wear masks and protective glasses if splashing or spraying of blood/body
fluids is expected.
z Mask of cotton wool, gauze, or paper masks are ineffective. Paper mask
with synthetic material for filtration are an effective barrier against
microorganism.
Caps
z Cover hair completely in aseptic units, operating rooms, or performing
selected invasive procedure.
Gown and Aprons

z Wear clean clothes made up of a material easy to clean.
z Change after exposure to blood and body fluids.
z Wear gown or apron of plastic or water resistant paper when splashes of
blood or other body fluids are likely to occur e.g. during surgery, obstetric
procedure, invasive procedure, post mortem and embalming.
74 MICROBIOLOGY
Occlusive Bandage Microbiology
z Cover all skin defects e.g. cuts, scratches or other breaks with waterproof
dressing before patient care.
Safe Handling of Sharps

z Take extreme care to avoid autoinoculation.
Notes
z Discard all chipped or cracked glassware in appropriate containers.
z Don’t manipulate disposable needles. Never bend, break, recap or remove
needle from syringe.
z Dispose your own sharps. Don’t pass used sharps directly from one person
to another.
z Discard needles in puncture proof rigid containers (Plastic or cardboard
boxes) after disinfection in 0.5-1% sodium hypochlorite solution. Use
needle shredder if available for needles or needles along with syringe
nozzle.
z Send sharp disposable containers for disposal when three-fourth full.
Safe Handling of Specimen

z Collect specimens, specially blood and body fluids in pre sterilized
containers properly sealed to prevent leakage or spillage.
z Use autoclaved/ pre-sterilized disposable syringes and needles for vene-
punture and lancets/ cutting needles foe finger pricks.
z Cover cuts in hands properly with waterproof adhesive bandages.
z Wear disposable gloves while collecting blood/body fluids and maintain
proper asepsis.
z Wash hands thoroughly with soap and water, particularly after handling
specimens.
Safe Handling of Blood/Body Fluid Spills

z Cover spills of infected or potentially infected material on the floor with
paper towel/ blotting paper/ newspaper.
z Pour 1% sodium hypochlorite solution on and around the spill area and cove
with paper for at least 30 minutes.
z After 30 minutes, remove paper with gloved hands and discard.
MICROBIOLOGY 75
Microbiology Use of Disposable Sterile Items

z Ensure proper handling of disposable/ sterile item before use.
z There should be no recirculation of disposable items.
WHAT HAVE YOU LEARNT

Notes
z The laboratory needs to be prepared for work both before and during the
work
z Laboratory fires can by caused by bunsen burners, runaway chemical
reactions, electrical heating units, failure of unattended or defective
equipment, or overloaded electrical circuits
z Fire cannot occur without an ignition source, fuel and an oxidizing
atmosphere (usually air), the three elements that comprise what is called
the “fire triangle”:
z Every effort should be made to prevent equipment from becoming
contaminated.
z Take appropriate measures to reduce the likelihood of equipment malfunction
that could result in leakage, spill or unnecessary generation of aerosolized
pathogens
z Glassware that has been rinsed with an organic solvent should be rinsed
with distilled water before it is placed in a drying oven.
z Standard precautions include Hand washing, Barrier protection, Safe
handling of sharp items, Safe handling of specimen (blood etc), Safe
handling of spillage of blood/body fluid, Use of disposable/ sterile items
z Hand Washing is an ideal safety precaution and gloves should not be
regarded as a substitute for hand washing.
z Wash hands thoroughly in running water with soap without missing any
area. For effective hand washing first wash palms with fingers followed by
back of hands, knuckles, thumbs, fingertips and wrists,. Rinse and dry
thoroughly.
z Wash hand immediately after accidental contamination with blood/body
fluid, before eating and drinking and after removing gowns/coats and
gloves.
z Use barrier protection like Gloves, Masks, Caps, Gown and Aprons,
Occlusive bandage
z Sharps must be handled safely and injury must be prevented
76 MICROBIOLOGY
z Safety precautions are to be followed while handling specimens and Blood/ Microbiology
body fluid spills
TERMINAL QUESTIONS
1. What are standard precautions
Notes
2. How would you handle blood/body fluid spills
3. Write measures to prevent fire accidents
4. Explain steps of hand washing
MICROBIOLOGY 77
MODULE Normal Flora of Human Body
Microbiology
7
Notes
NORMAL FLORA OF
HUMAN BODY
7.1 INTRODUCTION
In a healthy human, the internal tissues, e.g. blood, brain, muscle, etc., are
normally free of microorganisms. However, the surface tissues, i.e., skin and
mucous membranes, are constantly in contact with environmental organisms and
become readily colonized by various microbial species. The mixture of
organisms regularly found at any anatomical site is referred to as the normal
flora. The normal flora of humans consists of a few eucaryotic fungi and protists,
but bacteria are the most numerous and obvious microbial components of the
normal flora. A healthy foetus in utero is free from microorganisms. During birth
the infant in exposed to vaginal flora. Within a few hours of birth oral and
nasopharyngeal flora develops and in a day or two resident flora of the lower
intestine appears
OBJECTIVES
z describe normal flora
z enlist the Advantages and Disadvantages of flora
z describe the normal flora of various parts of the body
7.2 NORMAL MICROBIAL FLORA

The term “normal microbial flora” denotes the population of microorganisms
that inhabit the skin and mucous membranes of healthy normal persons. The skin
and mucous membranes always harbor a variety of microorganisms that can be
arranged into two groups:
78 MICROBIOLOGY
Normal Flora of Human Body MODULE
1. The resident flora consists of relatively fixed types of microorganisms Microbiology
regularly found in a given area at a given age; if disturbed, it promptly
reestablishes itself.
2. The transient flora consists of nonpathogenic or potentially pathogenic
microorganisms that inhabit the skin or mucous membranes for hours, days,
or weeks; it is derived from the environment, does not produce disease, and
does not establish itself permanently on the surface. Members of the Notes
transient flora are generally of little significance so long as the normal
resident flora remains intact. However, if the resident flora is disturbed,
transient microorganisms may colonize, proliferate, and produce disease.

1. Microorganisms that inhabit the skin and mucous membranes of healthy
normal persons is called as ...................
2. Normal flora can be grouped as ................... & ................... flora
3. Fixed type of microorganisms that are found in given area is described as
...................
4. Bacteria that inhabit the body surface or mucus membrane for a limited
period is described as ...................
7.3 RESIDENT FLORA

It consists of organisms which are regularly present in a particular area and when
disturbed it reestablishes itself like Esch.coli is a normal inhabitant of the
intestine.
Role of Resident flora

Microorganisms that are constantly present on body surfaces are commensals.
Their growth in a given area depends upon physiologic factors like temperature,
moisture, and the presence of certain nutrients and inhibitory substances.
Resident flora of certain areas plays a definite role in maintaining health and
normal function. Members of the resident flora in the intestinal tract synthesize
vitamin K and aid in the absorption of nutrients. On mucous membranes and
skin, the resident flora may prevent colonization by pathogens and possible
disease through “bacterial interference.” The mechanism of bacterial interference
is not clear. It may involve competition for receptors or binding sites on host
cells, competition for nutrients, mutual inhibition by metabolic or toxic
products, mutual inhibition by antibiotic materials or bacteriocins, or other
MICROBIOLOGY 79
Microbiology mechanisms. Suppression of the normal flora tends to be filled by organisms
from the environment or from other parts of the body and such organisms behave
as opportunists and may become pathogens.
On the other hand, members of the normal flora may themselves produce disease
under certain circumstances and if removed from the restrictions of that
environment and introduced into the bloodstream or tissues, these organisms
may become pathogenic. For example, streptococci of the viridans group are the
Notes most common resident organisms of the upper respiratory tract and if large
numbers of them are introduced into the bloodstream (eg, following tooth
extraction or tonsillectomy), they may settle on deformed or prosthetic heart
valves and produce infective endocarditis. Small numbers occur transiently in
the bloodstream with minor trauma (eg, dental scaling or vigorous brushing).
Bacteroides species are the commonest resident bacteria, if introduced into the
free peritoneal cavity or into pelvic tissues along with other bacteria as a result
of trauma, they cause suppuration and bacteremia.
There are many other examples, but the important point is that microbes of the
normal resident flora are harmless and may be beneficial in their normal location
in the host and in the absence of coincident abnormalities. They may produce
disease if introduced into foreign locations in large numbers and if predisposing
factors are present.
It has both advantages as well as disadvantages.
Advantages
(i) They prevent or suppress the entry of the pathogens.
(ii) These synthesize the vitamins especially Vit.-K and several B Group
vitamins.
(iii) The normal flora evokes the Antibodies production. These Antibodies cross
react with pathogens having related or shared antigens, thus raising the
immune status of the host against the invading pathogen.
(iv) Colonies produced by some organisms of normal flora have a harmful
effect on the pathogens.
(v) Endotoxins liberated by normal flora may help the defense mechanism of
the body.
Disadvantages
(i) They become pathogenic when the immunity is lowered.
(ii) They may act as pathogens in different issue (other than their normal
habitat) e.g. normal flora of intestine may cause urinary tract infection
(UTI).
80 MICROBIOLOGY
(iii) Normal flora may cause confusion in diagnosis due to their ubiquitous Microbiology
presence in the body and their resemblance to some of the pathogens.
Some Resident Microbiota
Notes
7.4 TRANSIENT FLORA

It consists of both non-pathogenic and potentially pathogenic bacteria that
inhabit the body surface or mucous membranes for a limited period. They can
be removed from the body surface by mechanical means like Pneumococcus and
Meningococcus can be removed from nasopharynx of the human beings from
time to time. Members of the normal flora form part of the host and include:
saprophytes, commensals, facultative pathogens and true pathogens.
Normal Flora of the Skin

Skin is constantly exposed to and is in contact with the environment, the skin
is particularly apt to contain transient microorganisms. The predominant
resident microorganisms of the skin are aerobic and anaerobic diphtheroid bacilli
(eg, corynebacterium, propionibacterium); nonhemolytic aerobic and anaerobic
staphylococci (Staphylococcus epidermidis, occasionally S aureus, and
peptostreptococcus species); gram-positive, aerobic, spore-forming bacilli that
are ubiquitous in air, water, and soil; alphahemolytic streptococci (viridans
streptococci) and enterococci (enterococcus species); and gram-negative coliform
bacilli and acinetobacter.
Low pH, fatty acids in sebaceous secretions and presence of lysozymes are
important factors for eliminating non-resident microorganisms from the skin.
Normal skin inhabits 102 - 104 organisms/sq. cm.
MICROBIOLOGY 81
Microbiology Microorganisms present on the skin surface are
Staph.epidermidis and Diphtheroids are the most common. Less common are
Peptococcus, Strept.viridens, Enterococcus, Micrococcus, Esch.coli, Candida,
etc.
Normal flora of Conjunctiva

The conjunctiva is relatively free from bacteria due to the presence of lysozyme
Notes in the tears which flushes the bacteria. Predominant organisms of the eyes are:
Moraxella sp
Diphtheroids
Straph epidermidis
Moraxella sp
Non hemolytic streptococci
Normal Flora of Nose and Nasopharynx

The nasopharynx of the infant is sterile at birth but in 2-3 days time it acquires
the flora carried by the mother and attendants. The nasopharynx is a natural
habitat of the common pathogenic bacteria causing infection of the nose, throat,
bronchi and lungs.
The flora of nose harbours
Diptheroids
Straphylococcus
Streptococcus
Haemophilus, and
Moraxella lacunata
Normal Flora of the Mouth

The mouth contains micrococci, gram positive aerobic spore bearing bacilli,
coliforms, proteus and lactobacilli. The gums pockets between the teeth and
crypts of the tonsils have a wide spectrum of anaerobic flora like fusiform bacilli,
treponemes, lactobacilli, etc. Candida is also found.
The mouth of infant is not sterile at birth. It generally contains the same types
of organisms as found in mother’s vagina. These bacteria diminish in number
and are replaced by similar bacteria present in the mouth of mother and nurse.
Normal Flora of Upper Respiratory Tract

Within 12 hours of birth alpha hemolytic streptococci are found in upper
respiratory tract and become the dominant organism of the oropharynx and
82 MICROBIOLOGY
remains so for the whole life. In the pharynx and trachea, similar flora is Microbiology
established. Smaller bronchi and alveoli are normally sterile.
Normal Flora of Gastrointestinal Tract:

The GI Tract of the foetus in utero is sterile. It becomes contaminated with
organisms shortly after birth. In breast fed infants, the intestine contains
lactobacilli, enterococci, colon bacilli and staphylococci.
In bottle fed infants the intestine contains anaerobic lactobacilli, colon bacilli Notes
and aerobic and anaerobic spore bearing organisms. With the change of food,
flora changes. Diet has a marked influence on the composition of the intestinal
and fecal flora.
In the stomach as pH is low, the stomach is sterile but as the pH increases in
small intestine the number of bacteria increases progressively beyond the
duodenum to the colon. The bacterial count is low in small intestine as compared
to large intestine. Lactobacilli and entrococci predominate in the duodenum and
proximal ileum. The bacterial flora is similar in lower ileum, caecum and rectum.
The anaerobic condition of colon is maintained by aerobic bacteria which
utilizes the free oxygen.
Normal Flora of the Genitourinary Tract

Mycobacterium smegmatis a harmless commensal is found in the secretions
(smegma) of both males and females genitalia. They may pose the confusion
with the tubercle bacilli.
Strains of mycoplasma and ureaplasma are frequently present as part of normal
flora. Gardnerella vaginalis, bacteroides and alpha streptococci have been
found in penile urethra.
Female urethra is either sterile or contains staphylococcus epidermidis. The
vagina of newly borne child is sterile and within 24 hours it colonizes with
micrococci, entrococci. In 2-3 days time doderlien’s bacillus appears. So the
flora keeps on changing depending upon the pH of the vagina. Doderlien bacilli
remain in the vagina till menopause. After menopause flora resembles that
before puberty.

1. Resident flora plays a definite role in ................ & ................
2. Resident flora of intestinal tract synthesize ................
3. Resident flora prevent colonization of pathogenic organism by ................
4. Non-resident microorganisms can be eliminated by ................, ................
& ................
MICROBIOLOGY 83
Microbiology

z Organisms found at any anatomical site is referred as normal flora
z A healthy fetus in utero is free from microorganisms and during brith the
infant is exposed to vaginal flora.
Notes z Normal microbial flora denotes the population of microorganism that
inhabit the skin and mucous membranes of healthy normal persons.
z The microorganisms can be arranged into two groups namely resident flora
and transient flora.
z Resident flora are fixed types of microorganism regularly found in a given
area at a given age
z Transient flora consists of nonpathogenic or potentially pathogenic
microorganisms that inhabit the skin or mucous membrane
z When the resident flora is disturbed, transient microorganism may colonize,
proliferate, and produce disease
z Microorganisms that are constantly present on body surfaces are commensals.
Their growth in a given area depends upon physiologic factors like
temperature, moisture and the presence of certain nutrients and inhibitory
substances.
z Resident flora of certain areas plays a definite role in maintaining health
and normal function
z Resident flora is believed to prevent colonization by pathogens and possible
disease through bacterial interference.
z Members of normal flora may themselves produce disease under certain
circumstances and if removed from the restrictions of that environment and
introduced into the bloodstream or tissues, these organisms may become
pathogenic.
z Resident flora prevents or suppresses the entry of the pathogens.
z They synthesize the vitamins especially vitamin k and several B group
vitamins
z Normal flora evokes the antibodies production and colonies of some
organisms of normal flora have a harmful effect on the pathogens.
z Endotoxins liberated by normal flora may help the defence mechanism of
the body.
z Low pH, fatty acids in sebaceous secretions and presence of lysozymes are
important factors for eliminating non-resident microorganism from the skin.
84 MICROBIOLOGY
Microbiology
TERMINAL QUESTIONS
1. Define normal flora.
2. Enumerate the normal flora of
(a) Skin
Notes
(b) Mouth
(c) Upper respiratory tract
(d) Gastrointestinal tract
(e) Genito-urinary tract
7.1
1. Normal flora
2. Resident and transient flora
3. Resident flora
4. Transient flora
7.2
1. Maintaining health and normal function
2. Vitamin k and several B group vitamins
3. Bacterial interference
4. Low pH, fatty acids in sebaceous secretions and presence of lysozymes
MICROBIOLOGY 85
MODULE Pathogenesis of Bacterial Infection
Microbiology
8
Notes PATHOGENESIS OF BACTERIAL
INFECTION
8.1 INTRODUCTION
In this chapter we would focus on how bacterias causes disease to human beings.
This process of causing disease is termed as Pathogenesis. Pathogenesis is a
multi-factorial process which depends on the immune status of the host, the
nature of the species or strain (virulence factors) and the number of organisms
in the initial exposure.
A limited number of bacterial species are responsible for the majority of
infectious diseases in healthy individuals. Due to the success of vaccination,
antibiotics, and effective public health measures, until recently, epidemics were
felt to be a thing of the past. Due to the development of antibiotic resistant
organisms, this situation is changing rapidly.
All humans are infected with bacteria (the normal flora) living on their external
surfaces (including the skin, gut and lungs). We are constantly also exposed to
bacteria (including air, water, soil and food). Normally due to our host defenses
most of these bacteria are harmless. In compromised patients, whose defenses
are weakened, these bacteria often cause opportunistic infectious diseases when
entering the bloodstream (after surgery, catheterization or other treatment
modalities). When initiated in the hospital, these infectious diseases are referred
to as nosocomial. Some common bacteria found in the normal flora
include Staphylococcus aureus, S. epidermidis and Propionibacterium
acnes (found on the skin)and Bacteroides and Enterobacteriaceae found in the
intestine (the latter in much smaller numbers).
OBJECTIVES
After reading this chapter, the student will be able to :
z describe the term pathogenesis.
z explain Koch’s postulates.
86 MICROBIOLOGY
Pathogenesis of Bacterial Infection MODULE
z differentiate colonization and pathogens Microbiology
z explain steps involved in the bacterial pathogenesis

z describe toxins
z differentiate endotoxins and exotoxins
z discuss the various diseases caused by bacteria
Notes
8.2 PATHOGENICITY
Pathogenicity is the capacity to initiate disease. It requires the attributes of
transmissibility or communicability from one host or reservoir to a fresh host,
survival in the new host, infectivity or the ability to breach the new host’s
defenses, and virulence, a variable that is multifactorial and denotes the capacity
of a pathogen to harm the host. Virulence in the clinical sense is a manifestation
of a complex bacterial–host relationship in which the capacity of the organism
to cause disease is considered in relation to the resistance of the host.
Types of bacterial pathogens

Bacterial pathogens can be classified into two broad groups, primary and
opportunistic pathogens.
Primary pathogens are capable of establishing infection and causing disease
in previously healthy individuals with intact immunological defenses. However,
these bacteria may more readily cause disease in individuals with impaired
defenses.
Opportunistic pathogens rarely cause disease in individuals’ with intact
immunological and anatomical defenses. Only when such defenses are impaired
or compromised, as a result of congenital or acquired disease or by the use of
immunosuppressive therapy or surgical techniques, are these bacteria able to
cause disease. Many opportunistic pathogens, e.g. coagulase negative
staphylococci and Escherichia coli, are part of the normal human flora and are
carried on the skin or mucosal surfaces where they cause no harm and may
actually have beneficial effects, by preventing colonization by other potential
pathogens. However, introduction of these organisms into anatomical sites in
which they are not normally found, or removal of competing bacteria by the use
of broad-spectrum antibiotics, may allow their localized multiplication and
subsequent development of disease.
The above classification is applicable to the vast majority of pathogens;
however, there are exceptions and variations within both categories of bacterial
pathogens. Different strains of any individual bacterial species can vary in their
genetic makeup and virulence capacity. For example, the majority of Neisseria
meningitidis strains are harmless commensals and considered opportunistic
MICROBIOLOGY 87
Microbiology bacteria, however, some hypervirulent clones of the organism can cause disease
in a previously healthy individual. Conversely, people vary in their genetic
make-up and susceptibility to invading bacteria. For example, Mycobacterium
tuberculosis is a primary pathogen but does not cause disease in every host it
invades.
Notes INTEXT QUESTIONS 8.1

1. The process of bacteria causing disease is termed as ..........................
2. Ability to affect the host’s disease is ..........................
3. Capacity of a pathogen to harm the host is ..........................
4. Pathogens which causes disease in healthy individual is ..........................
5. Pathogens that causes disease in immune compromised individual is
..........................
8.3 KOCH’S POSTULATES (MODIFIED)

Koch forwarded four criteria designed to establish a causal relationship between
a causative microbe and a disease. The postulates were formulated by Robert
Koch and Friedrich Loeffler in 1884 and refined and published by Koch in 1890.
Koch applied the postulates to establish the etiology of anthrax and tuberculosis,
and now have been generalized to other diseases.
1. The organism must always be found in humans with the infectious
disease but not found in healthy ones.
2. The organism must be isolated from humans with the infectious disease and
grown in pure culture.
3. The organism isolated in pure culture must initiate disease when re-
inoculated into susceptible animals.
4. The organism should be re-isolated from the experimentally infected
animals.
Postulates 3. and 4. are extremely important in definite proof of the role of agent
in human disease. However, this depends on the ability to develop animal models
that resemble the human disease. In many cases such models do not exist.
Pathogenesis
The process of pathogenesis involves various steps beginning with the
transmission of the infectious agent (bacterial) to the host, followed by
colonization of the site. After the colonization of host, the bacteria remain
adherent at the site of colonization then invades the host system. After surviving
the host immune system it is ready to cause the disease.
88 MICROBIOLOGY
Steps involved in the pathogenesis of the bacteria: Microbiology
1. Transmission
2. Colonization
3. Adhesion
4. Invasion
5. Survival in the host Notes
6. Tissue Injury
Transmission
Potential pathogens may enter the body by various routes, including the
respiratory, gastrointestinal, urinary or genital tracts. Alternatively, they may
directly enter tissues through insect bites or by accidental or surgical trauma to
the skin. Many opportunistic pathogens are carried as part of the normal human
flora, and this acts as a ready source of infection in the compromised host (e.g.
in cases of AIDS or when the skin barrier is breached). For many primary
pathogens, however, transmission to a new host and establishment of infection
are more complex processes.
Colonization
The establishment of a stable population of bacteria on the host’s skin or mucous
membranes is called colonization. For many pathogenic bacteria, the initial
interaction with host tissues occurs at a mucosal surface and colonization
normally requires adhesion to the mucosal cell surface. This allows the
establishment of a focus of infection that may remain localized or may
subsequently spread to other tissues.
MICROBIOLOGY 89
Microbiology Adhesion
Adhesion is necessary to avoid innate host defense mechanisms such as
peristalsis in the gut and the flushing action of mucus, saliva and urine, which
remove non-adherent bacteria. For bacteria, adhesion is an essential preliminary
to colonization and then penetration through tissues. Successful colonization
also requires that bacteria are able to acquire essential nutrients—in particular
iron—for growth. At the molecular level, adhesion involves surface interactions
Notes
between specific receptors on the mammalian cell membrane (usually
carbohydrates) and ligands (usually proteins) on the bacterial surface. The
presence or absence of specific receptors on mammalian cells contributes
significantly to tissue specificity of infection. Nonspecific surface properties of
the bacterium, including surface charge and hydrophobicity, also contribute to
the initial stages of the adhesion process. Several different mechanisms of
bacterial adherence have evolved, all utilizing specialized cell surface organelles
or macromolecules, that help to overcome the natural forces of repulsion that
exist between the pathogen and its target cell. Many bacteria express pili (or
fimbriae) which are involved in mediating attachment to mammalian cell
surfaces. Different strains or species of bacteria produce different types of pili
which can be identified on the basis of antigenic composition, morphology and
receptor specificity.
Invasion
Invasion is penetration of host cells and tissues (beyond the skin and mucous
surfaces), and is mediated by a complex array of molecules, often described as
‘invasins’. These can be in the form of bacterial surface or secreted proteins
which target host cell molecules (receptors).
Once attached to a mucosal surface, some bacteria, e.g. Corynebacterium
diphtheriae or Clostridium tetani, exert their pathogenic effects without
penetrating the tissues of the host. These produce biologically active molecules
such as toxins, which mediate tissue damage at local or distant sites. For a
number of pathogenic bacteria, however, adherence to the mucosal surface
represents only the first stage of the invasion of tissues. Examples of organisms
that are able to invade and survive within host cells include Mycobacteria,
Salmonella, Shigella and others. The initial phase of cellular invasion involves
penetration of the mammalian cell membrane and many intracellular pathogens
use normal phagocytic entry mechanisms to gain access. Inside the cell, they
become surrounded by host cell-derived membrane vesicles. Many intracellular
pathogens escape from these vesicles into the cell cytoplasm where they multiply
rapidly before spreading to adjacent cells and repeating the process of invasion.
The availability of specific receptors on host cells defines the type of host cells
90 MICROBIOLOGY
that are involved. As a result, some pathogens can invade a wide range of cell Microbiology
types whilst others have a much more restricted invasive potential. The receptors
for some of the invasive pathogens have been identified.
Virulence determinants
Both primary and opportunistic pathogens possess virulence determinants or
aggressins that facilitate pathogenesis. Possession of a single virulence Notes
determinant is rarely sufficient to allow the initiation of infection and production
of pathology. Many bacteria possess several virulence determinants, all of which
play some part at various stages of the disease process. In addition, not all strains
of a particular bacterial species are equally pathogenic. For example, although
six separate serotypes of encapsulated Haemophilus influenzae are recognized,
serious infection is almost exclusively associated with isolates of serotype b
(hence Hib vaccine). Moreover, even within serotype b isolates, 80% of serious
infections are caused by six out of > 100 clonal types.
Different strains of a pathogenic species may cause distinct types of infection,
each associated with possession of a particular complement of virulence
determinants. Different strains of E. coli, for example, cause several distinct
gastrointestinal diseases, urinary tract infections, septicemia, meningitis and a
range of other minor infections.
Many pathogens produce an impressive armoury of virulence determinants;
however, their expression is coordinated or regulated by several nutritional
and environmental factors. Among virulence regulators are the availability of
nutrition (e.g. iron), oxygen, suitable temperature or other growth requirements.
Importantly, differences in virulence between similar organisms may be due to
additional cryptic phenotypic or genotypic variations. For example, some
virulence factors are only expressed when indirect contact with host cells.
Virulence genes can move between bacteria via special genetic vehicles e.g.
plasmids, bacteriophage and transposons. The horizontally transferred virulence
factors (e.g. toxins) may or may not transform the recipient bacteria into better-
adapted or more virulent pathogens.
8.4 SURVIVAL IN THE HOST

Many bacterial pathogens are able to resist the cytotoxic action of plasma and
other body fluids involving antibody and complement (classical pathway) or
complement alone (alternate pathway) or lysozyme. Killing of extracellular
pathogens largely occurs within phagocytes after opsonization (by antibody and/
or complement) and phagocytosis. Circumvention of phagocytosis by extracellular
pathogens is thus a major survival mechanism. Capsules (many pathogens), protein
A (S. aureus) and M protein (S. pyogenes) function in this regard.
MICROBIOLOGY 91
Microbiology Protein A is a surface constituent of S. aureus as well as a secreted product and

binds to the Fc portion of immunoglobulins. Bacteria, on binding antibody,
activate the classical complement cascade which results in the attachment of
fragments of C3. Phagocytosis occurs after binding of the opsonized bacteria to
receptors for the Fc portion of IgG or C3 regions. Protein A is anti-
complementary (since, on binding to IgG, the complement cascade is activated,
depleting complement levels). Thus in the presence of protein A, interaction of
Notes bacteria (via bound complement) with C3 receptors will be inhibited. Free
protein A binds to the Fc portion of IgG, thus phagocytosis via Fc receptors may
not occur because of steric hindrance.
Peptidoglycan, like lipopolysaccharide, can activate the alternate complement
cascade. In S. pyogenes peptidoglycan is sufficiently exposed that it is able to
bind complement. The M protein of group A streptococci is the anti-phagocytic
component of the fimbriae. M protein binds fibrinogen from plasma which
blocks complement binding to the underlying peptidoglycan layer. Thus
streptococci in non-immune serum are not phagocytosed.
Intracellular pathogens (both obligate and facultative) must be able to avoid
being killed within phagolysozomes. This can occur from by-passing or lysing
these vesicles and then residing free in the cytoplasm. Alternatively, they can
survive in phagosomes (fusion of phagosomes with lysosomes may be inhibited
or the organism may be resistant to degradative enzymes if fusion with
lysosomes occurs).

1. ....................... is used to establish the etiology of diseases
2. The establishment of a population of bacteria on host’s skin is called
.................
3. ....................... is necessary to avoid innate host defense mechanism
4. ....................... is penetration of host cells & tissues
8.5 TISSUE INJURY

Bacteria cause tissue injury primarily by several distinct mechanisms involving:
z Exotoxins
z Endotoxins and non-specific immunity
z Specific humoral and cell mediated immunity
92 MICROBIOLOGY
Exotoxins Microbiology
Many bacteria produce proteins (exotoxins) that modify, by enzymatic action,

or otherwise destroy certain cellular structures. Effects of exotoxins are usually
seen acutely, since they are sufficiently potent that serious effects (e.g. death)
often result. Examples of this are botulism, anthrax, cholera and diphtheria. If
the host survives the acute infection, neutralizing antibodies (anti-toxins) are
often elicited that neutralize the affect of the exotoxin. Classes of exotoxins
include: Notes
Toxins that act on the extracellular matrix of connective tissuee.g. Clostridium

perfringens collagenase, Staphylococcus aureus hyaluronidase.
Toxins that have a cell binding “B” component and an active “A” enzymatic
component (A-B type toxins)
These include:
a) Those with ADP-ribosylating activity e.g. cholera toxin, E. coli heat labile
toxin, Pseudomonas aeruginosa and diphtheria toxins.
b) Those with a lytic activity on 28S rRNA e.g. shiga and shiga-like (vero)
toxins.
c) Those with a partially characterized site of action e.g. botulinum toxin,
tetanus toxin and anthrax lethal toxin.
MICROBIOLOGY 93
Microbiology Membrane Damaging Toxins e.g. Staphylococcus aureus delta toxin

Toxins which act extracellularly. These include proteases, collagenases and
hyaluronidases. For example, Clostridium perfringens produces a potent
collagenase, whilst Staphylococcus aureus produces a hyaluronidase. Damage
to the connective tissue matrix (by hyaluronidase and collagenase) can “loosen
up” the tissue fibers allowing the organism to spread through the tissues more
Notes readily. Also included in this group is the exfoliatin of Staphylococcus
aureus which causes separation of the layers within the epidermis and is the
causative agent of scalded skin syndrome in the newborn.
A - B Toxins. Such toxins consist of two components. One binds to cell surfaces
and the other passes into the cell membrane or cytoplasm where it acts. The
classical toxins demonstrated to act in this fashion are those of cholera and
diphtheria.
(i) ADP-ribosylating exotoxins
Diphtheria toxin (produced by Corynebacterium diphtheriae) is coded by the
phage tox gene. The toxin is synthesized as one polypeptide chain and readily
nicked into two chains held together by a disulfide bond. B binds to cells and
A has the enzymatic activity. A is endocytosed and from the endosome passes
into the cytosol. Diphtheria toxin ADP-ribosylates elongation factor (EF2) in
ribosomes, thus inhibiting protein synthesis. Pseudomonas exotoxin A has an
similar mode of action to diphtheria toxin.
Cholera toxin has several subunits which form a ring with one A subunit inserted
in the center. B binds to gangliosides on the cell surface and appear to provide
a channel through which A penetrates. A1 is formed by proteolytic cleavage and
after internalization ADP-ribosylates a cell membrane regulator complex (using
NADH as a substrate), in turn causing activation of adenylate cyclase. Activation
of adenylate cyclase causes an increase in cyclic AMP production with resulting
decrease in sodium chloride uptake from the lumen of the gut and active ion and
water secretion with a watery diarrhea resulting. E. coli labile toxin has a similar
mode of action.
(ii) Toxins that act on 28S rRNA
Shiga toxins (chromosomally encoded) are involved in the pathogenesis of
shigellosis, whilst shiga-like toxins (phage encoded) are primarily produced by
enterohemorraghic E. coli. They share a common mode of action. A fragment
of the A subunit passes to the ribosome where it has N-glycosidase activity on
a single adenosine residue; i.e. the bond between the base and ribose is lysed.
Diarrhea results not from active ion/water secretion, but poor water absorption
due to death of epithelial cells from inhibition of protein synthesis.
94 MICROBIOLOGY
(iii) Partially characterized site of action Microbiology
Botulinum neurotoxins, tetanospasmin and the lethal toxin of B. anthracis,

appear to be A-B type exotoxins. Botulinum toxin acts by causing inhibition of
release of acetylcholine at the neuromuscular junction. Tetanus toxin is taken
up at neuromuscular junctions and transported in axons to synapses. It then acts
by inactivating inhibitory neurons. The exotoxins of tetanus and botulism appear
to have B components, but the mode of action of their A subunits are not known. Notes
The B component of lethal toxin of B. anthracis is the protective antigen;
interestingly, this also serves as the B subunit for edema toxin.
Membrane Damaging Toxins: These toxins enzymatically digest the
phospholipid (or protein) components of membranes or behave as detergents.
In each case holes are punched in the cell membrane and the cytoplasmic
contents can leach out. The phospholipase (“toxin”) of C. perfringens is an
example of a membrane damaging toxin. It destroys blood vessels stopping the
influx of inflammatory cells. This also helps create an anaerobic environment
which is important in the growth of this strict anaerobe. The delta toxin of S.
aureus is an extremely hydrophobic protein that inserts into cell membranes and
is believed to have a detergent-like action.
Endotoxins
Despite the advances of the antibiotic era, around 200,000 patients will develop
Gram negative sepsis each year of whom around 25-40% will ultimately die of
septic shock. Septic shock involves hypotension (due to tissue pooling of fluids),
disseminated intravascular coagulation and fever and is often fatal from massive
system failure. This includes lack of effective oxygenation of sensitive tissues
such as the brain. There is no effective therapy to reverse the toxic activity of
lipid A or peptidoglycan in patients.
Endotoxins are toxic components of the bacterial cell envelope. The classical
and most potent endotoxin is lipopolysaccharide. However, peptidoglycan
displays many endotoxin-like properties. Certain peptidoglycans are poorly
biodegradable and can cause chronic as well as acute tissue injury. Endotoxins
are “non-specific” inciters of inflammation. For example, cells of the immune
system and elsewhere are stimulated to release cytokines (including interleukin
1 and tumor necrosis factor). Endotoxins also activate the alternate complement
pathway. The production of these cytokines results in attraction of
polymorphonuclear cells into affected tissues. PG and LPS and certain other cell
wall components (e.g. pneumococcal teichoic acid) are also activators of the
alternate complement cascade. Thus many bacteria will bind complement
encouraging their uptake and killing by phagocytes in the absence of antibody.
Certain complement by-products are also chemoattractants for neutrophils.
MICROBIOLOGY 95
Microbiology Endotoxins are also potent B cell mitogens, polyclonal B cell activators and
adjuvants (for both antibodies and cell mediated immunity); this plays a role in
the development of a suitable chronic immune response in handling the microbes
if they are not eliminated acutely.
In a “primary” infection during the acute phase ”non-antigen specific”
immunity will be of utmost importance in eradicating the infection. If the
organism persists (or in a reinfection at a later date), specific immunity will be
Notes
of greater significance in slowing growth of the organisms or in eliminating
infection. This is important in chronic infections such as tuberculosis, leprosy,
Lyme disease and syphilis.
Endotoxin
in small amounts
Target Kupffer cells Neutrophils

Neutrophils B lymphocytes Complement
Complement
Activation by
Activity Increase in Increase in
Activation alternative
IL-1, TNF kinins pathway
Increased
Effect Fever Vasodilation antibody Inflammation
synthesis
A
Endotoxin
in large amounts
(All of the above, plus)
Intravascular
B Shock
coagulation

1. Bacteria produce ........................ that modify cellular structures
2. Toxins that act extracelluarly are ......................, ...................... & ......................
3. ........................ are toxic components of bacterial cell envelope
4. Example of endotoxin is ........................
96 MICROBIOLOGY
Microbiology
8.6 IMMUNOPATHOLOGY
The infected tissue often serves as an innocent bystander and immunopathology
results. This can occur in acute and chronic infections. Over stimulation of
cytokine production and complement activation by endotoxins can cause tissue
injury in the absence of an immune response. Continuously generated antigens
released from persisting viable microbes will subsequently elicit humoral
antibodies and cell mediated immunity resulting in chronic immunopathology.
Notes
Certain poorly degradable antigens (e.g pneumococcal polysaccharide and
group A streptococcal cell walls) can maintain immunopathology even in the
absence of persistence of live agents. Other bacterial antigens cross-react with
host tissue antigens causing the development of autoimmunity (e.g. the M
protein of S. pyogenes cross-reacts with mammalian myosin). Thus
immunopathology can persist even after the infection and microbial antigens are
eliminated.
The immune system in resistance to infection - examples
1. Extracellular parasites. Antibodies cause lysis of the organism and/or their
opsonization by phagocytes at which point they are rapidly killed.
2. Intracellular parasites are primarily killed by cell mediated immunity.
3. Exotoxins can be neutralized by antitoxins. These can be elicited using
toxoid vaccines (toxoids are antigenic but not toxic). This occurs, for
example, in vaccination against diphtheria.
4. Certain organisms produce IgA proteases (including H. influenzae, S.
pneumoniae, N. gonorrhoeae and N. meningitidis) this helps survival on
external surfaces.
Some Organisms of Medical Interest
Gram negative aerobic cocci Gram positive cocci (facultative anaerobes)

Neisseria Streptococcus
Staphylococcus
Spirochetes Gram negative bacilli
Treponema Pseudomonas
Borrelia Bordetella
Leptospira Francisella
Spiral, Gram negative bacilli Gram positive bacilli
Campylobacter Listeria
Helicobacter Erysipelothrix
Gram negative bacilli Actinomycetes and related organisms
(a) Enterobacteriaceae Corynebacterium
Escherichia Mycobacterium
MICROBIOLOGY 97
Microbiology
Salmonella Nocardia
Shigella Actinomyces
Yersinia Corynebacterium-like in appearance
Enterobacter Propionibacterium
Proteus Fastidious Gram negative bacteria
Serratia Brucella
Edwardsiella Rochalimeae/Bartonella
Notes (b) Others Chlamydia
Vibrio Rickettsia
Hemophilus Mycoplasma
Pasteurella
(c) Legionellaceae
Legionella
Tatlockia
Some major Exotoxins

Organism Disease Toxin
Bacillus anthracis Anthrax Edema toxin
Lethal toxin
Clostridium botulinum Botulism Botulism .toxin
Clostridium difficile Pseudo membranous colitis Enterotoxin
Clostridium perfringens Gas gangrene Alpha toxin Hyaluronidase
Food poisoning Enterotoxin
Clostridium tetani Tetanus Tetanospasmin
Corynebacterium diphtheria Diphtheria Diphtheria toxin
Escherichia coli Diarrhea (ETEC) Heat labile toxin
Heat stable toxins
Hemorrhagic colitis Vero toxin
Pseudomonas aeruginosa Diseases of compromised host Exotoxin A
Staphylococcus aureus Opportunistic infections Alpha-gamma toxins,
leucocidin
Toxic shock Toxic shock toxin
Food poisoning Enterotoxin
Scalded skin syndrome Exfoliatin
Streptococcus pyogenes Scarlet feverToxic shock Erythrogenic/pyrogenic
toxin
Shigella dysenteriae Bacillary dysentery Shiga toxin
Vibrio cholera Cholera Choleragen
98 MICROBIOLOGY
Microbiology

Match the following
Organism Toxin
1. Bacterial anthrasis (a) leucocidin
2. Clositridium botulinum (b) erythrogenic toxin
Notes
3. Staphylococcus aureus (c) Edema toxin
4. Streptococcus pyogens (d) Botulism toxin

z The capacity to initiate disease is called pathogenesis
z Pathogenesis depends on the immune status of host, nature of species or
strain (Virulence factor) & number of organisms in the initial exposure
z Bacterial pathogens are of two types namely primary and opportunistic
pathogens
z Primary pathogens are capable of establishing infection and cause disease
in previously healthy individuals with intact immune defense
z Opportunistic pathogens cause disease in individuals with impaired or
compromised defenses
z Kochs postulate establishes a casual relationship between a microbe and
disease
z The process of pathogenesis involves various steps beginning with the
transmission of the infectious agent (bacterial) to the host, followed by
colonization of the site.
z After the colonization host the bacteria remain adherent at the site of
colonization then invades the host system.
z After being survived from host immune system it is ready to cause the
disease.
z Pathogens possess virulence determinants or aggressins that facilitate
pathogenesis
z Bacteria cause tissue injury by Exotoxins, Endotoxins & Non-specific
immunity, specific humoral and cell mediated immunity.
TERMINAL QUESTIONS
1. What are pathogenic bacteria. Explain with suitable example?
2. What do you understand by the term opportunistic infections. Enlist some
opportunistic infection seen in human being?
MICROBIOLOGY 99
Microbiology 3. What are the reasons for opportunistic infections in human beings?
4. Enlist the steps involved in the pathogenesis of bacteria?
5. Explain every step involved in the pathogenesis of bacteria with suitable
example?
6. Differentiate between endotoxin and exotoxins?
Notes
8.1
1. Pathogenesis
2. Infectivity
3. Virulence
4. Primary pathogens
5. Opportunistic pathogen
8.2
1. Koch postulate
2. Colonization
3. Adhesion
4. Invasion
8.3
1. Exotoxins
2. Proteases, collagenases & hyaluroindes
3. Endotoxins
4. Lipopolysaccharide
8.4
1. (c)
2. (d)
3. (a)
4. (b)
100 MICROBIOLOGY
Bacterial Culture Media MODULE
Microbiology
9
Notes
BACTERIAL CULTURE
MEDIA
9.1 INTRODUCTION
Why bacteria have to be grown (cultured) in the laboratory on artificial culture
media?
1. One of the most important reasons being its utility in diagnosing infectious
diseases. Isolating an organism from sites in body normally known to be
sterile is an indication of its role in the disease process. Indeed, isolating
an organism from the clinical specimen is the first step in proving its role
as an etiologic agent.
2. Culturing bacteria is also the initial step in studying its morphology and its
identification.
3. Bacteria have to be cultured in order to obtain antigens from developing
serological assays or vaccines.
4. Certain genetic studies and manipulations of the cells also need that bacteria
be cultured in vitro.
5. Culturing bacteria also provide a reliable way estimating their numbers
(viable count).
6. Culturing on solid media is another convenient way of separating bacteria
in mixtures.
This lesson deals with culture media.
OBJECTIVES
z enlist the common ingreditents of culture medium
z describe about history of culture medium in brief
MICROBIOLOGY 101
MODULE Bacterial Culture Media
Microbiology z classify the culture media

z describe the preparation and storage of Culture media
When culturing bacteria, it is very important to provide similar environmental
and nutritional conditions that exist in its natural habitat. Most culture medium
contains water, a source of carbon & energy, source of nitrogen, trace elements
and some growth factors. Besides these, optimum pH, oxygen tension and
osmolarity too have to be taken into consideration. Some of the ingredients of
Notes culture media include:
While tap water is suitable for culture media, it must not be used if it contains
high amount of minerals. In such situations, distilled or demineralised water
should be used. Peptone is a byproduct of protein digestion. Proteins are often
obtained from heart muscle, casein, fibrin or soya flour and is digested using
proteolytic enzymes such as pepsin, trypsin or papain. The final product contains
peptones, proteoses and amino acids besides a variety of inorganic salts
including phosphates, potassium and magnesium. Casein hydrolysate is
obtained from hydrolysis of milk protein casein using HCl or trypsin. Meat
extract is obtained by hot water extraction of lean beef and then concentrated by
evaporation. Yeast extract is prepared from washed cells of bakers’ yeast and
contains wide range of amino acids, growth factors and inorganic salts.
9.2 BRIEF HISTORY

Robert Koch realized the importance of solid media and used potato pieces to
grow bacteria and agar was used to solidify culture media. Before the use of agar,
attempts were made to use gelatin as solidifying agent. Gelatin had some
inherent problems; it existed as liquid at normal incubating temperatures (35–
37oC) and was digested by certain bacteria.
Classification
Bacterial culture media can be classified in at least three ways; Based on
consistency, based on nutritional component and based on its functional use.
1. Classification based on consistency
z liquid media
z semi-solid media
z solid media
Liquid media
In liquid medium, bacteria grow producing turbidity/ surface pellicle (Vibrio &
Bacillus)/ granular deposits (Streptococci). Culturing bacteria in liquid media
102 MICROBIOLOGY
has some drawbacks. Properties of bacteria are not visible in liquid media and Microbiology
presence of more than one type of bacteria cannot be detected.
Solid media
Any liquid medium can be rendered solid by the addition of certain solidifying
agents. Agar agar (simply called agar) is the most commonly used solidifying
agent. It is an unbranched polysaccharide obtained from the cell membranes of
some species of red algae such as the genera Gelidium. Agar is composed of two Notes
long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95°C
and solidifies at 42oC, doesn’t contribute any nutritive property, it is not
hydrolysed by most bacteria and is usually free from growth promoting or growth
retarding substances. Agar is available as powders. New Zealand agar and
Japanese agar are most commonly used at concentration of 2% and 4%
respectively to make a solid agarmedium.
Semi-solid media
Reducing the amount of agar to 0.2-0.5% renders a medium semi-solid. Such
media are fairly soft and are useful in demonstrating bacterial motility (U-tube
and Cragie’s tube). Certain transport media such as Stuart’s and Amies media are
semi-solid in consistency. Hugh & Leifson’s oxidation fermentation test medium
as well as mannitol motility medium are also semi-solid.
Biphasic media
Sometimes, a culture system comprises of both liquid and solid medium in the
same bottle. This is known as biphasic medium (Castaneda system for blood
culture). The inoculum is added to the liquid medium and when subcultures are
to be made, the bottle is simply tilted to allow the liquid to flow over the solid
medium. This obviates the need for frequent opening of the culture bottle to
subculture.
Other solidifying agents

Besides agar, egg yolk and serum too can be used to solidify culture media.
Serum containing medium such as Loeffler’s serum slope and egg containing
media such as Lowenstein Jensen (LJ) medium and Dorset egg medium are
solidified as well as disinfected by a process of inspissation.

1. The by-product of protein digestion is .................
2. ................. is the most commonly used solidifying agent
MICROBIOLOGY 103
Microbiology 3. Culture system having both liquid & solid medium in the same container
is called as .................
4. ................. media are useful in demonstrating bacterial motility
2. Classification based on nutritional component

Media can be classified as simple, complex and synthetic (or defined). Those
Notes bacteria that are able to grow with minimal requirements are said to non-
fastidious and those that require extra nutrients are said to be fastidious. Simple
media such as peptone water, nutrient agar can support most non-fastidious
bacteria.
Complex media such as blood agar have ingredients whose exact components
are difficult to estimate.
Synthetic or defined media such as Davis & Mingioli medium are specially
prepared media for research purposes where the composition of every component
is well known.
3. Classification based on functional use or application

Basal media are basically simple media that supports most non-fastidious
bacteria. Peptone water, nutrient broth and nutrient agar considered basal
medium
Enriched media are used to grow nutritionally exacting (fastidious) bacteria
Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal
medium makes them enriched media. Blood agar, chocolate agar, Loeffler’s
serum slope etc are few of the enriched media.
Blood agar is prepared by adding 5-10% (by volume) to a basal medium such as
nutrient agar or other blood agar bases. Since blood cannot be sterilized, it has to
be collected aseptically from the animal. Animals have to be bled and the blood
is collected in sterile containers with anticoagulant or glass beads. While sheep
blood is preferred, blood from rabbit, horse and ox can also be collected. Human
blood must be avoided since it may contain inhibitory substances including
antibiotics. After the blood agar base is autoclaved, blood is added to the medium
at temperature just above the solidifying point of agar. The mixture is then
poured on to the plates and allowed to solidify. Blood agar is useful in
demonstrating hemolytic properties of certain bacteria.
Chocolate agar is also known as heated blood agar or lysed blood agar. The
procedure is similar to that of blood agar preparation except that the blood is
added while the molten blood agar base is still hot. This lyses the blood cells and
releases their contents into the medium. This process turns the medium brown,
hence the name. This medium is especially useful in growing Hemophilus sp and
104 MICROBIOLOGY
Neisseria sp. Serum for medium can be obtained from animal blood but must be Microbiology
filtered through membrane or seitz filter before use.
Selective and enrichment media are designed to inhibit unwanted commensal
or contaminating bacteria and help to recover pathogen from a mixture of
bacteria. While selective media are agar based, enrichment media are liquid in
consistency. Various approaches to make a medium selective include addition of
antibiotics, dyes, chemicals, alteration of pH or a combination of these. Notes
Thayer Martin Agar used to recover N. gonorrhoeae contains Vancomycin,
Colistin and Nystatin.Mannitol Salt Agar and Salt Milk Agar used to recover
S.aureus contain 10% NaCl. Potassium tellurite medium used to recover
C.diphtheriae contains 0.04% potassium tellurite. McConkey’s Agar used for
Enterobacteriaceae members contains Bile salt that inhibits most gram positive
bacteria. Pseudosel Agar (Cetrimide Agar) used to recover P.aeruginosa
contains cetrimide. Crystal Violet Blood Agar used to recover S.pyogenes
contains 0.0002% crystal violet. Lowenstein Jensen Medium used to recover
M.tuberculosis is made selective by incorporating malachite green. Wilson &
Blair’s Agar for recovering S.typhi is rendered selective by the addition of dye
Brilliant green. TCBS Agar and Monsur’s Tellurite Taurocholate Gelatin Agar
used for isolating V. cholerae from fecal specimens have elevated pH (8.5-5.6),
which inhibits most other bacteria.
Enrichment media are liquid media that also serves to inhibit commensals in
the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone
water are used to recover pathogens from fecal specimens.
Differential/Indicator media
Differential media or indicator media distinguish one microorganism type from
another growing on the same media. This type of media uses the biochemical
characteristics of a microorganism growing in the presence of specific nutrients
or indicators (such as neutral red, phenol red or methylene blue) added to the
medium to visibly indicate the defining characteristics of a microorganism.
When a particular substrate (carbohydrate) is incorporated into a medium and a
mixture of bacteria inoculated on it, only that bacterium that can ferment it
produces acid. This change in pH is detected by using a pH indicator
incorporated in the medium and the bacterium that can ferment the sugar appears
in a different colour. This approach is used in MacConkey’s agar, CLED agar,
TCBS agar, XLD agar etc.
MacConkey’s agar is the most commonly used media to culture and identify
gram negative bacilli (especially enterobacteriaceae members). It contains bile
salts (selective agent), lactose (sugar), peptone and neutral red (pH indicator),
MICROBIOLOGY 105
Microbiology agar and water. Those bacteria that can ferment lactose produce pink coloured
colonies where non-lactose fermenting colonies produce colourless colonies.
Similarly, Vibrio cholerae produces yellow coloured colonies on sucrose

containing TCBS medium.Reduction of potassium tellurite to metallic tellurium
by Corynebacterium diphtheriae results in production of black coloured
colonies on KT agar. Production of H2S by Salmonella typhi results in
Notes production of black coloured colonies on Wilson & Blair’s medium.
Enterococcus fecalis produces black coloured colonies on bile esculin agar due
to reduction of esculin to esculetin.
Transport media
Clinical specimens must be transported to the laboratory immediately after
collection to prevent overgrowth of contaminating organisms or commensals.
This can be achieved by using transport media. Such media prevent drying
(desiccation) of specimen, maintain the viability of all organisms in the
specimen without altering their concentration . Some of these media (Stuart’s &
Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize
inhibitory factors. Cary Blair medium and Venkatraman Ramakrishnan medium
are used to transport feces from suspected cholera patients. Sach’s buffered
glycerol saline is used to transport feces from patients suspected to be suffering
from bacillary dysentery. Pike’s medium is used to transport streptococci from
throat specimens.
Anaerobic media
Anaerobic bacteria need reduced oxidation –reduction potential and extra
nutrients. Such media may be reduced by physical or chemical means. Boiling
the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1%
thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can
render a medium reduced.
z Robertson cooked meat that is commonly used to grow Clostridium spps

medium .
z Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast

extract and casein hydrolysate.
z Methylene blue or resazurin is an oxidation-reduction potential indicator

that is incorporated in the medium. Under reduced condition, methylene
blue is colourless.
106 MICROBIOLOGY
Preparation and Storage of Culture Media Microbiology
Care must be taken to adjust the pH of the medium before autoclaving. Various
pH indicators that are in use include phenol red, neutral red, bromothymol blue,
bromocresol purple etc. Dehydrated media are commercially available and must
be reconstituted as per manufacturers’ recommendation. Most culture media are
sterililized by autoclaving. Certain media that contain heat labile components
like glucose, antibiotics, urea, serum, blood are not autoclaved. These components
Notes
are filtered and may be added separately after the medium is autoclaved. Certain
highly selective media such as Wilson and Blair’s medium and TCBS agar need
not be sterilized. It is imperative that a representation from each lot be tested for
performance and contamination before use. Once prepared, media may be held at
4-5oC in the refrigerator for 1-2 weeks. Certain liquid media in screw capped
bottles or tubes or cotton plugged can be held at room temperature for weeks.

1. Bacteria that require extra nutrients for growth are called as .............organism
2. Blood agar is a type of ............. media
3. Chocolate agar is specially useful in growing .............
4. Media used to inhibit commensals are ............. media
5. Robertson Cooked meat is commonly used to grow ............. species

z Culture Media are used in diagnosing infections diseases.
z Culture of bacteria is carried out for studying its morphology and its
identification.
z Most culture media contains water, a source of carbon and energy, source
of nitrogen, trace elements and some growth factors, optimum pH, oxygen
and osmolarity.
z Based on consistency culture media is classified as liquid, semi-solid and
solid media.
z Agar is used for solidifying liquid media into solid media.
z Semi-solid media are useful in demonstrating bacterial motility.
z Biphasic media comprises of both liquid and solid medium in the same
bottle.
MICROBIOLOGY 107
Microbiology z Based on nutritional component, culture media are classified as simple,

complex and synthetic.
z Bacteria that grow with minimum requirements are called non-fastidions.
z Bacteria that require extra nutrients are called fastidious.
z Based on functional use or application, culture media are classified as Basal
media, Enriched media, Blood sugar, chocolate agar, selective & enrichment
Notes media.
z Enrichment media serves to inhibit commensals in clinical specimen.
z Differential media/indicator media distinguish are microorganism from
another growing on the same media.
z Transport media prevent drying of specimen and maintain viability of
organisms in the specimen.
TERMINAL QUESTIONS
1. Classify Culture media
2. Describe the preparation & storage of culture media
3. Explain transport & differential media
ANSWERS TO INTEXT QUETIONS
9.1
1. Peptone
2. Agar
3. Biphasic medium
4. Semi solid
9.2
1. Fastidious
2. Enriched
3. Hemophilus & Neisseria
4. Enrichment
5. Clostridium species
108 MICROBIOLOGY
Methods of Isolation of Bacteria MODULE
Microbiology
10
METHODS OF ISOLATION OF Notes
BACTERIA
10.1 INTRODUCTION
We have learned in earlier chapters that there exist so many bacteria that cause
human disease.so now our task is to isolate these bacteria and identify them. The
identification is required so as to cure the illness or the infection caused due to
these bacteria, using appropriate antibiotics. Identification also holds significance
for epidemiological purposes.
This chapter would focus on various methods used for isolation of bacteria.
While in subsequent chapters we would learn about identification of bacteria and
the ways to contain the infections caused by them.
OBJECTIVES
After reading this chapter, you will be able to :
z Expalin the steps involved in the isolation of bacteria.
z describe the significance of Specimen collection.
z describe the significance of Preservation and transportation of specimen.
z explain the role of microscopy in isolation of bacteria.
z explain various methods for isolation of bacteria.
10.2 ISOLATION OF BACTERIA

Isolation of bacteria forms a very significant step in the diagnosis and
management of the illness. Isolation of bacteria involves various steps –
z Specimen collection
z Preservation and transportation of specimen
MICROBIOLOGY 109
MODULE Methods of Isolation of Bacteria
Microbiology z Microscopic examination of sample

z Various methods used for isolation of bacteria
Specimen collection
Many different specimens are sent for microbiological examination from
patients with suspected bacterial infection. Common specimens include urine,
Notes faeces, wound swabs, throat swabs, vaginal swabs, sputum, and blood. Less
common, but important specimens include cerebrospinal fluid, pleural fluid,
joint aspirates, tissue, bone and prosthetic material (e.g. line tips).
Some types of specimen are normally sterile e.g. blood, CSF. These samples are
usually obtained via a percutaneous route with needle and syringe, using
appropriate skin disinfection and an aseptic technique. The culture of bacteria
from such specimens is usually indicative of definite infection except if they are
skin contaminants (bacteria inhabitants of normal skin).
Fig. 10.1: Universal container.
In contrast, many microbiological specimens are obtained from non-sterile sites

e.g. vaginal or throat swabs, urine sample, stool sample. Such samples often
contain bacteria of no clinical relevance in addition to possible pathogens,
making the interpretation of culture results more difficult. In general it is
preferable to send samples from sterile sites if available.
It is preferred to obtain the samples for bacteriological culture before antibiotic
therapy is started. This maximizes the sensitivity of the investigations and
reduces false-negative results. Similarly, samples of tissue or pus are preferred
over swabs, to maximize the recovery of bacteria in the laboratory.
Specimens must be accurately labelled and accompanied by a properly
completed requisition form, indicating the nature of the specimen, the date of
sample collection, relevant clinical information, the investigations required, and
details of antibiotic therapy, if any.
110 MICROBIOLOGY
This allows the laboratory to perform the correct range of tests, and helps in the Microbiology
interpretation of results and reporting. Along with clinical specimens, medical
microbiology laboratories also process samples of food, water and other
environmental samples (e.g. air sampling from operating theatres) as part of
infection control procedures.
High-risk samples
Notes
Certain bacterial infections are a particular hazard to laboratory staff, and
specimens that might contain these pathogens should be labelled as ‘high risk’
to allow for additional safety measures if necessary. For example - blood cultures
from suspected typhoid (Salmonella typhi) or brucellosis (Brucella species), and
samples from suspected Mycobacterium tuberculosis.
Preservation and Transport of specimen

Most specimens are sent to the laboratory in sterile universal containers. Swabs
are placed in a suitable transport medium (eg. charcoal medium) otherwise it
leads to false negative reporting.
Fig. 10.2: Charcoal laden transport media
Specimens should be transported as soon as possible to the laboratory. In case

a delay is anticipated the specimen should be stored at 4° C.
Immediate transport is necessary in order to:
(i) Preserve the viability of the ‘delicate’ bacteria, such as Streptococcus
pneumoniae or Haemophilus influenzae (delays in processing can cause
false-negative culture results);
(ii) Minimize the multiplication of bacteria (e.g. coliforms) within specimens
before they reach the laboratory. In particular urine and other specimens
that utilize a semiquantitative culture technique for thier detection, as
delays in transport can give rise to falsely high bacterial counts when the
specimen is processed.
Microscopy
A Gram stain helps with the visualization of bacteria, and gives an indication
of the type of bacteria present, based on the shape of the bacteria and the staining
MICROBIOLOGY 111
Microbiology properties (Gram positive: purple; Gram negative: pink/red). A Gram stain also
helps to identify mixtures of bacteria, helps to determine the appropriate range
of agar plates to be used for subsequent culture, and helps with the interpretation
of culture results.
Notes
Fig. 10.3: Gram positive cocci
Fig. 10.4: Gram negative bacilli

For liquid specimens e.g. CSF, the sample is first centrifuged to concentrate any
bacterial cells in the deposit, and Gram stain and culture is performed from the
deposit after the supernatant is decanted. This helps increase the sensitivity of
both microscopy and culture.
Ziehl-Neelsen (ZN) stain is used to demonstrate the presence of Mycobacteria.
Mycobacteria can also be visualized using the fluorescent dye auramine and a
fluorescence microscope. Direct immunofluorescence is employed to detect
certain pathogens (e.g. Legionella, Pneumocystis) using specific antibodies
conjugated to a fluorescent dye.
Another microscopic technique is dark ground microscopy. This is mainly used
to detect the thin spirochaetal cells of Treponema pallidum (syphilis bacteria).

1. Specimens that contain pathogens which are hazardous to laboratory staff
should be labeled as ..................
2. Swabs are sent to laboratory in .................. medium
112 MICROBIOLOGY
3. If delay is anticipated in transporting the specimen, it should be stored at Microbiology
.................. temperature
4. .................. gives an indication of bacteria present in the sample
5. .................. stain is used in demonstration of mycobacteria
6. .................. microscopy is used to detect syphilis organism
10.3 METHODS OF ISOLATION OF BACTERIA Notes
Methods of isolation of bacteria can be broadly classified into two

z Culture methods
z On Solid media
z On Liquid media
z Automated systems
z Non-culture methods
Culture methods
The specimens received in the laboratory are plated on the culture media. The
appropriate culture media is selected depending upon the bacteria suspected. The
following precautions need to be taken into consideration when the culture
methods are processed
z Optimal atmospheric conditions
z Optimal temperature
z Growth requirement of the bacteria
Atmospheric conditions:
Colonies of bacteria are usually large enough to identify after 18–24 hours of
incubation (usually at 37°C), but for some bacteria longer incubation times are
required (from 2 days to several weeks). Culture plates are incubated (1) in air,
(2) in air with added carbon dioxide (5%), (3) anaerobically (without oxygen)
or (4) micro-aerophilically (a trace of oxygen) according to the requirements of
the different types of bacteria that may be present in specimens.
In case of Mycobacteria especially the scotochromogen the culture bottles are
placed in dark or the bottles are covered with black paper and kept for incubation
at 37°C.
Temperature:
Most of the bacteria requires a temperature of 37°C for optimal growth. This
temperature is provided placing the inoculated culture plates in the incubator set
at 37°C temperature.
MICROBIOLOGY 113
Microbiology
Notes
Fig. 10.5: Incubator
Growth requirement of the bacteria

Different bacteria have different growth requirements. For eg Streptococcus
pneumoniae requires factor V and factor X for its growth, which are found in
chocolate agar. Thus for sample suspected of S. pneumoniae the samples are
plated on chocolate agar. Similarly depending upon the growth requirements
the appropriate culture media are used.

1. .................. & .................. methods are commonly used methods for
bacterial isolation
2. Colonies of bacteria can be identified after .................. hours of incubation
3. The optimum temperature most bacteria require to grow are ..................
4. Chocolate agar has .................. & .................. which is used in the diagnosis
of streptococci Pneumonia
114 MICROBIOLOGY
Microbiology
10.4 CULTURE ON SOLID MEDIA
The principal method for the detection of bacteria from clinical specimens is by
culture on solid culture media. Bacteria grow on the surface of culture media
to produce distinct colonies.
Different bacteria produce different but characteristic colonies, allowing for
early presumptive identification and easy identification of mixed cultures. There
are many different types of culture media. Agar is used as the gelling agent to Notes
which is added a variety of nutrients (e.g. blood, peptone and sugars) and other
factors (e.g. buffers, salts and indicators).
Some culture media are nonselective (e.g. blood agar, nutrient agar) and these
will grow a wide variety of bacteria. While some e.g. MacConkey agar are more
selective (in this case through the addition of bile salts selecting for the ‘bile-
tolerant’ bacteria found in the large intestine such as Escherichia coli and
Enterococcus faecalis). MacConkey agar also contains lactose and an indicator
system that identifies lactose-fermenting coliforms (e.g. Escherichia coli,
Klebsiella) from lactose-non fermenting coliforms (e.g. Morganella Salmonella).
Media can be made even more selective by the addition of antibiotics or other
inhibitory substances, and sophisticated indicator systems can allow for the easy
detection of defined bacteria from mixed populations.
Method of inoculating the solid culture media

Method used for inoculating the solid media depends upon the purpose of
inoculation- whether to have isolated colonies or to know the bacterial load of
the sample (quantitative analysis).
For obtaining the isolated colonies streaking method is used, the most common
method of inoculating an agar plate is streaking.
Fig. 10.6: Streaking method
MICROBIOLOGY 115
Microbiology Streak plates

1. A small amount of sample is placed on the side of the agar plate (either
with a swab, or as a drop from an inoculating loop).
2. A sterile loop is then used to spread the bacteria out in one direction from
the initial site of inoculation. This is done by moving the loop from side
to side, passing through the initial site.
Notes 3. The loop is then sterilised (by flaming) again and the first streaks are then
spread out themselves.
4. This is repeated 2-3 times, moving around the agar plate as shown in the
figure.
In this method single bacterial cells get isolated by the streaking, and when the
plate is incubated, forming discrete colonies that will have started from just one
bacterium each.
For quantitative analysis or semi quantitative analysis of the sample for example
in case of urinary tract infection. In fact E.coli is implicated as the causative
organism in urinary tract infection only if there are >105Colony forming units
per millilitre of urine. The method of inoculating the solid culture media is as
shown in the figure.
Fig. 10.7: Inoculation methods
Fig. 10.8: Uninoculated Mac conkey Agar and Blood agar plate
116 MICROBIOLOGY
Microbiology
Notes
Fig. 10.9: Lactose fermenting (pink coloured) colonies on mac conkey agar
Culture in liquid media

Bacteria can also be grown in liquid media (broth). Like agar plates, broth
cultures may be non selective or selective. Bacterial growth is easy to detect as
the clear liquid turns turbid, usually within 24–48 hr, but incubation may need
to be extended to 14 days or more.
The advantage of broth culture is that it is significantly more sensitive than direct
culture on agar. The disadvantage is that, by itself, it is not easy to determine
the type of bacteria present or whether a mixed growth has occurred, and in most
cases the broth must be subcultured onto solid agar plates. This causes an
additional delay in culture results. Broth cultures are also prone to contamination.
Broth enrichment media are used when high sensitivity is required e.g. for
detection of bacteria from CSF, or to detect small numbers of Salmonella in a
stool sample containing many millions of other bacteria.
Fig. 10.10: Liquid media
Automated system
Automated blood culture systems eg. BACTEC, BacteAlert utilize liquid
culture. Bacterial growth may be detected by a variety of methods (e.g. detection
of bacterial CO2 production).
MICROBIOLOGY 117
Microbiology
Notes
Fig. 10.11: Bactec
Fig. 10.12: Bactec
Automated liquid culture systems are also available for the culture of
Mycobacteria, and similar technology can be used to automate sensitivity
The advantage of automated system are
Rapidity : they aid in faster growth of bacteria. Thus less time consuming.
The incidence of contamination during the processing of sample are minimised
Real time monitoring of the growth
One of the main limitations is the commercial viability.
Non culture methods

Isolation of bacteria can also be carried out by non-culture methods. In particular
the more advanced Amplification techniques like Polymerase chain reaction
(PCR), ligase chain reaction (LCR), strand displacement amplification (SDA),
and nucleic acid sequence based amplification (NASBA) are being used in
clinical laboratories for isolation and identification of bacteria.
118 MICROBIOLOGY
The following are some of the factors that are considered in interpreting Microbiology
bacteriological culture results:
z type of specimen
z any delays in processing
z types of bacteria recovered
z knowledge of the normal human flora at different sites Notes
z clinical information provided on the request form
z details of recent antibiotic therapy
There must be good liaison between healthcare workers and the microbiology
laboratory, in order to ensure that the most appropriate investigations are
performed, results are interpreted correctly, and clinically relevant bacteriological
reports are produced.

1. .................... is used as gelling agar in culture media
2. .................... culture media grow a wide variety of bacteria
3. .................... is an example of selective media
4. For obtaining the isolated colonies .................... method is common method
of inoculating
5. .................... is the liquid medium in which bacteria may be grown
6. Examples of Amplication techniques are ...................., .................... &
....................

z Isolation of bacteria forms a very significant step in the diagnosis and
management of the illness. Isolation of bacteria involves various steps –
Specimen collection, Preservation and transportation of specimen,
Microscopic examination of sample.Various methods used for isolation of
bacteria culture methods which includes culture on solid or liquid media
and automated system. Non culture methods include the molecular techniques
eg PCR, SDA, NASBA.
MICROBIOLOGY 119
Microbiology
TERMINAL QUESTIONS
1. What is the need for isolation of bacteria?
2. Describe in brief various steps involved in the isolation of bacteria.
3. What is difference between blood agar and chocolate agar
Notes 4. Explain the term selective and non selective media with proper examples.
5. Draw a labeled diagram of inoculation of solid culture media for isolation
of bacteria.
6. Draw a labeled diagram for inoculation of solid media for processing the
urine sample of a patient suspected of urinary tract infection.
7. Describe in brief the advantages and the limitation of use of liquid culture
media for isolation of bacteria.
8. Mention the advantages and the disadvantages of automated system for
isolation of bacteria.
9. Name some non culture methods for isolation of bacteria
10.1
1. High-risk
2. Charcoal
3. 4oC
4. Gram stain
5. Ziehl-Neelson
6. Dark Ground
10.2
1. Direct culture & Non-culture
2. 18-24
3. 37oC
4. Factor V & Factor X
120 MICROBIOLOGY
10.3 Microbiology
1. Agar
2. Non-selective
3. MacConkey
4. Streaking
5. Broth Notes
6. Polymerase Chain Reaction, Ligase Chain Reaction, Nucleic Acid Sequence
Based Amplification
MICROBIOLOGY 121
MODULE Bacterial Identification Tests
Microbiology
11
Notes BACTERIAL IDENTIFICATION
TESTS
11.1 INTRODUCTION
In the previous chapter we have discussed various methods of isolation of
bacteria. The bacteria thus isolated needs to be further identified to genus and
species level. The identification is required so as to cure the illness or the
infection caused due to the bacteria by using appropriate antibiotics. Identification
also holds significance for epidemiological purposes.
OBJECTIVES
z describe the processes involved in the identification of bacteria.
z explain the significance of microscopy in the process of identification of
bacteria.
z explain the significance of biochemical test in the process of identification
of bacteria.
z describe the significance of serology in the process of identification of
bacteria.
z describe the significance of phage typing in the process of identification of
bacteria.
z explain the significance of antimicrobial susceptibility testing in the process
of identification of bacteria
11.2 BACTERIAL IDENTIFICATION

The isolated bacteria are further processed through one or few of the procedures
mentioned below so as to identify the bacteria
z Staining of the isolated bacteria
z Motility testing
122 MICROBIOLOGY
Bacterial Identification Tests MODULE
z Biochemical testing Microbiology
z Serological tests
z Phage typing
z Identification disc testing
z Semiautomated and Automated identification systems
z Molecular techniques
Notes
(i) Staining of the isolated bacteria
Staining of the bacteria forms the foremost and the most important step in the
identification of bacteria. The isolated bacteria are stained by various methods
depending upon the bacteria in focus. Various staining techniques are as follow
1. Gram staining: differentiates bacteria into two types
Gram positive and Gram negative bacteria
Gram positive bacteria can be either cocci or bacilli or vibrios. Gram
positive pathogenic bacteria are staphylococci, streptococci, pneumococci,
etc
Gram negative bacteria can be either cocci or bacilli. Gram negative
pathogenic bacteria commonly encountered are E.coli, Klebsiella, Salmonella
spp, shigella, etc
2. Albert staining: is performed in case if one suspects a Corynebacterium
spp.
3. Acid fast staining: is performed in cases suspected of Mycobacterial
infection. Eg. Tuberculosis, leprosy, etc.
4. Special staining is necessary in case of spirochetes and other organisms.

1. ......................... of bacteria is the important step in identification of bacteria
2. Gram stain differentiates bacteria as ......................... & .........................
3. ......................... staining is used in Identification of Corynebacterium spp
4. ......................... staining is used in identification of Mycobacterial infection
5. ........................., ......................... & ......................... are examples of Gram
Positive Bacteria
6. ........................., ......................... & ......................... are examples of Gram
Negative Bacteria
MICROBIOLOGY 123
Microbiology (ii) Motility testing

Motility testing is performed by preparing a wet mount and is then observed
under the microscope. Motility of bacteria can also be tested by inoculating the
bacteria in the semisolid motility medium.
(iii) Biochemical tests

Notes The staining is followed by use of various biochemical reagents and tests to get
closer to the identification of bacteria. There are many biochemical tests
available for bacterial identification. Few of them are required to be carried out
depending upon the bacteria. The commonly used biochemical tests are as
mentioned below
(a) Catalase test
(b) Coagulase test
(c) Oxidase test
(d) Sugar fermentation test
(e) Indole test
(f) Citrate test
(g) Urease test
(a) Catalase test

Purpose
The catalase test facilitates the detection of the enzyme catalase in bacteria. It
is essential for differentiating catalase-positive Micrococcaceae from catalase-
negative Streptococcaceae. While it is primarily useful in differentiating between
genera, it is also valuable in speciation of certain gram positives such
as Aerococcus urinae (positive) from Aerococcus viridians (negative) and gram-
negative organisms such as Campylobacter fetus, Campylobacter jejuni,
and Campylobacter coli (all positive) from other Campylobacter species.
Procedure:
Place a microscope slide inside a petri dish. Keep the petri dish cover available.
Using a sterile inoculating loop or wooden applicator stick, collect a small
amount of organism from a well-isolated 18- to 24-hour colony and place it onto
the microscope slide. Be careful not to pick up any agar. This is particularly
important if the colony isolate was grown on agar containing red blood cells.
Carryover of red blood cells into the test may result in a false-positive reaction.
Using a dropper or Pasteur pipette, place 1 drop of 3% H2O2 onto the organism
on the microscope slide. Do not mix. Immediately cover the petri dish with a
124 MICROBIOLOGY
lid to limit aerosols and observe for immediate bubble formation (O2 + water Microbiology
= bubbles). Observing for the formation of bubbles against a dark background
enhances readability.
Notes
Fig. 11.1
Catalase positive bacteria: Staphylococcus spp

Catalase negative bacteria: Streptococcus spp
b. Coagulase test
Purpose
The coagulase test differentiates strains of Staphylococcus aureus from other
coagulase-negative species. S. aureus strains are capable of coagulating plasma
in the tube test and will produce clumps of cells in the slide test.
The coagulase test can be performed using two different procedures - Slide test
and tube test. The slide test is simple, giving results within 10 seconds, but it
can give false negatives. The tube test is the definitive test, however, it can take
up to 24 hours to complete. For both tests, clumping or clots of any size indicate
a positive response. While S. aureus is the most commonly isolated coagulase-
positive organism, there are several other species of Staphylococcus which are
positive for coagulase activity. S. schleiferi and S. lugdunensis may give positive
results in the slide test for bound coagulase, and S. schleiferi and S.
intermedius may give positive results in the tube coagulase test .
Procedure:
The slide test is performed by preparing a suspension of bacterial cells mixed
into a drop of rabbit plasma on a microscope slide. If bound coagulase is present
on the bacterial cells, then the presence of plasma will cause the bacterial cells
to clump. The clumping will occur because the clumping factor is an adhesin,
which causes the cells to bind to fibrinogen in the plasma. This will result in
visible clumping of bacterial cells on the microscope slide. Figure given below
illustrates the visible clumping of cells on the microscope slide.
MICROBIOLOGY 125
Microbiology
Notes
Fig. 11.2: Slide coagulase test.
The tube coagulase test is performed by mixing bacterial cells into a larger
volume of plasma in a small test tube. As the bacteria multiply in the plasma,
they secrete staphylocoagulase. Staphylocoagulase initiates blood coagulation
by activating prothrombin. Staphylocoagulase adheres to fibrinogen, forming a
complex that cleaves fibrinogen into fibrin, bypassing the blood clotting cascade
and directly causing a clot of fibrin to form. Formation of a clot will be noted
within 24 hours for a positive response. Figure shows a negative reaction and
a positive reaction.
Fig. 11.3: Tube coagulase test.
Coagulase positive bacteria: Staphylococcus aureus

Coagulase negative bacteria: Staphylococcus epidermis, Staphylococcus
saprophyticus

1. Motility of bacteria can be tested by inoculating the bacteria in .............
medium
2. Catalast test is primarily useful in differentiating between .............
3. Example of catalase positive bacteria is .............
126 MICROBIOLOGY
4. In coagulast test ............. is formed in slide test and ............. is produced Microbiology
in tube test.
5. ............. is the most common coagulase positive organism
(c) Oxidase test

Purpose
The oxidase test is a biochemical reaction that assays for the presence of Notes
cytochrome oxidase, an enzyme sometimes called indophenol oxidase. In the
presence of an organism that contains the cytochrome oxidase enzyme, the
reduced colorless reagent becomes an oxidized colored product.
Procedure
There are many method variations to the oxidase test. These include, but are not
limited to, the filter paper test, filter paper spot test, direct plate method, and test
tube method.
Filter Paper Test Method
1. Soak a small piece of filter paper in 1% Kovács oxidase reagent and let
dry.
2. Use a loop and pick a well-isolated colony from a fresh (18- to 24-hour
culture) bacterial plate and rub onto treated filter paper.
3. Observe for color changes.
4. Microorganisms are oxidase positive when the color changes to dark purple
within 5 to 10 seconds. Microorganisms are delayed oxidase positive when
the color changes to purple within 60 to 90 seconds. Microorganisms are
oxidase negative if the color does not change or it takes longer than 2
minutes.
Fig. 11.4
Oxidase positive bacteria : Pseudomonas, Vibrio cholera

Oxidase negative bacteria: E. coli, Klebsiell, Salmonella.
MICROBIOLOGY 127
Microbiology (d) Indole test

Purpose
The indole test screens for the ability of an organism to degrade the amino acid
tryptophan and produce indole. It is used as part of the IMViC (indole, MR-Vp
Citrate) procedures, a battery of tests designed to distinguish among members
of the family Enterobacteriaceae.
Notes
Procedure
Inoculate the tube of tryptone broth with a small amount of a pure culture.
Incubate at 37°C for 24 to 48 hours.
To test for indole production, add 5 drops of Kovác's reagent directly to the tube.
A positive indole test is indicated by the formation of a pink to red color (“cherry-
red ring”) in the reagent layer on top of the medium within seconds of adding
the reagent.
If a culture is indole negative, the reagent layer will remain yellow or be slightly
cloudy.
Indole positive bacteria : E. coli, Vibrio cholera
Indole negative bacteria : Klebsiella, Salmonella, Shigella spp.
Fig. 11.5
(e) Citrate Test

Purpose
The citrate test screens a bacterial isolate for the ability to utilize citrate as its
carbon and energy source. A positive diagnostic test rests on the generation of
alkaline by-products of citrate metabolism. The subsequent increase in the pH
of the medium is demonstrated by the color change of a pH indicator.
128 MICROBIOLOGY
The citrate test is often part of a battery of tests used to identify gram-negative Microbiology
pathogens and environmental isolates.
Procedure
Use a fresh (16- to 18-hour) pure culture as an inoculation source. Pick a single
isolated colony and lightly streak the surface of the slant. A needle is the
preferred sampling tool in order to limit the amount of cell material transferred
to the agar slant. Avoid using liquid cultures as the inoculum source. Citrate Notes
utilization requires oxygen and thus screw caps, if used, should be placed loosely
on the tube. Incubate at 35oC (+/- 2oC) for 18 to 48 hours. Some organisms may
require up to 7 days of incubation due to their limited rate of growth on citrate
medium.
Citrate positive: growth will be visible on the slant surface and the medium will
be an intense Prussian blue. The alkaline carbonates and bicarbonates produced
as by-products of citrate catabolism raise the pH of the medium to above 7.6,
causing the bromothymol blue to change from the original green color to blue.
Citrate negative: trace or no growth will be visible. No color change will occur;
the medium will remain the deep forest green color of the uninoculated agar.
Only bacteria that can utilize citrate as the sole carbon and energy source will
be able to grow on the Simmons citrate medium, thus a citrate-negative test
culture will be virtually indistinguishable from an uninoculated slant.
Citrate positive bacteria: Klebsiella spp.
Citrate negative bacteria: E. coli.
Fig. 11.6
(f) Urease test

Purpose
The urease test identifies those organisms that are capable of hydrolyzing urea
to produce ammonia and carbon dioxide. It is primarily used to distinguish
urease-positive bacteria from other Enterobacteriaceae.
MICROBIOLOGY 129
Microbiology Procedure
Christensen’s Urea Agar (4, 5)
Use a heavy inoculum from an 18- to 24-hour pure culture to streak the entire
slant surface. Do not stab the butt as it will serve as a color control . Incubate
tubes with loosened caps at 35oC. Observe the slant for a color change at 6 hours,
24 hours, and every day for up to 6 days. Urease production is indicated by a
Notes bright pink (fuchsia) color on the slant that may extend into the butt. Note that
any degree of pink is considered a positive reaction. Prolonged incubation may
result in a false-positive test due to hydrolysis of proteins in the medium. To
eliminate protein hydrolysis as the cause of a positive test, a control medium
lacking urea should be used.
Rapidly urease-positive Proteeae (Proteus spp., Morganella morganii, and
some Providencia stuartii strains) will produce a strong positive reaction within
1 to 6 hours of incubation. Delayed-positive organisms (e.g., Klebsiella or
Enterobacter) will typically produce a weak positive reaction on the slant after
6 hours, but the reaction will intensify and spread to the butt on prolonged
incubation (up to 6 days). The culture medium will remain a yellowish color if
the organism is urease negative.
Fig. 11.7
Urease positive bacteria : Proteus spp., Morganella morganii
Urease negative bacteria : E. coli,

1. Example of oxidase negative bacteria ...............
2. Positive indole test is indicated by formation of ............... in the reagent
layer
3. Indole test is used to distinguish among members of the family ...............
4. Citrate test is commonly used to identify ............... pathogens
5. Example of Urease positive bacteria is ...............
130 MICROBIOLOGY
Serology Microbiology
It forms an important step in bacterial identification. It usually involves detection

of antigens by enzyme or fluorescence immunoassays. Serology is also used to
confirm identification obtained by other methods. For example, salmonella
species identified by biochemicals tests is processed for serotyping by slide
agglutination. Another example being Vibrio cholera.
Notes
Phage typing
Phage typing is a method used for detecting single strains of bacteria. It is used
to trace the source of outbreaks of infections. The viruses that infect bacteria are
called bacteriophages (“phages” for short) and some of these can only infect a
single strain of bacteria. These phages are used to identify different strains of
bacteria within a single species.
A culture of the strain is grown in the agar and dried. A grid is drawn on the base
of the petri dish to mark out different regions. Inoculation of each square of the
grid is done by a different phage. The phage drops are allowed to dry and are
incubated: The susceptible phage regions will show a circular clearing where the
bacteria have been lysed, and this is used in differentiation.
Fig. 11.8
Identification discs
Kirby Baur disc diffusion method is primarily aimed to identify the antibiotic
susceptibility of the bacteria. It is also helpful in identification of some bacteria
for eg Micrococci spp, Streptococci spp, Morexalla spp, etc
Semiautomated and Automated identification systems

The isolated colonies obtained, are processed by these system. The system
identifies the bacteria and also carries out the antibiotic susceptibility testing for
MICROBIOLOGY 131
Microbiology the same. Microscan walkaway system, Vivtek system, Sensititre Gram-
Negative Auto identification system, the Phoenix system are some of the
Semiautomated and Automated identification systems available for bacterial
identification.
Bactec AFB system, Mycobacteria Growth Indicator Tube (MGIT), and MGIT
960 aresome automated identification systems available for Mycobacterial
identification.
Notes
Molecular techniques
Molecular methods includes G+C % content, DNA-DNA hybridisation and
DNA base sequencing. These methods are not used routinely used in hospital
laboratories. Amplification techniques like Polymerase chain reaction, ligase
chain reaction, strand displacement amplification, and nucleic acid sequence
based amplification are being used in clinical laboratories for direct detection
of bacteria. Eg. Neisseeria gonorrhoea, Leptospirosis,etc.

1. Serology involves detection of antigens by ................... or ...................
2. Serology is used in confirmation of ................... & ...................
3. ................... is used for detecting single strains of bacteria
4. Viruses that infect bacteria are called ...................

z Techniques like straining of isolated bacteria, motility testing, Biochemical
testing, Serological tests, Phage typing, identification disc testing,
Semiautomated and Automated identification system & Molecular techniques
are used for bacterial identification.
z Various staining techniques like Gram stain, Albert stain, Acid fast Stain
& Special Staining are used for bacterial identification
z Catalase test, coagulase test, Oxidase test, Sugar fermentation test, Indole
test, Citrate Test, Urease test are the Biochemical tests used for bacterial
identification
z Serology tests like Enzyme or Fluorescence immunoassays are used to
confirm identification obtained by other methods
132 MICROBIOLOGY
z Phage typing is used for detecting single strains of bacteria and also to trace Microbiology
the source of outbreaks of infections
z Kirby bayer disc diffusion method is used to identify antibiotic susceptibility
of bacteria
z Semiautomated & automated identification systems identify bacteria and
also carry out antibiotic susceptibility testing.
Notes
TERMINAL QUESTIONS
1. Describe in brief the various staining techniques
2. Enlist biochemical test performed for identification of bacteria
3. Describe in brief the role of serology in identification of bacteria
4. What do you understand by the term phage typing. Explain
5. Explain the role of antimicrobial susceptibility testing in identification of
bacteria with suitable examples.
6. Name the molecular techniques used for the identification of bacteria.

11.2
1. Staining
2. Gram Positive and Gram Negative
3. Albert
4. Acid fast
5. Staphylococci, Streptococci & Pneumococcia
6. Ecoli, Klebsiella & Salmonella
11.2
1. Semisolid motility
2. Genera
3. Staphylococci
4. Clumping & clots
5. Staphylococcus aureus
MICROBIOLOGY 133
Microbiology 11.3
1. E.coli
2. Cherry-red ring
3. Enterobacteriaceae
4. Gram negative
Notes 5. Proteus spp
11.4
1. Enzyme, Fluorescence
2. Salmonella & Vibrio Cholera
3. Phage typing
4. Bacteriophages
134 MICROBIOLOGY
Antibiotic Susceptibility Testing MODULE
Microbiology
12
ANTIBIOTIC SUSCEPTIBILITY Notes
TESTING
12.1 INTRODUCTION
Once we have identified the bacterium which is causing the infection we need
to find out the antibiotics that would be effective against it. This is done by
antibiotic sensitivity testing. there are various methods which can be employed
for this purpose
OBJECTIVES
z describe various terminologies related to Antibiotic susceptibility testing
z escribe principle for Antibiotic susceptibility testing.
z describe the procedure for performing Antibiotic susceptibility testing
z describe different methods used for Antibiotic susceptibility testing
12.2 TERMINOLOGY
Selectivity
All Clinically effective antimicrobial agents exhibit selective toxicity towards
the bacterium rather than the host. It is this characteristic that distinguishes
antibiotics from disinfectants. The basis for selectivity will vary depending on
the particular antibiotic. When selectivity is high the antibiotics are normally non
toxic. However, even highly selective antibiotics can have side effects.
Therapeutic Index
The therapeutic index is defined as the ratio of the dose toxic to the host to the
effective therapeutic dose and the higher the therapeutic index the better the
antibiotic.
MICROBIOLOGY 135
MODULE Antibiotic Susceptibility Testing
Microbiology Categories of Antibiotics

Antibiotics are categorized as bactericidal, if they kill the susceptible bacteria
or bacteriostatic, if they reversibly inhibit the growth of bacteria. In general the
use of bactericidal antibiotics is preferred but many factors may dictate the use
of a bacteriostatic antibiotic. When a bacteriostatic antibiotic is used the duration
of therapy must be sufficient to allow cellular and humoral defense mechanisms
to eradicate the bacteria. If possible, bactericidal antibiotics should be used to
Notes treat infections of the endocardium or the meninges. Host defenses are relatively
ineffective at these sites and the dangers imposed by such infections require
prompt eradication of the organisms.
In vitro sensitivity tests

Bacterial pathogens are tested for their susceptibility to antibiotics to guide
antibiotic treatment. Sensitivity tests are generally performed from single pure
bacterial colonies on an agar plate. Direct sensitivity tests are set up directly from
specimens or liquid cultures, producing quicker, but less standardized results.
Disk sensitivity tests

Antibiotic diffuses out of a disk placed on the surface of the agar. If bacteria are
sensitive to the antibiotic, then a zone of growth inhibition forms around the disk
after incubation. The zone size depends on several factors and two methods are
available to control this process, comparative disk testing (where both a test and
control organism are tested on the same plate), and standardized disk testing.
Breakpoint sensitivity tests

Antibiotic is incorporated into the agar at a uniform concentration and bacteria
inoculated onto the agar surface. Only bacteria resistant to the antibiotic at the
breakpoint concentration will then grow. Using multipoint inoculators, many
bacterial strains can be tested simultaneously on each agar plate.
Minimum inhibitory concentration (MIC)

The MIC is the minimum (lowest) concentration of an antibiotic that will inhibit
the growth of a bacterial strain. This can be determined by several methods
including macro- and micro dilution tests, extended breakpoint sensitivity tests,
and e-test strips. Determination of MIC is important in the management of
certain infections (e.g. Endocarditis).
Minimum bactericidal concentration (MBC)

The MBC is the lowest concentration of the antibiotic that will kill a bacterial
strain. The MBC is less clinically relevant than the MIC, as MBC tests are harder
to standardize.
136 MICROBIOLOGY
Detection of bacterial resistance mechanisms Microbiology
Various bacterial resistance mechanisms (e.g. ß-lactamase production, antibiotic

resistance genes) can be detected in the laboratory, providing a quick method
of predicting in vitro sensitivity results.
Automated sensitivity tests

Automated systems can reduce the technical time required to perform sensitivity Notes
tests. These systems often utilize liquid culture, producing faster results than
conventional agar based tests.
Clinical relevance of in vitro antibiotic sensitivity test

In vitro sensitivity test results should only be used as a guide to treatment, and
the results do not always correlate with clinical response. The success of
antibiotic treatment can be affected by many factors including immune
responses, pharmacological factors and other biological variables, and the
presence of biofilms.
In vitro sensitivity tests

In order to guide the appropriate antibiotic treatment of bacterial infections,
bacterial pathogens isolated from clinical specimens are usually tested against
a selection of antibiotics to assess their degree of susceptibility. This is usually
done with bacteria that have been grown on solid media. Sensitivity tests are
performed from single pure colonies and require a further 18–24 hrs of
incubation. Thus while culture results may be available within 24 hrs of receipt
of a specimen, sensitivity results usually take an additional day.
In some situations, direct sensitivity tests are performed, either from the
specimen itself (e.g. Urine) or from a liquid broth with bacterial growth (e.g.
Blood culture bottle). In this case, sensitivity tests are setup at the same time as
the specimen is subcultured to agar plates. Although this speeds up the process,
there are several disadvantages:
(i) it is difficult to ensure the correct inoculum (the number of bacteria spread
onto the agar surface)
(ii) the inoculum may be mixed (more than one type of bacteria), making the
results difficult to interpret and requiring the test to be repeated
(iii) the selection of antibiotics tested may be inappropriate for the bacterium
subsequently grown.
MICROBIOLOGY 137
Microbiology

Match the following
1. Selectivity (a) Kills bacteria
2. Therapeutic index (b) Minimum concentration for
Notes inhibiting bacterial growth
3. Bacteriocidal (c) Selective toxicity to
antimicrobial agents
4. Bacteriostatic (d) Minimum concentration that
kills bacteria
5. Minimum Inhibitory Concentration (e) Ratio of toxic and effective
dose
6. Minimum Bactericidal Concentration (f) Inhibits bacterial growth
Several different methods are available for assessing the susceptibility of

bacteria to antibiotics.
Disk sensitivity tests

Disk sensitivity tests are performed on agar plates. A small disk of filter paper,
pre-impregnated with a defined quantity of antibiotic, is placed on the surface
of an agar plate that has already been inoculated with a suspension of bacteria.
The antibiotic diffuses out of the disk into the agar, along a concentration
gradient, as the plates are incubated (for 18–24 h). If the bacterial strain is
sensitive to the antibiotic, then a zone of inhibition (no growth) occurs around
the disk (Fig. 12.1).
The diameter of the zone depends on a number of factors including
(i) the quantity of antibiotic within the disk
(ii) the degree of susceptibility of the bacteria to the antibiotic
Fig. 12.1: Disk sensitivity test. A – agar; B – antibiotic disc; C – antibiotic diffuses into
agar along concentration gradient; D – bacterial growth on surface of agar after 18 hours
of incubation; E – zone (diameter) of inhibition.
138 MICROBIOLOGY
(iii) the physicochemical properties of the antibiotic; Microbiology
(iv) the depth (in mm) of the agar plate;

(v) the concentration of bacteria in the inoculum (semiconfluent growth is
required).
There are two methods employed to determine the sensitivity pattern. The
comparative disk test (stokes’ method) uses both a test organism and a control
organism on the same plate (fig. 12.2). The control organism is of defined Notes
sensitivity to the antibiotics being tested, and this method allows a direct
comparison of the diameter of the zones of inhibition between the test and
control organisms.
Fig. 12.2: Schematic representation of comparative disk sensitivity test (stokes′ method).
Control—control bacterial strain (known sensitivity to antibiotics);test—bacterial strain
under test; A-F- six different antibiotic disks. In this figure, the test organism is
sensitive to antibiotics B, C & E, but resistant to antibiotics A (>3 mm
reduction in zone diameter compared to control), D & F.
Standardized disk testing

This uses carefully standardized agar plates and inocula. A standardized
inoculum of the test organism is plated out across the whole surface of the agar
plate (control organisms are tested on a separate plate). The diameter of the zones
of inhibition are measured in mm, and the organism
Reported as sensitive or resistant based on defined cut-off points (for example
<18 mm=resistant).
One disadvantage of disk testing is that it is usually only possible to have a
maximum of six different antibiotic disks on a standard agar plate.
MICROBIOLOGY 139
Microbiology Breakpoint sensitivity tests

Breakpoint sensitivity tests use a different principle to disk testing. A defined
concentration of antibiotic (the ‘breakpoint’) is incorporated into the agar during
production of the agar plates. Bacteria are then inoculated onto a small part of
the surface of the agar (usually with ‘multipoint’ inoculators that allows many
different stains to be tested on the same plate) and the agar plate incubated for
18–24 hrs. Bacteria that are resistant to the antibiotic (at the breakpoint
Notes concentration) will grow, whilst those that are sensitive will not. A control agar
plate with no added antibiotic is used to check for viable bacterial growth (Fig.
12.3).
Fig. 12.3: Breakpoint sensitivity tests. Control – sensitivity agar plate with no added
antibiotic; Ampicillin—sensitivity agar plate incorporating Ampicillin at a defined
(uniform) concentration 1 to 10- different bacterial strains inoculated on to the
surface of the agar plates with a multipoint inoculator. In this example,
all 10 strains have grown on the control plate. Strains 1, 3, 6, 7, 9
& 10 are sensitive to Ampicillin. Strains 2, 4, 5 & 8 are resistant.
Using a multipoint inoculator, >30 different strains of bacteria can be tested

against a wide range of different antibiotics in one batch. This process is less
technically time-consuming than the equivalent number of disk tests.
Sometimes the same antibiotic is used at two different concentrations in separate
agar plates (e.g. 1 and 4 mg/1). Using these low and high breakpoint
concentrations, bacteria can be classified as ‘sensitive’, ‘intermediate’ or
‘resistant’. By including a whole range of concentrations of the same antibiotic
in separate plates, the minimum inhibitory concentration of the antibiotic can
be determined for each of the strains being tested (see below).
Minimum inhibitory concentration (MIC)

The MIC is the minimum (lowest) concentration of an antibiotic that will inhibit
the growth of a bacterial strain. Conventionally, this is determined using a series
of doubling dilutions of the antibiotic in liquid culture medium, to produce a
range of concentrations in test tubes (macrodilution) or in a microtiter tray
(microdilution). After inoculation of the test strain into each antibiotic
concentration, bacterial growth is determined by visible turbidity after 18–24 h
of incubation (Fig. 12.4). The MIC is the lowest concentration of antibiotic with
no visible bacterial growth.
140 MICROBIOLOGY
Microbiology
Notes
Fig. 12.4: Broth Dilution test
Fig. 12.5: Microbroth dilution Test
MIC tests can also be done by extended breakpoint sensitivity tests (see
above). These methods are technically time-consuming and relatively expensive.
An alternative method is by use of commercially available E-test strips. These
are specialized antibiotic-impregnated strips which, like disk testing, are placed
on the surface of inoculated agar plates. During incubation, antibiotic diffuses
into the agar forming a zone of inhibition. There is a manufactured concentration
gradient within the strip, and numerical gradations are marked along the edge
of the strip to reflect this. The MIC is determined by measuring the point at which
the edge of the zone of inhibition crosses the e-test strip (fig. 12.5).
Antibiotic MIC tests are usually performed only in certain situations in a clinical
bacteriology laboratory. They are most commonly used when a very precise
assessment of the in vitro susceptibility of a bacterial strain is required, for
instance in the treatment of pneumococcal meningitis (topic f3) or Streptococcal
endocarditis. MIC tests are also used to assess the overall degree of activity of
antibiotics against different strains of the same bacterial species, particularly
when evaluating or developing new antimicrobial agents. A simple way of
describing the relative activity of an antibiotic against a group of organisms, is
by using the terms mic50 and mic90. These are the lowest concentrations of the
antibiotic that inhibit 50 and 90% of the bacterial strains tested, respectively.
MICROBIOLOGY 141
Microbiology
Notes
Fig. 12.6: MIC and MBC testing. In this example, the minimum inhibitory concentration
(MIC) of the antibiotic is 0.5 mg/l (tube a). The Minimum Bactericidal Concentration
(MBC) is 2.0 mg/l (tube B).
Fig. 12.6: Determination of MIC by E-test. A – Zone of inhibition; B – Bacterial growth;

C – E-test strip; D – the MIC is the point at which the edge of the zone
crosses the E – test strip – in this example it is 3 mg/l.
142 MICROBIOLOGY
Minimum bactericidal concentration (MBC) Microbiology
The MBC is the lowest concentration of the antibiotic that will ‘kill’ a bacterial
strain. The definition of ‘killing’ is a 99.9% (3 Iog10) reduction in viable
bacteria. The MBC test is an extension of an MIC test (fig. 12.6). The simplest
method for determining the MBC is to perform a subculture from antibiotic
concentrations with no visible growth in the MIC test on to antibiotic-free agar.
This will determine whether the bacteria have been inhibited from growing but
are still viable, or whether they have been killed. Notes
Some antibiotics are highly bactericidal. In this case the MIC and MBC are
usually very similar. Bacteristatic antibiotics on the other hand have much
higher MBC than MIC. Occasionally a bacterial strain may have a high MBC
but low MIC with a normally bactericidal antibiotic (e.g. Penicillin). This is
described as bacterial ‘tolerance’ to the antibiotic.
MBC tests are very difficult to standardize and are often not entirely reproducible.
The clinical relevance of MBC tests and the demonstration of tolerance is less
clear than with MIC determinations, but they are occasionally performed to
guide antibiotic therapy in some difficult cases of infection.
Detection of bacterial resistance mechanisms
An alternative method for guiding appropriate antibiotic therapy is through the
detection of bacterial resistance mechanisms. These can be used to predict the
results of conventional sensitivity tests, especially when a specific resistance
mechanism is detected. Often these tests do not require overnight incubation,
and thus the results may be available at an earlier stage to guide treatment.
Some common examples of bacterial resistance detection used in clinical
laboratories are given in table 12.1. This type of approach is likely to become
increasingly used, especially as molecular techniques become more widely
available.
Table 12.1: Examples of the detection of bacterial resistance mechanisms
Resistance Organism Method of detection Comment
mechanism
ß-Lactamase Haemophilus Rapid ‘stick’ test Predicts resistance to
production influenza neisseria (hydrolysis of ampicillin and
gonorrhoeae nitrocefin)Rapid ‘stick’ test amoxicillin predicts high
(hydrolysis of nitrocefin) level resistance to
penicillin
Methicillin Staphylococcus Latex agglutination for Predicts resistance to ?-
resistance aureus pbp2?detection of meca Lactam antibiotics
(altered pbp2?) gene by pcr (mrsa)
Rifampicin Mycobacterium Detection of rpo b gene Detects 95% of

resistance tuberculosis mutations by pcr rifampicin-resistant m.
Tuberculosis strains
PBP- Penicillin Binding Protein; MRSA - Methicillin Resistant Staphylococcus aureus.
MICROBIOLOGY 143
Microbiology Automated sensitivity tests

There are a variety of commercially available automated systems available to
help reduce the technical time required to perform and record routine sensitivity
tests. For example, the results of disk sensitivity tests and breakpoint sensitivity
tests can be read using a camera interfaced to a computer system. Other
Systems utilize liquid cultures, and detect the effect of antibiotics on the rate
Notes of bacterial growth through measurement of turbidity (nephelometry) or the
production of co2. These automated systems can significantly shorten the
necessary incubation time, with the possibility of some results being available
within the same working day. They can also significantly reduce the time taken
to produce sensitivity results for slow-growing organisms, notably mycobacterium
tuberculosis.
Clinical relevance of in vitro antibiotic sensitivity tests

It must be remembered that in vitro sensitivity tests are only a guide to the
appropriate antibiotic treatment. A laboratory report indicating that organism A,
is resistant to antibiotic B does not necessarily mean that antibiotic B will not
work, and vice versa. Whilst in vitro tests are designed to try and reflect the in
vivo situation (e.g. through utilization of appropriate breakpoints to reflect
antibiotic pharmacokinetic parameters), they can never take account of all the
human and bacterial biological variables.
There are a number of factors to consider when interpreting laboratory reports

that include sensitivity test results:
(i) many infections will resolve spontaneously (assuming a normal immune

system). If a patient has already responded clinically to a certain antibiotic
treatment, then it is not always necessary to change the antibiotic if the
laboratory report indicates that the organism isolated is ‘resistant’.
(ii) the organism identified on the laboratory report may not be the primary
pathogen.
(iii) an organism reported with sensitivity results does not always require
treatment. A good example of this is with catheter-specimens of urine.
Treatment is generally required only if the patient is symptomatic (i.e. Treat
the patient not the result!).
(iv) an antibiotic with apparent in vitro activity may not work clinically, as there
are many pharmacokinetic and other factors to consider in choosing the
most appropriate antibiotic therapy
144 MICROBIOLOGY
Microbiology

1. ß lactamase production is detected by ....................... test
2. Methcillin resistance is detected by ....................... test
3. Rifampicin resistance is seen in ....................... infection
Notes
4. Bacterial stains having high MBC with low mic is described as ...................
to antibiotics

z Clinically effective antimicrobial agents exhibit selective toxicity towards
bacterium
z Therapeutic index is the ratio of dose toxic to host to the effective
therapeutic dose.
z Antibiotics are bactericidal, if they kill the susceptible bacteria or
bacteriostatic, if they inhibit the growth of bacteria
z Antibiotics diffuses out of a disc placed on the surface of agar
z Bacteria resistant to antibiotic at breakpoint concentration will then grow
z Minimum inhibitory concentration is the lowest concentration of antibiotic
that will inhibit the growth of bacterial strain.
z Minimum bactericidal concentration is the lowest concentration of antibiotic
that will kill a bacterial strain.
TERMINAL QUESTIONS
1. Describe the difference between MIC and MBC.
2. What are various culture media that can be used for Antibiotic Susceptibility
Test
3. Describe in brief various methods for antibiotic susceptibility testing.
MICROBIOLOGY 145
Microbiology
12.1
1. (c)
2. (e)
Notes
3. (a)
4. (f)
5. (b)
6. (d)
12.2
1. Rapid stick
2. Latex agglutination
3. Mycobacterium tuberculosis
4. Tolerance
146 MICROBIOLOGY
Quality Control in Microbiology MODULE
Microbiology
13
Notes
QUALITY CONTROL IN
MICROBIOLOGY
13.1 INTRODUCTION
Quality: Quality means meeting the pre-determined requirements of users for a
particular substance or service.
ISO: International standard Organization
The ISO is one of the leading International Bodies that has brought together
International Community in developing uniform standards for quality in
manufacturing and service sectors.
OBJECTIVES
z escribe terms related to Quality Assurance
z enlist the phases of Quality assurance
z describe the phases of quality Assurance
z appreciate the benefits of quality assurance program
Quality includes the following
Total Quality Management (TQM)
Continuous Quality Improvement (CQI)
Quality Assurance (QA)
MICROBIOLOGY 147
MODULE Quality Control in Microbiology
Microbiology TQM evolved as an activity to improve patient care by having the laboratory
monitor its work to detect deficiency and subsequently correct them.
CQI and PI seek to improve patient care by placing the emphasis s on not
mistakes in the first place.
QA is associated with the three phases of quality assurance
Pre- analytical
Notes Analytical
Post –analytical
Pre-analytical
Specimen collection: The material must be from the actual site of infection. It
should be properly collected with minimum chances of contamination. It should
be collected in a adequate sized sterile container.e.g. In case of tonsillitis the
throat swab should be taken from the inflamed peri-tonsillar fossae. Pus should
be collected from the inflamed area near the margins of the abscess and should
not be collected from the centre of the abscess where the dead and necrotic
material is likely to be there.
Optimal time of Collection of sample must be established to provide the best
chance of recovering the causative micro-organism from the specimen. E.g. In
typhoid fever the blood culture is recommended to be done in the first week of
fever. The WIDAL test should be done in the end of second week of fever. The
specimen should be collected before the administration of any antibiotic. If the
patient is on antibiotics then the specimen should be collected before the next
dose of antibiotic is administered.
A sufficient quantity of the specimen should be collected to perform the tests
required. E.g. 5-10 ml of blood should be collected in the blood culture bottle.
Appropriate collection devices and specimen containers should be used for
the collection of specimen. All containers used for collection of culture specimen
should be sterile. The handling of the containers, while collection of the
specimen, should also be such that the sterility of the container is maintained at
all times.
Labeling of the specimen should be proper to ensure there is no mixing up of
specimen.
Proper selection of culture media should be made to ensure that the pathogenic
organisms are isolated from the specimen. Fastidious organisms like Streptococci
and Meningococci may require blood agar and chocolate agar to be used for
isolation.
Specimen should also be collected for direct microscopy and proper smears
should be made.
148 MICROBIOLOGY
Specimen transportation: The primary objective of the transport of diagnostic Microbiology
specimen, whether within the hospital, from the clinic or externally by mail, or
transportation to a distant reference laboratory, is to maintain the sample in as
near its original state as possible. If prolonged delay is expected before the
specimen can be processed, it is generally preferable to freeze the specimen at -
70°C. Freezing at -20°C may be used for many specimens if the period of storage
is brief. Storage should not be in a frost free refrigerator.
Notes
Transport media: Some transport media’s are available for microbiology
specimen e.g. Stuart’s media, Cary-Blair media.
Specimen receipt and Preliminary observations: Initial observation and
handling of specimen should be performed carefully. While handling the
specimen universal safety precautions should be observed at all times. Personal
protective equipment like gloves and masks should be worn whenever necessary.
The acceptance of specimens includes the following:-
Documentation of essential data in a log book
Visual examination of the specimen for adequacy. Samples which do not meet
the acceptance criteria should be rejected. It is always a good idea do define the
rejection criteria. For example saliva is rejected when sputum sample is
supposed to be collected. A well formed stool is not the proper sample for
hanging drop preparation to look for darting motility of suspected Vibrio cholera
bacteria.
Analytical
Analytical phase includes the following:-
(a) Training of the staff: The quality system is only as good as the staff who
actually work with it. No matter how good the quality system is on paper, if
the theory cannot be translated into practice, quality cannot be achieved.
Training of the staff is essential to achieve the goals of the quality system.
The training must include an understanding of the importance of quality.
Post training support is also essential to ensure continued competence of the
staff.
(b) Microscopic examination of specimen: The microscopic examination of
the clinical specimen is done to assess the presence of pathogenic bacteria,
neutrophils etc. It may also be used to assess the suitability of the specimen
for acceptance or rejection.
(c) Processing of specimen: The proper processing of microbiology specimen
includes the proper selection of culture media, maintaining the optimal
MICROBIOLOGY 149
MODULE Quality Control in Microbiology
Microbiology temperature and atmosphere of incubation and proper characterization of

the isolated pathogen by appropriate biochemical reactions and antibiotic
sensitivity testing.
(d) Monitoring and evaluation: The laboratory management must develop
and implement quality indicators to systematically monitor and evaluate
laboratory’s contribution to the patient care. Assessment of quality through
audits (Internal or External) is a must. The laboratory must participate in an
Notes External quality assurance program. It is also possible to do inter – laboratory
comparisons of test results. Internal quality is also essential to evaluate the
technician competence and the performance of automated equipments.
Post - analytical
a) Reporting of results: Reports of microbiology culture results should be
issued as soon as useful information becomes available. Each laboratory
must establish those results that will be considered “Urgent” or “critical”. In
addition some results may be considered as important but not necessarily
urgent. For example when a pathogenic bacteria is observed in the direct
microscopy examination of cerebro spinal fluid then this is to be considered
as a critical result. The detection of metachromatic granules in a Gram
positive bacilli on Albert’s stain is suggestive of Corynebacterium diphtheria
and hence is considered as a critical result.
b) Analysis of results: It is incumbent on the laboratory director to provide
feedback to the clinician on some parameters of laboratory performance.
Studies on the Turn around time (TAT) and anti microbial susceptibility
patterns is helpful to the clinicians.
Benefits of Quality assurance programs include the following
z Production of quality products and reliable services.
z Motivation factor for the staff to work better.
z Creation of good reputation for the laboratory.
z Prevention of legal suits and associated complications

1. Meeting pre-determined requirement of users is ……………..
2. Uniform standards of quality is developed by ……………..
3. Phases of quality assurance program are …………….., …………….. and
……………...
4. Training of staff is part of …………….. phase of quality assurance program.
150 MICROBIOLOGY
Microbiology

z Quantity is meeting pre-determined requirements of user for a particular
device.
z International standard organization, develops uniform standards for quality
in manufacturing and service sectors.
Notes
z Quality Assurance has three phases namely pre-analytical, Analytical and
post-analytical.
z Pre-analytical phase involves activities during specimen collection, of time
of collection, quantity, collection devices and containers, Labelling of
specimen, selection of culture media, transportation of specimen and
transport media used.
z Analytical phase includes training of staff, microscopic examination of
specimen, processing of specimen, monitoring and evaluation of laboratories.
z Post analytical phase includes reporting of results and analysis of results.
z Main benefit of Quality assurance program is to provide quality products
and reliable services.
TERMINAL QUESTIONS
1. Define quality.
2. Describe briefly the phases of Quality Assurance.
3. Enlist the benefits of Quality Assurance Program.

1. Quality
2. International Standard Organisation (ISO)
3. Pre-analytical, Analytical and post-analytical
4. Analytical
MICROBIOLOGY 151
MODULE Staphyloccous
Microbiology
14
Notes
STAPHYLOCCOUS
14.1 INRODUCTION
Staphylococci are gram positive cocci that occur in groups in cluster. They are
ubiquitous and most common cause of localized lesions in human beings. They
develop resistance to pencillin and other antibiotics
OBJECTIVES
z classify staphylococcus
z describe the morphology of staphylococcus
z discuss the characteristics of staphylococcus
z describe the laboratory diagnosis of staphylococcus
Staphylocci was first observed in human by Von Recklinghausen. Sir Alexander

Oysten established the causative role of coccus in abscesses and other lesions.
He named in staphylococcus which means, staphylo – bunches of grapes, kokkos
means a berry because of the grape like clusters in cultures. Staphylococcae
strains from pyogenic lesions produce yellow colonies and white colonies from
normal skin.
Classification
1. Staphylococcus aureus – gives positive coagulase-test, ferments mannitol
and mostly pathogenic
152 MICROBIOLOGY
Staphyloccous MODULE
2. Staphylococcus epidermidis contains coagulase negative non ferments with Microbiology
mannitol and mostly nonpathogenic
14.2 STAPHYLOCOCCUS AUREUS

A. Morphology
They are spherical in shape which are approximately 1μm in diameter arranged Notes
in grape like clusters. These are non-motile and non-sporing. They are uniformly
Gram Positive
Fig. 14.1
Fig. 14.2
B. Cultural characteristics
They grow readily on ordinary media with temperature ranging from 10-42°C,
optimum being 37°C with pH of 7.4 – 7.6 and they are aerobes
MICROBIOLOGY 153
Microbiology On nutrient agar, the colonies are large (2-4 diameter) circular, convex, smooth,
opaque and easily emulsifiable. Most strains produce pigment optimally at 22°C
and in aerobic cultures which is enhanced by adding 1% glycerol monacetate or
milk in the medium. Colonies on blood agar are similar to that of nutrient agent.
Several selective media containing (8-10% NaCl) like salt-milk agar, salt broth,
Lithium chloride and tellurite helps in isolating S.aeures from specimen of
Notes faeces
Fig. 14.3
C. Biochemical reactions
They ferment many sugars producing acid but not gases. S.auere ferments
mannitol mostly. They are Catalast positive, reduces nitrates to nitrites
Characteristics
Coagulase positive
Greater biochemical activity, ferment mannitol
Produce clear hemolysis on blood agar
Produce a golden yellow pigment
Liquefy gelatin
Produce phosphatase
D. Resistance
They are more resistant nonsporing bacteria. They retain their viability for 3-6
months. Staphylococci may withstand 60oc for minutes, with thermal death point
154 MICROBIOLOGY
of 62oC for 30 minutes. Heat resistant strains may grow even at high Microbiology
temperatures as 45oC. Most strains grow in the presence of 10% NaCl and some
even in 15% NaCl
Staphylococci were uniformly sensitive to penicillin and some strains produce
pencillinase. Pencillinase resistant are of three types namely
z Produce beta lactamase (pencillinase) which inactivates penicillin by
splitting the beta lactam ring. Staphylococci produce four types of pencilinases
A to D & hospital stains are usually type A pencillinase Notes
z Changes the bacterial surface receptors reducing binding of beta lactam

antibiotics to cells. This also covers beta lactamase resistant pencillins such
as Methicillin and Cloxacillin. They are called Methicillin Resistant
Staphylococcus Aureus (MRSA). As methicillin is a unstable drug cloxacillin
is used for sensitivity testing
z Development of tolerance to pencillin, by which the bacterium is only
inhibited but not killed
z Staphylococci shows resistances to all clinical useful antibiotics like
erythromycin, tetracycline, aminoglycosides and hence vancomycin is
found useful

1. Staphylococci are gram ................ cocci
2. Staphylococci are facultative ................
3. Staphylococci occur in ................
4. Staphylococci produce ................ colour colonies in pyogenic lesions
5. Staphylococcus aureus are coagulase ................ and ................ mannitol
6. Staphylococcus epidermidis are coagulase ................ and ................ mannitol
14.3 PATHOGENICITY AND VIRULENCE

Staphylococci produce two types of disease infections and intoxication
The virulence factors include
(i) Cell associated polymers – cell wall polysaccharide offeres rigidity and
structural integrity to bacterial cell
(ii) Cell surface proteins
Protein A present on S.aureus strains induces platelet damage and
hypersensitivity. Protein A binds to Fc terminal of IgG molecule, leaving fab
MICROBIOLOGY 155
Microbiology region free to combine with its specific antigen. Protein A bearing staphylo-
cocci coated with any IgG antiserum will be agglutinated if mixed with its
corresponding antigen. This is known as coagulation.
Clumping factor
Surface protein, bound coagulase is responsible for slide coagulase test.
When a saline suspension of S.aureus is mixed on a slide with a drop of
Notes human plasma the cocci are clumped. Slide coagulase test is routinely used
for identification of S. aureus
(iii) Extracellular enzymes
Lipases-lipd hydrolases helps S.aureus infect the skin and subcutenous
tissues. Hyaluronidase breaks down the connective tissue. Staphylokinase
helps in initiating and spread of infection.
Nuclease a heat stable nuclease is a characteristic feature of Staphylococcus
aureus
Protein receptors, Staphylococci possess receptors for many mammalian
proteins such as fibronectin, fibrinogen, IgG and C1q. these facilitate
staphylococcal adhesion to host cells and tissues.
(iv) Toxins
Cytolytic toxins are membrane active substance consisting of heamolysin
namely Alpha hemolysin, Beta, Gamma, and Delta & Leucocidin.
Enterotoxin
This is responsible for manifestations of Staphlococcal food poisoning like
nausea, vomiting and diarrhea. The toxin is heat stable resisting at 100oc for 10-
40 minutes. Nearly 2/3 strains frowing in carbohydrate & protein secrete toxins.
Meat, fish, milk and milk products are common items of source of infection. The
source of infection is usually food handlers who are carriers. The illness is
usually self limiting.
Toxic Shock syndrome Toxin (TSST)

Toxic Shock syndrome Toxin is a positively fatal multisystem disease presented
with fever, hypotension, myalgia, vomiting, diarrhea, mucosal hyperemia and an
erythematous rash.
Exfoliative (epidemolytic) toxin

This causes staphylococcal scalded skin syndrome (SSSS), a exfoliative skin
disease in which the outer layer of the epidermis gets separated from the
156 MICROBIOLOGY
underlying tissues. The severe form of the disease is known as Ritter’s disease in Microbiology
the newborn and toxic epidermal necrolysis in older patients. Milder forms are
pemphigus neonatorum and bullous impetigo.

1. Virulence factors of staphylococci are ............., ............., ............. & Notes
.............
2. ............. causes staphylococcal food poisoning
3. ............. toxin causes staphylococcal scalded skin syndrome
4. Severe form of staphylococcal scalded skin syndrome is ............. in children
14.4 STAPHYLOCOCCAL DISEASE

Staphylococcal infections are among the most common of bacterial infections
and range from the trivial to the fatal. They are characteristically localized
pyogenic lesions, in contrast to the spreading nature of streptococcal infection.
Common staphylococcal infections are as follows
Region Infections
Skin and soft tissue Folliculitis furuncle(boil), abscess, wound
infection, carbuncle, impetigo, paronychia
Muscloskeletal Osteomyelitis, arthritis, bursitis, pyomyositis
Respiratory Tonsillitis, pyaryngitis, sinusitis, otitis,
bronchopneumonia, lung abcess, empyema,
rarely pneumonia
Central nervous system Abscess, meningitis, intracranial
thrombophlebitis
Endovascular Bacteremia, septicemia, pyemia, endocarditis
Urinary Instrumentation, implants and bacteria related
Bacteremia
Bacteriophage typing
Staphylococci may be typed, based on their susceptibility to bacteriophages and
the typing is done in pattern method. The strain is inoculated on a plate of
nutrient agar to form a lawn culture. After drying, the phages are applied over
marked squares in a fixed dose. After overnight incubation, the culture will be
observed to be lysed by some phages but not by others. The phage type of the
MICROBIOLOGY 157
Microbiology strain is expressed by the designations of all the phages that lyse it. Phage typing
is of great importance in epidemiological studies of staphylococcal infections.
Laboratory Diagnosis
Specimen Collection
The specimens to be collected depend on the type of lesion, like pus from
Notes suppurative lesions, sputum from respiratory infection. In case of food poisoning,
feces and the remains of suspected food should be collected. For detection of
carriers, nasal swab is the usual specimen. Swabs from perineum, pieces of hair
and umbilical stump are taken.
Direct Microscopy
Direct microscopy with Gram stained smears is useful in the case of pus, where
cocci in clusters may seen. Diagnosis may be readily made by culture. The
specimens are plated on blood agar. Staphylococcal colonies appear after
overnight incubation. Specimens where staphylococci are expected to be scanty
and outnumbered by other bacteria, selective media like Ludlams or salt-milk
agar or Robertson’s cooked meat medium containing 10 percent sodium chloride
may be used for inoculation. Smears are examined and coagulase test done when
staphylococci are isolated
Biochemical Test
The coagulase test can be done using two methods, tube and slide. The tube
coagulase test detects free coagulase. About 0.1ml of a young broth culture or
agar culture suspension of the isolate is added to about 0.5ml of human or rabbit
plasma in a narrow test tube. EDTA, oxalate or heparin may be used as the
anticoagulant for preparing the plasma. The tubes are incubated in water bath at
37oc for 3- 6 hours. If positive, the plasma clots and does not flow when the tube
is tilted.
The slide test detecting bound coagulase is much simpler and usually gives
results parallel with the tube test. When there is divergence, the tube test will be
the deciding factor. For the slide test, the isolate is emulsified in a drop of saline
on a slide. After checking for absence of autoagglitination, a drop of human or
rabbit plasma is added and mixed. Prompt clumping of the cocci indicated a
positive test. Positive and negative controls also are set up. Antibiotic sensitivity
tests should be performed as a guide to treatment.
Coagulase Negative Staphylococci

Coagulase negative staphylococci constitute a major component of the normal
flora of the human body, whereas some like staph epidermidis, staph haemolyticus
and staph saprophyticus cause disease. Staph epidermidis is a normal flora of the
158 MICROBIOLOGY
skin but may cause disease when the host defences are compromised. It Microbiology
commonly causes stitch abcesses, and may grow on foreign bodies such as
artificial heart valves, intravascular catheters and prosthetic appliances causing
bacteremia.
Staph saprophyticus is also a normal flora present on normal skin and
periurethral area and can cause urinary tract infection in sexually active young
women. Notes
Characteristics Staph aureus Staph epidermidis Staph saprophyticus
Coagulase + - -
Novobiocin sensitivity S S R
Acid from mannitol + - -

Anaerobically
Phosphatase + + -
S – Sensitive R – Resistant

1. ............... typing is of great importance in epidemiological studies of
staphylococcal infections
2. ............... medium is used for inoculation of staphylococcal infections
3. ............... coagulose test detects free coagulase
4. ............... coagualose test detects bound coagulase

z Staphylococcus are spherical shaped, nonmotile, and facultative anaerobes
z Positive to catalase test by Gram stain,
z Coagulase positive are staphylococcus aureus and coagulase negative
staphylococcus saprophyticus, staphylococcus epidermidis.
z Staphylococci are susceptible to penicillinase resistant penicillins such as
methicillin and cloxacillin and to aminoglycosides and macrolides. Methicillin
resistant staphylococcus aureus cause nosocomial infections.
MICROBIOLOGY 159
Microbiology
TERMINAL QUESTIONS
1. Describe the morphological characteristics of Staphylococcus
2. Discuss the laboratory diagnosis of Staphylococcus
3. Explain the pathogenecity of Staphylococcus
Notes 4. Describe Methicillin Resistant Staphylococcus Aureus
ANSWERS TO INTEXT QUESTION
14.1
1. Positive
2. Anaerobes
3. Clusters
4. Yellow
5. Positive & ferments
6. Negative & non-ferments
14.2
1. Cell polymers, cell surface protein, toxins & extracellular enzymes
2. Enterotoxin
3. Exfoliative
4. Ritter’s disease
14.3
1. Phage
2. Robertson’s cooked meat
3. Tube
4. Slide
160 MICROBIOLOGY
Streptococcus MODULE
Microbiology
15
Notes
STREPTOCOCCUS
15.1 INTRODUCTION
Streptococci
Streptococci are Gram-positive cocci arranged in chains or pairs. They are part of
the normal flora of humans and animals. Some of them are human pathogens.
The most important of them is Streptococcus pyogenes causing pyogenic
infections, with a characteristic tendency to spread,
as opposed to staphylococcal lesions, which are
typically localized. It is also responsible for the
nonsuppurative lesions, acute rheumatic fever
and glomerulonephritis which occur as sequelae
to infection.
Cocci in chains were first seen in erysipelas and
wound infections by Billroth (1874), who called
them Streptococci (streptos, meaning twisted or
Fig. 15.1
coiled). Ogston (1881) isolated them from acute
abscesses, distinguished them from staphylococci and established their
pathogenicity by animal inoculation. Rosenbach (1884) isolated the occci from
human suppurative lesions and gave them the name Streptococcus pyogenes.
OBJECTIVES
z describe the morphological and physiological characteristics of bacteria in
the genus Streptococcus
z list the feature by which Streptococcus pyogenes, Streptococcus agalactiae,
Streptococcus mutans and Entercoccus (formerly Streptococcus) faecalis
can be identified.
MICROBIOLOGY 161
MODULE Streptococcus
Microbiology z describe the spectrum of diseases caused by the various streptococci.·

z describe & compare suppurative & nonsuppurative Infections of streptococci.
15.2 CLASSIFICATION
STREPTOCOCCI
O2 requirement
Notes
Aerobes and Obligate anaerobes
facultative Peptostreptococci
anaerobes
Hemolysis
1. Alpha (the viridans group) 2. Beta (the hemolytic 3. Gamma (the

Classified into species by streptococcoi enterococcus group)
physiological and Classified into species
biochemical properties by Physiological and
Serological Group-specific C biochemical properties
carbohydrate antigen
20 Lancefield groups (ABCDEFGHKL
MNOPQRSTUV)
Group A - Streptococcus pyogenes
Serological typing (M protein)

Griffith types (1, 2, 3, etc.)
Several systems of classification have been employed but in medical bacteriology

the following method is useful.
Streptococci are first divided into obligate anaerobes and facultative anaerobes.
The former are designated peptostreptococci and are discussed in a later chapter.
The aerobic and facultative anaerobic streptococci are classified on the basis of
their hemolytic properties. Brown (1919) categorized them into three varieties
based on the growth in 5% horse blood agar pour plate cultures.
α) hemolytic streptococci
1. Alpha (α
Alpha (α) hemolytic streptococci produce a greenish discolouration with partial

hemolysis around the colonies. The zone of lysis is small (1 or 2 mm wide) with
indefinite margins, and unlysed erythrocytes can be made out microscopically
within this zone. These are known as ‘viridans streptococci’ or Streptococcus
viridians (from ‘viridis’ meaning green). The alpha streptococci are normal
commensals in the throat, but may cause opportunist infections rarely.
Pneumococcus (Streptococcus pneumonia) is also an alpha hemolytic
streptococcus.
162 MICROBIOLOGY
β) hemolytic streptococci
2. Beta (β Microbiology
Beta (β) hemolytic streptococci produce a sharply defined, clear, colourless zone
of hemolysis, 2-4mm wide, within which red cells are completely lysed. The
term ‘hemolytic streptococci’ strictly applied only to beta lytic strains. Most
pathogenic streptococci belong to this group.
3. Gamma (γγ) or nonhemolytic streptococci
Notes
Gamma (γ) or nonhemolytic streptococci produce no change in the medium and
so are sometimes referred to as ‘indifferent streptococci’. They include the fecal
streptococci (enterococci, Str faecalis) and related species. They are called the
‘enterococcus group’.
Hemolytic streptococci were classified by Lancefield (1933) serologically into
groups based on the nature of a carbohydrate (C) antigen on the cell wall. These
are known as Lancefield groups, twenty of which have been identified so far and
named A-V (without I and J). The great majority of hemolytic streptococci that
produce human infections belong to group A. Hemolytic streptococci of group A
are known as Streptococcus pyogenes. These may be further subdivided into
types based on the protein (M, T and R) antigens present on the cell surface
(Griffith typing). About eighty types of Str pyogenes have been recognized as far
(types 1,2,3 and so on).
Table 1 shows the medically important streptococci and their chacteristics.
15.3 STREPTOCOCCUS PYOGENES

Morphology : The individual cocci varies in size from 0.5 – 1.00 mm in
diameter whereas they are spherical and oval in shape. They are arranged in
chains because chain formation is due to cocci dividing in one plane & daughter
cell failing to separate completely. Streptococcus has been classified as Str.
longus (long chain) & Str. breyis (short chain.). Streptococcus are gram positive,
non-motive, non-sporing and capsulated.
Culture Characteristics : It is an aerobes & facultative anaerobes growing best
at 37°C (range 22 – 42). It is exacting in nutritive requirements, growth occurring
in media containing fermentable carbohydrate & enriched with blood & serum.
When cultured on blood agar it gives colonies which are circular, semitransparent,
low convex disc with area of clear hemolysis around it. Virulent strain on fresh
isolation from lesion, produce a ‘matt’ (finely granular) colony, while avirulent
strains from ‘glossy’ colonies. Strains with well marked capsules produce
‘mucoid’ colonies, corresponding in virulence to the matt type. When cultures in
liquid medium like glucose or serum broth it shows granular turbidity with
powdery deposit. No pellicle is formed.
MICROBIOLOGY 163
Microbiology Table 15.1: Medically important streptococci and their characteristics
Species or Lancefield Hemolysis Habitat in Laboratory Common

Common group human hosts tests diseases caused
name
Str. pyogenes A Beta Throat, Bacitracin URTI,

Skin sensitive; Pyoderma, RF,
PYR test Glomerul-
Notes positive; onephritis
Ribose not
fermented
Str. agalactiae B Beta Female Nenonatal

genital tract, meningitis,
rectum septicemia
Str. equisimilis C Beta Throat Pharyngitis,

endocarditis
Str. anginosus A,C,F,G Beta Throat, Pyogenic

Untypable (alpha, colon, female infections
gamma) genital tract
Enterococcus D Gamma Colon UTI,

sp (Str faecalis (alpha, endocardities
and other beta) suppurative
enteroccci) infections
Nonenter-
ococcal Group D Gamma Colon Endocardities
D species (str Alpha
bovis) (gamma)
Viridans Not types Endocarditis

Mouth, colon, Optochin
streptococci (Str. sanguis);
female resistant,
(many species) dental caries (str.
genital tract species
classsification mutans)
biochemical
properties

1. Cocci in chains were first seen in which infections ?
2. What is the basis for classification of Hemolytic streptococci ?
3. Which type of colonies are produced by Beta Hemolytic Streptococci ?
164 MICROBIOLOGY
4. What type of colonies formed by Virulent & Avirulent strain of streptococcus? Microbiology
5. Which is the Common Causative URTI Organism for ?
Biochemical reaction : Streptococci ferments all the sugars like Sorbitol,

Maltose, Lactose & Mannitol producing acid but no gas. It is Catalase negative.
Resistance : Str. Pyogenes is a delicate organism, easily destroyed by heat (54°C
for 30 minutes). It dies in a few days in cultures, unless stored at a low Notes
temperature (4°C), preferably in Robertson’s cooked meat medium. It can,
however, survive in dust for several weeks if protection from sunlight. It is
rapidly inactivated by antiseptics. It is more resistant to crystal violet than many
bacteria, including Staph aureus.
Toxins, Enzymes & other virulence factors : Streptococcus pyogenes
produces several types of exotoxins & enzymes those act as virulence factors.
Also young protein act as a virulence factor by inhibiting phagocytosis. The C
polysaccharide has been shown to have a toxic effect on connective tissue in
experimental animals.
Hemolysins : Streptococci produce two hemolysins, streploysin ‘O and ‘S’.
Streptolysin O is so called because it is oxygen labile. It is inactive in the
oxidized form but may be reactivated by treatment with mild reducing agents. On
blood agar, streptolysin O activity is seen only in pour plates and not in surface
cultures. It may be obtained in the active state by growing streptococci in broth
containing reducing agents such as sodium hydrosulphite. It is also heat labile. It
appears to be important in contributing to virulence. It is lethal on intravenous
injection into animals and has specific cardiotoxic activity. It has leucotoxic
activity also. In its biological action, streptolysin O resembles the oxygen labile
hemolysins of Cl. perfringens, Cl. tetani and the pneumococcus.
Streptolysin O is antigenic and antistreptolysin O appears in serum after
streptococcal infection, which is very important in diagnosis. Streptolysin S is so
called because it is soluble in serum. It shows stability with oxygen, dry heat. It
is responsible for hemolysis seen on the surface of the blood agar plates. It also
has leucocidal activity.
Pyrogenic exotoxin (erythrogenic or dick or scarletinal toxin) is a toxin named
erythrogenic because its intradermal inoculation in susceptible individual causes
erythmatous reaction. This test is known as “dick test’. This test is used to
identify the children susceptible to scarlet fever so, named as “Scarletinal toxin”.
This toxin induces fever so named as streptococcal pyrogenic exotoxin. It has
three types A, B, C. It is a super antigen so act as T-cell mitogens and causes rapid
release of inflammatory cell which cause wide spread manifestation.
MICROBIOLOGY 165
Microbiology Streptokinase is a enzyme which act as a toxin which promotes the lysis of
human fibrin clot by activating plasminogen to plasmin. It act as a diagnostic
marker as it antigenic in nature so antibodies are produced in convalescent area.
This is helpful in retrospective study. It shows biological significance during
infection, by breaking down the fibrin barrier around tissue & thus helps in
spread of infection. It shows therapeutic significance in myocardial infarction &
other thromboembolic disorders.
Notes Deoxyribonuclease (streptodornase, DNAase) : It is a enzyme which causes
depolymerisation of DNA also showing diagnostic significance as Streptokinase.
It shows biological significance because pyogenic exudates contain large
amount of DNA. DNAase causes liquefaction of pus & its serus character. It also
liquefy thick pus in empyema which is a therapeutically important.
NADase (Diphosphopyridine necleotidase) : It is a enzyme which acts on
NAD & liberates nicotinamide from it. It is a diagnostic as it is a antigenic in
nature. Biological significance is not known.
Hyaluronidase : It is a enzyme which breaks down the hyaluronic acid of tissue.
It is a biological significance it helps in spread of infection.
Serum opacity factor : Some M types of Str. Pyrogen produce lipoproteinase
that produces opacity when applied to agar gel containing the horse serum. That
is known as Serum opacity factory.
Antigenic Structure : The capsule when present inhibitis phagcocytosis. It is
not antigenic in human beings. The cell wall is composed of an outer layer of
protein and lipoteichoic acid, a middle layer of group-specific carbohydrate and
in inner layer of peptidoglycan. The peptidoglycan (mucoprotein) is responsible
for cell was rigidity. It also has some biological properties such as pyrogenic and
thrombolytic activity. Serological grouping of streptococci depends on the C
carbohydrate. Str pyogenes belongs to group A. As this antigen is an integral part
of the cell wall, it has to be extracted for grouping by a precipitation test with
group antisera. Several protein antigens have been identified in the outer part of
the cell wall. Str pyogenes can be typed based on the surface proteins M, T and
R. The M Protein is the most important of these. It acts as a virulence factor by
inhibiting phagocytosis. It is antigenic. The antibody to the M protein promotes
phagocytosis of the coccus and is therefore protective. The M protein is heat and
acid stable but susceptible to tryptic digestion. It can be extracted by the
Lancefield acid extraction method and typing is done with type-specific sera.
The T protein is an acid labile, trypsin resistant antigen present in many serotypes
of Str pyogenes. It may be specific but many different M types possess the same
T antigen. It is usually demonstrated by the slide agglutination test using trypsin-
treated whole streptococci. Hair-like pili (fimbria) project through the capsule of
group A streptococci. The pili consist partly of M protein and are covered with
lipoteichoic acid which is important in the attachment of streptococci to
epithelial cells.
166 MICROBIOLOGY
Pathogenecity Microbiology
Str. Pyrogen produces pyrogenic infection that spread locally along with
lymphatic & blood serum. They produce mainly two types of lesions.
Suppurative infection Non-suppurative

Notes
Respiratory Infection Acute rheumatic fever
Skin and soft issue Acute glomerulonepheritis
Genital (puerperal sepsis)
Abscess in liver, lung.
Kidney and brain
Respiratory Infection: The primary site of invasion of the human body of Str
pyogenes is the throat. Sore throat is the most common of the streptococcal
diseases. It may be localized as tonsillitis of may involve the pharynx more
diffusely (pharyngitis). Virulent group A streptococci adhere to the pharyngeal
epithelium by means of lipoteichoic acid covering the surface pili. The
glycoprotein fibronectin on the epithelial cells probably serves as the lipoteichoic
acid ligans. Tonsillitis is more common in older children and adults than in
younger children, who commonly develop diffuse pharyngitis. Localisation is
believed to be favoured by hypersensitivity due to prior contact.
Chronic Tonsilitis
From the throat, streptococci may spread to the surrounding tissues, leading to
suppurative complications such as otitis media, mastoidities, quinsy, Ludwig’s
angina and suppurative adenitis.
Skin and soft tissue infections

Str pyogenes causes a variety of suppurative infections of the skin, including
infection of wounds or burns, with a predilection to produce lymphangitis and
cellulitis. Infection of minor abrasions may at times lead to fatal septicemia.
The two typical streptococcal infections of the skin are Erysipelas and Impetigo.
Erysipelas : It is a diffuse infection involving the superficial lymphatics. The
affected skin, which is red, swollen and indurated, is sharply demarcated from
the surrounding healthy area. While erysipelas is rare and seen only in older
patients, impetigo is found mainly in young children. The skin area shows
erythema with edema. One attack does not give protection & recurrent infection
in same area occurs in some person.
Impetigo : It is caused by Str pyogenes it is a superficial crushed spot, especially
in children usually less than 1 Inch in diameter. Impetigo and streptococcal
infection of scabies lesions are the main causes of acute glomerulonephritis in
MICROBIOLOGY 167
Microbiology children in the tropics. It last for 1-2 week. It heals spontaneously without
leaving scar.
Genital Infections : Both aerobic & anerobic streptococci are normal inhabitants
of female genital tract. They are important causative organism of puerperal
sepsis.
Notes
Endogenous route Exogenous route
from outside the body from inside the body
Other suppurative infections : Str pyogenes may cause abscesses in internal

organs such as the brain, lungs, liver and kidneys, and also septicemia and
pyemia.
Nonsuppurative complications : Str pyogenes infections lead to two important
nonsuppurative sequelae – acute rheumatic fever and acute glomerulonephritis.
Acute rheumatic fever Acute Glomerulonephritis

1 Site of infection Throat Skin or throat
2 Latent period Longer (> 2-5 week) Shorter (1-3 weeks)
3 Prior sensitization Essential Not necessary
4 Serotypes Any Pyodermal types
(49, 52, 53, 54, 57, 61)
Throat infection types
(12, 21, 25)
5 Repeated attack Common Absent
6 Manifestation Swelling of joints & pancarditis. Hematuria, albuminuria & Edema.
7 Pathology Connective tissue disease Increases cellularity of Glomerulus
Typical lesion : with larger Deposit on outer
Aschoff's nodules membrane & smaller deposit on
inner Membrance of GBM.
8 Pathogenesis Antibody produced against Mechanism is not clear.
protein & polysaccharide,
which cross-react with
myocardial & heart valve
tissue.
9 ASO titer Markedly raised Moderately raised or low
10 Complement level Unaffected Spontaneous resolution
11 Course Progressive/static Spontaneous resolution
12 Prognosis Variable Good
13 Penicillin Essential Not indicated
Prophylaxis
168 MICROBIOLOGY
Laboratory diagnosis of streptococci : In acute infections, diagnosis is Microbiology
established by culture, while in the nonsuppurative complications, diagnosis is
mainly based on the demonstration of antibodies.
Notes
Fig. 15.2: Fluorescent antibody technique
The sample collection require for acute conditions are throat swab, pus or blood
for isolation of Str. Pyogen & Vaginal Swab, blood, CSF, ear swab for Str.
Agalactiae, Urine & blood for enterococci.
There are different methods of demonstrating organisms in direct method of
organisms.
In Microscopy Gram Staining is done for Gram Positive Cocci which formed
chain and non motile which is indicative of Streptococci. Microscopy don’t have
any value in throat & genetal infections because these streptococci are part of
resident flora.
Culture : In this method different media is used. In Spikes (pike’s) medium it is
blood agar containing in 10,00,000 crystal violet & 1 in 16,000 sodium azide. It
is a transport medium. While Blood agar is a most common culture medium used
for isolation. A specimen is collected under Aseptic Precaution. It is transported
a lab in pike’s medium. It is plated on blood agar & incubated at 37°C
anaerobically with CO2. Colonies & hemolysis appear. Seriological testing for
definite Lancefield group & Griffith typing is done. Sheep blood agar is
preferable as it is inhibitory to hemophilus hemolyticus, which may be confused
with colonies of streptococci. For rapid identification of Str . pyrogen Maxtod’s
method is used. In Maxtod method Str. progen is more sensitive to bacitracin
than others in which filter paper disc soaked in solution of bacitracin is applied
on the inoculated blood agar, which shows wide zone of inhibition.
MICROBIOLOGY 169
Microbiology Antigen detection, Serologic test & Typing are used as indirect method for
demonstrating organisms. In antigen detection rapid diagnostic test kits are used
in which streptococci A antigen is available but Serologic test is not helpful in
acute infection for detection of antibody. Typing is required for epidemiological
purposes.
For Diagnosis of Nonsuppurative conditions like Acute rheumatic fever & Acute
Glomerulonephritis. Aim is Demonstration of high levels of antibody against
Notes streptococcal toxins. Streptococci produce two types of hemolysin streptolysin
O & streptolysin S. Streptolysin O is antigenic & antibody against it
(antistreptolysin O) appears in serum. Estimation of ASO titer in serum is the
standard procedure for diagnosis. ASO titer is raised in few nonsuppurative
conditions like Penumococcal pneumonica, Tuberculosis, Gonorrhea, Hepatitis
& Rheumatoid arthritis.
Estimation of Anti-DNAase B titer is considered significant when is it a more
than 300-350.
Anti-Hyaluronidas test is important in pyoderma where ASO is not important.
Streptozyme test is passive slide hemagglutination in which erythrocytes
sensitize with extracellular antigen of streptococci added to patient’s serum. It
produces agglutination, which is taken as positive test. This test is positive in all
types of streptococcal infections.
15.4 OTHER HEMOLYTIC STREPTOCOCCI

Group B (Str. agalactiae)
It is single most cause of Neonatal meningitis is west.
Their ability to hydrolyze hippurate acts as presumptive identification method.
Group C
Streptococci of this group are mainly animal pathogen & divided into four
species.
Group C pathogens from human sources are mainly str. quisimilis species.
It causes upper Respiratory tract infection as well as deep infection.
It differs from Str. pyrogen that it ferments ribose
It is commercial source of thrombolytic therapy.
Group F
These group poorly on blood agar unless incubated under CO2 they have been
called “minute streptococci”. One member of this group is Streptococcus MG
which is an alphalytic strain isolated from cases of primary atypical pneumonia.
170 MICROBIOLOGY
Group G Microbiology
These are commensals in the throats of human beings, monkeys or dogs. They
may occasionally cause tonsillitis, endocarditis and urinary infections in human
beings.
Group D
They mainly of two types Notes
Entercocci (E.faecalis) & Non-enterococci ( Str. bovis, Str. equines)
Entercocci shows Distinctive features is ability to grow in presence of 40% bile,
6.5% sodium chloride, At pH 9.6 & temperature 45OC and in 0.1% methylene
blue milk.
E faecalls is most commond species isolated from human.
It can be indentified by its ability to ferment mannitol, sincrose, sorbitol and
aesculin & to grow on tellurite blood agar producing black colony. It mainly
causes UTI, Wound infection & endocarditis.
Non-enterococci are inhibited by 6.5% sodium chloride & bile they case UTI &
endocarditis.
15.5 THE VIRIDANS GROUP

This group, formerly called Streptococcus viridians, is a miscellany of streptococci
normally resident in the mouth and upper respiratory tract, and typically
producing greening (alpha lysis) on blood suger – hence the name viridians.
Some of them may be nonlytic. They cannot be categorized under the Lancefield
antigenic groups.
They are ordinarily nonpathogenic but can on occasion cause disease. In persons
with preexisting cardiac lesions, they may cause bacterial endocarditis, Str
sanguis being most often responsible. Following tooth extraction or other dental
procedures, they cause transient bacterremia and get implanted on damaged or
prosthetic valves or in a congenitally diseased heart, and grow to form
vegetation. Prophylactic antibiotic cover is advisable in such persons before
tooth extraction or similar procedures. While viridians streptococci are generally
penicillin sensitive, some strains may be resistant. It is therefore essential that in
endocarditis.
Str mutans (so called because it assumes a bacillay form in acid environments) is
important in the causation of dental caries. It breaks down dietary sucrose,
producing acid and a tough adhesive dextran. The acid damages dentine and the
dextrans bind together food debris, epithelial cells, mucus and bacteria to form
dental plaques, which lead to caries.
MICROBIOLOGY 171
Microbiology

1. Which type of Hemolysin produced by Streptococcus ?
2. What is the most common causative organism for Pharyngitis ?
3. Acute & Chronic infection diagnosed by which method ?
Notes
4. Which hemolytic streptococci is known as Minute Streptococci ?
5. Which streptococci is causative of dental caries ?

z Streptococci are Gram-positive cocci arranged in chains or pairs. They are
non-motile and non-sporing organisms
z Streptococci are divided into obligate anaerobes and facultative anaerobes
based on their oxygen requirement.
z Based on hemolytic properties Streptococci are classified into alpha
hemolytic Streptococci, beta hemolytic Streptococci, gamma or non-
hemolytic Streptococci
z Most pathogenci Streptococci belong to beta hemolytic Streptococci group.
z Hemolytic Streptococci of group are known as str. pyogenes
z Streptococus are classified as str. Longus and str Breyis and they are aerobes
and facultative anaerobes
z Biochemically Streptococci fermetts cell organss
z Str pyogenes is a delicate organisms easily destroyed by heat and rapidly
inactivated by antiseptics
z Streptococci produce two hemolysis streptolysi O and S. Streptolysin O is
oxygen labice and streptolysin S is souble in serum.
z Str pyrogen produces supporative infections of respiratory tract, skin and
soft cesians, genital tract, abcess of liver, lung, kidney and brain.
z Non-supporative infections are acute rheumatic fever and glomerubnephritis.
TERMINAL QUESTIONS
1. Describe Morphology & physiological characteristics of streptococcus?
2. Classify the streptococcus on the basis of Hemolysis?
3. Describe medically important Streptococci & their characteristics?
172 MICROBIOLOGY
4. Describe antigenic structure of Streptococci? Microbiology
5. Compare Rheumatic Fever & Acute Glomerulonephritis?

6. Describe Lab Diagnosis of Streptococci?
7. Describe Etiopathogenesis of Soft Tissue Infection?
8. Describe other Hemolytic Streptococci & write their clinical significance?
Notes
15.1
1. Erysipelas and Wound infections
2. Nature of Carbohydrates C
3. Sharply, Clear, Colourless Zone of hemolysis. 2-4mm wide, within which
red cells are completely lysed.
4. Virulent – Matt (Finely Granular), Avirulent – (Glossy Colonies)
5. Str. Pyogenes
15.2
1. Streptolysin ‘O’ & ‘S’
2. Streptococci
3. Acute Infection by culture & Chronic Infection by Demontration of
antibodies.
4. Group ‘F’.
5. Str. Mutans
MICROBIOLOGY 173
MODULE Pneumococcus
Microbiology
16
Notes
PNEUMOCOCCUS
16.1 INTRODUCTION
Pneumococcus, earlier known as Diplococcus pneumoniae (as it occurs in pairs)
is now called Streptococcus pneumoniae in 1974 because it is related to
Streptococcus (growth in chains in liquid media). Pneumococci normally inhabit
the mucosa of the upper respiratory tract which is kind of the natural habitat of
these bacteria. Healthy adults are carriers (approximately 40–70 %) of
Pneumococci. Most of Pneumococcal diseases are endogenous infections i. e
from the mucosa of respiratory tract the pneumococci invade the carrier host and
cause disease.
OBJECTIVES
z describe the classification of Pneumococcus;
z describe the morphological characteristics of Pneumococcus;
z discuss the biochemical and other specific characteristics;
z explain mechanism of virulence, pathogenicity;
z enumerate the diseases caused by Pneumococcus;
z culture and identify Pneumococcus from specimen;
z describe the vaccines available to prevent Pneumococcal infections;
z discuss the vaccine schedule, doses and when to give vaccine.
174 MICROBIOLOGY
Pneumococcus MODULE
Microbiology
16.2 HISTORY
Louis Pasteur and George Sternberg independently discovered Pneumococci in
1888.However, the relationship between Pneumococci and pneumonia was
discovered in 1886 by Fraenkel and Weichselbaum. Organism was named as
Diplococcus pneumoniae because of its paired cocci appearance in Gram
stained smear from sputum. However, later it was found that this organism is
related to Streptococci as explained earlier so the organism was named Notes
Streptococcus pneumoniae.
Frederik Griffith in 1928 demonstrated a phenomenon called “transformation”
wherein he injected a mixture of non-virulent Strept. pneumoniae and killed
virulent Strept. pneumoniae in mice and found that the mice died due to infection
with virulent pneumococci. Later 1944) it was found that the DNA of killed
pneumococci in the mixture entered the non virulent pneumococci and
transformed them into virulent pneumococci. This phenomenon was named
transformation and it marked the beginning of molecular genetics.
16.3 CLASSIFICATION
Pneumococcus belongs to the kingdom bacteria. The classification is given
below:
Class: Bacilli;
Order: Lactobacillales;
Family: Streptococcaceae;
Genus: Streptococcus;
Species: Streptococcus pneumoniae;
Serotypes: I, II, III and heterogeneous group IV (More than 90 different
serotypes are recognized in this group).
16.4 MORPHOLOGY
Pneumococci are Gram-positive, slightly elongated, oval to lanceolate-shaped
diplococci (0.5 and 1.25 micrometers in diameter), usually occur in pairs or short
chains surrounded by a thick capsule. One end of the Pneumococcus is broad and
the other end is pointed giving it the typical lanceolate shape. The broad end of
the cocci in pair is in apposition and pair of cocci is surrounded by a big capsule.
The capsule is most apparent in smears made from exudates (patient sample),
capsule is usually lost in culture.
MICROBIOLOGY 175
MODULE Pneumococcus
Microbiology
16.5 CULTURAL CHARACTERISTICS
Pneumococci are fastidious organisms to grow i. e. they require enriched
medium (blood agar) to grow. The optimum temperature for growth is 37° C
(range is 25-42° C) and pH is 7.8. Pneumococci grow better in an atmosphere
with 5-10% CO2 (culture plates kept in candle jar and incubated).
Specimen is cultured on Blood agar and Chocolate Agar and plates are incubated
Notes as above. Plates are examined for growth after 18 hrs and more. The colonies on
Blood agar are alpha –hemolytic, dome shaped, mucoid (smooth, shiny). The
mutants without capsules produce colonies with a rough surface (“R” form).
Smooth (S) to Rough (R) variation can occur on repeated culture.
Under anaerobic conditions colonies may be surrounded by clearing of medium,
beta haemolysis (due to oxygen labile haemolysin) instead of green discolouration
–the alpha haemolysis. Streptococcus pneumoniae is a very fragile bacterium,
contains within itself the enzymatic (autolysin- autolytic enzyme, Lyt A) ability
to disrupt and to disintegrate the cells. The physiological role of this autolysin is
to cause the culture to undergo a characteristic autolysis that kills the entire
culture when grown to stationary phase. Bile salt enhances autolysis.
Fig. 16.1: Blood agar plate showing alpha haemolysis (greenish colouration)
typical of Pneumococci.
Most clinical isolates of pneumococci undergo lysis mediated by autolysin

between18-24 hours after culture under optimal conditions. Autolysis changes
the colony character from plateau-type morphology to colony with lysed/
depressed center.
16.6 BIOCHEMICAL AND SPECIFIC IDENTIFICATION

CHARACTERISTICS
Pneumococcus is an aerotolerant anaerobe and ferments many sugars. Hiss’s
serum sugars are used for fermentation reaction. Pneumococci hydrolyze inulin
176 MICROBIOLOGY
Pneumococcus MODULE
and this test is used to differentiate Pneumococci from Streptococci. Pneumococci Microbiology
are oxidase and catalase test negative. They do not display an M protein like
some other Streptococci.
The specific characteristics of Pneumococci include bile solubility, optochin
sensitivity and Quellung phenomenon or Capsule swelling reaction. Let us
discuss these one by one.
Notes
16.6.1 Bile Solubility Test
A few drops of 10% sodium deoxycholate solution are added to 1 ml of overnight
broth culture of pneumococci. Clearing of broth culture is seen within few
minutes due to lysis of pneumococci. Other method to do this test is to place a
loopful of 10% deoxycholate solution on the colony of pneumococci on blood
agar-the lysis of colony is seen within few minutes. The test is used to
differentiate Pneumococci from other alpha haemolytic streptococci like Strept
viridans.
Fig. 16.2: Bile solubility test-showing clearing of turbidity due to destruction of

Pneumococci.
16.6.2 Optochin Sensitivity Test

Optochin discs (5 mg ethyl hydrocuprein hydrochloride) are available
commercially. Blood agar plate is inoculated with Pneumococci; the disc is
placed in the center of the plate and is incubated in a CO2 incubator overnight.
The plate is examined the next day and the zone of inhibition around the disc is
measured. An inhibition zone of 15 mm or more means the organism is sensitive
to optochin. The test differentiates Pneumococci from other alpha haemolytic
streptococci.
MICROBIOLOGY 177
MODULE Pneumococcus
Microbiology
Notes
Fig. 16.3: Blood agar plate showing zone of inhibition of Pneumococcal

growth around optochin disc.
16.6.3 Quellung Test or Capsule Swelling Reaction

This test can be performed on specimen like sputum or on plate showing mixture
of organisms. The specimen or material is mixed with a drop of Pneumococcal
polyvalent antiserum ; smear is made and stained. The Pneumococcal capsule
appears to be swollen. Another way to do the test is mix one loopful of bacterial
suspension/specimen with one loopful of polyvalent or type specific anti serum
and a drop of methylene blue staining solution. Mix all three and examine under
microscope. Highly refractile swollen capsule surrounding Pneumococci will be
seen in the presence of specific antiserum. This test is used to identify and
serotype Pneumococci.

1. Process of converting non circular pneumococci into virulent organism is
...........
2. Pneumococci are gram ........... and ........... cocei occuring in pairs.
3. Puenmococi are ........... organisms.
4. ........... serum sugar are used for fermentation reaction.
5. ........... test is used for differectiating pneumococci from Streptococi.
6. Pneumococci are oxidase and catalase ...........
16.7 ANTIGENS
The outermost structure is capsule made of polysaccharide. This polysaccharide
diffuses into medium and into host tissues during infection and is called “specific
soluble substance” (SSS). Capsule plays an important role in virulence which we
will discuss later.
178 MICROBIOLOGY
Pneumococcus MODULE
On the basis of type of polysaccharide Pneumococci are classified into: Microbiology
z Type I;
z Type II;
z Type III;
z Heterogenous group IV. This group has more than 90 different serotypes.
Notes
Other antigens include somatic “C” carbohydrate antigen and the nucleoprotein.
The antigen “C” is used to detect C reactive protein, a beta globulin which is
raised in sera of patients of pneumonia and other diseases where there is
inflammation and breakdown of tissue.
16.8 VIRULENCE AND PATHOGENICITY

Pneumococci are normally present in naso-pharynx of humans; may become
invasive and spread to the surrounding organs like sinuses, middle ear,
respiratory tract and meninges to cause infections of these organs. Pneumococci
produce some weak toxins like haemolysin and leucocidin which are not
virulent; Pneumococci produce a virulent toxin named pneumolysin. Pneumolysin
damages cell membrane, is cytotoxic and may activate complement . This
combined with the anti-phagocytic property of the capsular polysaccharide; all
contribute in pathogenesis of infections and diseases caused by Pneumococci.
Autolysin of Pneumococci lyses the bacteria present in tissues and the bacterial
products released on lysis may also cause harm to tissues and thus may be
involved in causing disease.
So you see the capsule protects the pneumococci from phagocytosis and is the
most important determinant of pneumococcal virulence. Un encapsulated
variants are not capable of causing disease. Other potential virulence factors
include pneumolysin and probably bacterial products released on lysis of
bacteria as already indicated.
16.9 INFECTIONS AND DISEASES CAUSED BY STREPT

PNEUMONIAE
There are certain factors which may predispose to pneumococcal infections.
These include: primary cardiopulmonary diseases, primary respiratory viral
infections (e.g., influenza), extirpation of the spleen (splenectomy) and/or some
complement system defects.
Pneumococci can cause from simple infections like sinusitis to serious, invasive
type of pneumococcal infections (septicemia and meningitis). Respiratory
infections including pneumonia are most commonly caused by pneumococci.
MICROBIOLOGY 179
MODULE Pneumococcus
Microbiology The various infections caused by Pneumococci are listed below;

z Sinusitis;
z Otitis media
z Mastoiditis;
z Lobar pneumonia;
z Bronchopneumonia;
Notes z Acute exacerbation of chronic bronchitis;
z Joint infections;
z Endocarditis;
z Meningitis;
z Bactreamia;
z Septicaemia;
z Abscesses in organs following septicaemia.
z Conjuctivitis
The common symptoms of respiratory Pneumococcal infection are cough, high
fever, difficulty in breathing, rapid breathing and pain in the chest area. The signs
include headache, fatigue, muscle ache, nausea and vomiting. Laboratory
diagnosis for Pneumococci and treatment should be carried out in all suspected
cases of infection with Pneumococci.
16.10 LABORATORY DIAGNOSIS AND

IDENTIFICATION OF PNEUMOCOCCI
Clinical diagnosis of an infection is easy; however, to decide whether the
infection is caused by Pneumococci, we have to do the aetiological diagnosis.
For this purpose the appropriate sample is collected and processed as detailed
below to detect Pneumococci.
Laboratory diagnosis of pneumococcal infection is done as below:

z Collect the appropriate sample from clinically suspected cases of
pneumococcal disease;
z The appropriate sample in respiratory infection is sputum; otitis media-pus/
aspirate from middle ear; blood in case of septicaemia; CSF from a case of
meningitis and so on;
z Perform gram staining on smear prepared from the sample;
z Examine the smear microscopically and look for typical Lancet shaped Gram
positive diplococcic surrounded by a thick capsule;
180 MICROBIOLOGY
Pneumococcus MODULE
z Do slide agglutination test by mixing a drop of the CSF/aspirate, etc. with a Microbiology
drop of commercially available polyvalent and/or locally prevalent serotype
specific antiserum to detect the presence of specific soluble substance (SSS)
in the specimen which points to Pneumococcal serotype causing infection
and treatment can be started right away;
z Culture the sample on blood agar and chocolate agar plates, incubate in 5-10
% CO2, at 37° C overnight (18 hrs); Notes
z Examine the plates for growth, in case of Pneumococci typical colonies
surrounded by greenish discolouration due to alpha haemolysis will be seen
as described above;
z Prepare a smear from the plate, do the Gram staining and examine for typical
Gram positive diplococcic;
z Carry out the bile solubility test, optochin sensitivity test and inulin
fermentation test to confirm the identity of Strept pneumonia;
z Carry out the Latex slide agglutination test by mixing a drop of the culture
suspension with a drop of commercially available polyvalent or locally
prevalent serotype specific antiserum helps to confirm the serotype of
Pneumococci causing infection;
16.11 RESISTANCE
Pneumococci are sensitive to heat (52°C) and commonly used antiseptics. It is
difficult to maintain Pneumococci for long in culture. Pneumococci in the lab
can be maintained by culture on semisolid blood agar and by lyophilization.
16.12 EPIDEMIOLOGY AND PROPHYLAXIS
16.12.1 Epidemiology
The reservoir of Pneumococci is the healthy human carriers and patients
suffering from pneumococcal infections. Pneumococcal infections are endemic
and occur in all seasons, more frequently at extremes of ages, in the elderly and
small children. Infections are more common during the outbreaks of respiratory
viral infections like influenza. Pneumococcus causes secondary infections in
patients suffering from influenza. Outbreaks of Pneumococcal pneumonia can
occur in overcrowding and closed communities.
The incidence of infection also depends on the prevalent serotype of

Pneumococcus. Type 3 is the most virulent so if it is prevalent in the community
then there may be more infections.
MICROBIOLOGY 181
MODULE Pneumococcus
Microbiology 16.12.2 Prophylaxis

Pneumococcal Vaccine is used for prevention of pneumococcal infections in
extremes of ages; individuals with chronic lung, heart and renal diseases;
individuals with non/dysfunctional spleen, celiac disease and so forth. Two types
of Pneumococcal vaccines are available and used. These are polysaccharide and
conjugated pneumococcal vaccines.
Notes The purified polysaccharide vaccine (PPV 23) is a 23 valent vaccine containing
the serotypes - 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C,
19F, 19A, 20, 22F, 23F, 33F. This vaccine is poorly immunogenic in children
below the age of 2 years, has low immune memory, does not reduce
nasopharyngeal carriage and does not provide herd immunity. Efficacy is around
70 % only in the high-risk population. The dose is 0.5 ml administered
subcutaneous/intramuscularly. Vaccine is safe with occasional local side effects.
To improve the immunogenicity and efficacy of pneumococcal vaccine
polysaccharide conjugate vaccines (PCV) was developed by conjugating
Pneumococcal polysaccharide with different proteins.

1. ........... of capsule plays a major role in the virulence of the organism.
2. Pneumococci produce virulence toxin known as ...........
3. ........... Text is used to identify the seotype of pneumococci.
4. Pneumococci are named inhabitat of ..........

z Pneumocci are gram-positive, slightly elongated, oval to lanceolate shaped
diplococci, ocuring in pairs or short chains surronded by capsule
z Pnuemococci are fasitidious organism
z Speciemn is cultured on Blood agar and Chocolate Agar
z Pneumococcus ferments many sugars. Pneumocci hydrolyze inulin, oxidase
& catalase test negative
z Spccific characteristics of pneumococcus are bile solubility, optochin
sensitivity & Quellung phenomenon or capsule swelling reaction
z Capsule is made of Ploysaccharide which is called as Specific Solubel
Substance (SSS)
182 MICROBIOLOGY
Pneumococcus MODULE
z Pneumococci are sensitive to heat and commonly used antiseptics Microbiology
z Pneumococcal vaccine is used for prevention of pneumococcal infections.
TERMINAL QUESTIONS
1. Enumerate the biochemical and other tests used to differentiate Strept Notes
pneumoniae from other Streptococci.
2. Describe Quellung test.
3. Describe optochin sensitivity test.
4. Briefly describe the mechanism of pathogenesis of pneumococcal infection.
5. Enumerate the diseases caused by Strept. Pneumonia.
6. Briefly describe the steps in lab diagnosis of infections caused by Strept
pneumonia.
7. Discuss the types of pneumococcal vaccines available. Describe the dosing
schedule and frequencyANSWERS TO INTEXT QUETIONS
16.1
1. Transformation
2. Positive; diplo
3. Fastidions
4. Hiss's
5. Inculin
6. Negative
16.2
1. Sepcific source substance
2. Penumolysin
3. Quellung
4. Upper respiratory
MICROBIOLOGY 183
MODULE Enterococcus
Microbiology
17
Notes
ENTEROCOCCUS
17.1 INTRODUCTION
Enterococcus is a genus of lactic acid bacteria. Enterococci are catalase negative
Gram-positive cocci that often occur in pairs (diplococci) or short chains, and are
difficult to distinguish from streptococci on physical characteristics alone.
OBJECTIVES
z describe Enterococci
z identify Enterococci in the laboratory
z explain the pathogenesis
z discuss the antibiotic resistance
17.2 ENTEROCOCCI
Enterococci are facultative anaerobic organisms, i.e., they are capable of cellular
respiration in both oxygen-rich and oxygen-poor environments. Though they are
not capable of forming spores, enterococci are tolerant of a wide range of
environmental conditions: extreme temperature (10-45°C), pH (4.5-10.0) and
high sodium chloride concentrations. They are part of the normal enteric flora.
Two species are common agents causing infection among humans: E. faecalis
(90-95%) and E. faecium (5-10%). Rare clusters of infections occur with other
species, including E. casseliflavus, E. gallinarum, and E. raffinosus. The
enterococci were previously classified as group D streptococci.
184 MICROBIOLOGY
Enterococcus MODULE
Microbiology
Notes
Fig. 17.1
Enterococci can exhibit beta/gamma-haemolysis on sheep’s blood agar.

Identification: Because the group D cell wall specific antigen is a teichoic acid,
it is not an antigenically good marker; enterococci are usually identified by
characteristics other than immunologic reaction with group-specific antisera.
z They are usually non-haemolytic, but occasionally á-haemolytic.
z The enterococci are gram positive cocci, occuring in singles, pairs, and short
chains.
z Cells are sometimes coccobacillary when gram stains are prepared from
agar plate growth. Cells are more oval and in chains when gram stains are
prepared from thioglycolate broth.
z The enterococci are facultative anaerobe and optimum growth occurs at
35°C.
z They grow well at between 10°C and 45°C whereas streptococci generally
grow at a much narrower temperature range.
z All strains grow in broth containing 6.5% NaCl and hydrolyze esculin in the
presence of 40% bile salts (bile-esculin medium).
z Enterococci hydrolyze pyrrolidonyl-ß-naphthylamide (PYR), the exceptions
to this are E. cecorum, E. columbae, and E. saccharolyticus.
z Most strains produce leucine aminopeptidase (LAP). Some strains belonging
to Group I enterococci give negative LAP tests.
z They are more resistant to penicillin G than the streptococci, and rare
isolates have plasmids that encode for â-lactamase.
z Many isolates may be vancomycin-resistant.
MICROBIOLOGY 185
MODULE Enterococcus
Microbiology
z Presumptive identification of a catalase negative gram positive cocci as an
Enterococcus can be accomplished by demonstrating that the unknown
strain is PYR and LAP positive, and grows in 6.5% NaCl and at 45°C.
INTEXT QUESTION 17.1

Notes
1. Enterococci are .............. cocci
2. Culturally Enterococci are .............. anerobes
3. Enterococci most frequently cause .............. Infection
4. .............. is highly antibiotic resistance
17.3 SPECIES IDENTIFICATION

Once established that the unknown catalase-negative gram-positive coccus is an
Enterococcus the tests can be used to identify the species. The species are
divided into 5 Groups based on the reactions in acid formation in mannitol,
sorbitol, and sorbose broths and hydrolysis of arginine.
The pigmentation test aids in the identification of E. casseliflavus, E. mundtii, E.

pullins, E. gilvus and E. sulfureus. These enterococci produce a yellow pigment
that can be detected on several different media.
The pyruvate utilization test aids in the differentiation of E. faecalis and E.

faecium. This test is also used to help differentiate between E. faecalis variant
strains and E. hirae.
The tellurite tolerance test aids in the differentiation of E. faecalis and E.

faecium.
E. haemoperoxidus is variable in the mannitol reaction and may be in group II

or III.
Diseases caused: There are at least 12 species of enterococci. Enterococcus

faecalis is the most common and causes 85–90% of enterococcal infections,
while Enterococcus faecium causes 5–10%, while E. faecalis remains the
predominant species in clinical infection, E.faecium isolates are increasing in
proportion. The trend is particularly true for blood isolates.
The enterococci are among the most frequent causes of nosocomial infections,
particularly in intensive care units, and are selected by therapy with cephalosporins
and other antibiotics to which they are resistant.
186 MICROBIOLOGY
Enterococcus MODULE
Enterococci are transmitted from one patient to another primarily on the hands of Microbiology
hospital personnel, some of whom may carry the enterococci in their
gastrointestinal tracts. Enterococci occasionally are transmitted on medical
devices.
In patients, the most common sites of infection are the urinary tract, wounds,
biliary tract, and blood. Enterococci may cause meningitis and bacteraemia in
neonates. In adults, enterococci can cause endocarditis. Notes
However, in intra-abdominal, wound, urine, and other infections, enterococci
usually are cultured along with other species of bacteria, and it is difficult to
define the pathogenic role of the enterococci.
Antibiotic Resistance
A major problem with the enterococci is that they can be very resistant to
antibiotics. E faecium is usually much more antibiotic-resistant than E faecalis.
Intrinsic Resistance
Enterococci are intrinsically resistant to cephalosporins, penicillinase-resistant
penicillins, and monobactams. They have intrinsic low-level resistance to many
aminoglycosides, are of intermediate susceptibility or resistant to
fluoroquinolones, and are less susceptible than streptococci (10- to 1000-fold) to
penicillin and ampicillin. Enterococci are inhibited by â-lactams (eg, ampicillin)
but generally are not killed by them.
Vancomycin Resistance
The glycopeptide vancomycin is the primary alternative drug to penicillin (plus
an aminoglycoside) for treating enterococcal infections. Enterococci that are
resistant to vancomycin have increased in frequency. Vancomycin resistance has
been most common in E faecium, but vancomycin-resistant strains of E faecalis
also occur.

1. Bile aesculin positive Gram positive coccus is ...............
2. Vancomycin resistant catalase negative gram positive cocci can be ...............
3. Two important species of Enterococci are ............... & ...............
4. Most common route of transmission of Enterococci from one to another is
by ...............
MICROBIOLOGY 187
MODULE Enterococcus
Microbiology

z Enterococci are catalase negative Gram-positive cocci
z Enterococci can exhibit beta/gamma-haemolysis on sheep’s blood agar
z They grow well at between 10°C and 45°C
Notes z All strains grow in presence of 6.5% NaCl and are bile-aesculin test positive
z Most strains are PYR and LAP positive
z They can be very much resistant to antibiotics
TERMINAL QUESTIONS
1. Define genus enterococci?
2. What are the diseases caused by enterococci?
3. How will you identify Enterococcus in laboratory?
17.1
1. Gram positive
2. Facultative anaerobes
3. Nosocomial
4. E. Faecium
17.2
1. Enterococcus
2. Enterococcus
3. E. faecalis and E. Faecium
4. Contaminated hands
188 MICROBIOLOGY
Nisseriae MODULE
Microbiology
18
Notes
NISSERIAE
18.1 INTRODUCTION
The Neisseria are Gram-negative cocci that usually occur in pairs. They are
aerobic, nonsporulating, nonmotile, oxidase-positive cocci typically arranged in
pairs. N.meningitidis and N. gonorrheae are medically important pathogens, and
are found associated with or inside polymorphonuclear cells. Some Neisseriae
sp are normal inhabitants of the human respiratory tract.
OBJECTIVES
z explain the characteristics of Neisseria species
z discuss the diagnosis of disease caused by Neisseria species
18.2 NEISSERIA MENINGITIDIS
Morphology
Meningococci are Gram-negative, oval or spherical cocci, 0.6 – 0.8 µm in size,
typically arranged in pairs, with the adjacent sides flattened.
Cultural characteristics
Meningococci have exacting growth requirements and do not grow on ordinary
media. Growth occurs on media enriched with blood, serum or ascetic fluid,
which promote growth by neutralizing certain inhibiting substances in culture
media rather than by providing additional nutritional needs.
MICROBIOLOGY 189
MODULE Nisseriae
Microbiology They are strict aerobes, no growth occurs anaerobically. The optimum temperature
for growth is 35-36oC. no growth takes place below 30oC. Optimum pH is 7.4-
7.6. Growth is facilitated by 5-10 percent CO2 and high humidity.
On solid media after incubation for 24 hrs, the colonies are small translucent,
round, convex, bluish grey, with a smooth glistering surface and with entire
edges. Blood agar, chocolate agar and Mueller-Hinton starch casein hydrolysate
Notes agar are the media commonly used for culturing meningococci.
Biochemical reactions
They are catalase and oxidase positive, the prompt oxidase reaction helps in the
identification of neisseria in mixed cultures. When a freshly prepared 1%
solution of oxidase reagent is poured on the culture media, the neisseria colonies
turn deep purple. Indole and hydrogen sulphide are not produced and nitrates
are not reduced. Glucose and maltose are utilized, but not sucrose or lactose,
producing acid but no gas.
Antigenic properties and classification

Meningococci are capsulated, unlike other neisseriae. Based on their capsular
polysaccharide antigens, meningococci are classified into at least 13 serogroups,
of which Groups A,B and C are most important. Group A is usually associated
with epidemics and Group C mostly with localized outbreaks, while Group B
caused both epidemics and outbreaks.
Resistance
Meningococci are very delicate organisms being highly susceptible to heat,
dessication, alterations in pH and to disinfectants. They are sensitive to
penicillin and other antibiotics, but resistance strains have emerged and become
common in many areas.
Pathogenicity
Cerebrospinal meningitis and meningococcal septicemia are the two main types
of meningococcal disease. Meningococci are strict human parasites inhabiting
the nasopharynx. Infection is usually asymptomatic. In some, local inflammation
ensues, with rhinitis and pharyngitis. Dissemination occurs only in a small
proportion. Most common complication include Waterhouse-Friderichsen
syndrome, a massive, usually bilateral hemorrhage into the adrenal glands
caused by fulminant meningococcemia, adrenal insufficiency and disseminated
intravascular coagulation.
190 MICROBIOLOGY
Nisseriae MODULE
Laboratory diagnosis Microbiology
In meningococcal meningitis, the cocci are present in large numbers in the spinal
fluid and, in the early stage in the blood as well. Demonstration of meningococci
in the nasopharynx helps in the detection of carriers.
(a) Examination of CSF
The fluid will be under pressure and turbid, with a large number of pus
cells. For bacteriological examination, if a sufficient quantity is available, Notes
the CSF is divided into three portions. One portion is centrifuged and
Gram- stained smears are prepared from the deposit. Meningococci will
be seen mainly inside polymorphs but often extracellularly also. The
second portion of the CSF is inoculated iin blood agar or chocolate agar
plates and incubated at 35-36oC under 5-10% CO2. Colonies appear after
18-24 hrs which may be identified by morphological and biochemical
reactions. The third portion of the CSF is incubated overnight either as it
is or after adding an equal volume of glucose broth and then subcultured
on chocolate agar.
(b) Blood culture
Meningococcemia and in early cases of meningitis, blood culture is often
positive. Cultures should be incubated for 4-7 days, with daily subcultures.
(c) Nasopharyngeal swab
This is useful for the detection of carriers. The swab should be held in a
suitable transport medium like stuart’s medium
(d) Petechial lesions
Meningococci may sometimes be demonstrated in petechial lesions by
microscopy and culture.
(e) Molecular diagnosis
Group-specific diagnosis of infection can be made by detection of
meningococcal DNA sequence in CSF or blood by PCR amplifications.
Treatment
Prompt treatment is essential to ensure recovery without sequelae. Intravenous
penicillin G is the treatment of choice. Chloramphenicol is equally effective.

1. Neisseria are gram ...................... cocci which occur in ......................
2. ...................... & ...................... are pathogenic strains of Neisseria
MICROBIOLOGY 191
MODULE Nisseriae
Microbiology 3. N.gonorrhoeae commonly causes ...................... in neonates

4. Common CNS infection Neisseria causes is ......................
18.3 NEISSERIA GONORRHOEAE (GONOCOCCUS)

Morphology
Notes The organism appears as a diplococcus with the adjacent sides concave, being
typically kidney shaped. It is predominantly within the polymorphs. Gonococci
possess pilli on their surface. Pili facilitate adhesion of the cocci to the mucosal
surfaces and promote virulence by inhibiting phagocytosis.
Gonococci are more difficult to grow than meningococci. They are aerobic but
may grow anaerobically also. Growth occurs best at pH 7.2-7.6 and at a
temperature of 35-360c with 5-10% CO2. They grow well on chocolate agar and
Mueller-Hinton agar. A popular selective medium is the Thayer-Martin medium
which inhibits most contaminants including nonpathogenic neisseria. Colonies
are small, round, translucent, convex and slightly umbonate, with a finely
granular surface and lobate margins.
Gonococci resemble meningococci except in the effect of maltose. Gonococci
acidify only glucose and not maltose.
Antigenic properties
Gonococci are antigenically heterogeneous. They are capable of changing their
surface structures in vitro. Pili, which are hair like structures act as virulence
factors by attaching to host cells and inhibiting phagocytosis. The trilaminar
outer membrane of gonococci contains protein I and II which acts as ligands
attaching the coccus to the host cells. The outer membrane also contain
lipopolysaccharide which may be responsible for the toxicity in gonococcal
infections.
Resistance
The gonococcus is a very delicate organism, readily killed by heat, drying and
antiseptics. In cultures, the coccus dies in 3-4 days but survives in slant cultures
at 35oC.
192 MICROBIOLOGY
Nisseriae MODULE
Pathogenicity Microbiology
Gonorrhea is a venereal disease which has been known since ancient times. The
name gonorrhea, meaning flow of seed. The disease is acquired by sexual
contact. Infection of the lower genital tract can result in a purulent or pus like
discharge from the genitals which may be foul smelling. N.gonorrhoeae can also
cause conjunctivitis, pharyngitis, proctitis or urethritis, prostatitis and orchitis.
Conjunctivitis is common in neonates and silver nitrate or antibiotics are often
Notes
applied to their eyes as a preventive measure against gonorrhea. Infection of
the genitals in females with N.gonorrhoeae can result in pelvic inflammatory
disease if left untreated, which can result in infertility.
Fig. 18.1
Laboratory diagnosis
Specimens
Pus and secretions are taken from the urethra, cervix, rectum, conjunctiva,
throat, or synovial fluid for culture and smear.
Smears
Gram-stained smears of urethral or endocervical exudates reveal many diplococci
within pus. These give a presumptive diagnosis.
Cultures
Immediately after collection, pus or mucus is streaked on enriched selective
medium like modified Thayer-Martin medium and incubated in an atmosphere
containing 5% CO2 (candle jar) at 36oc. to avoid overgrowth by contaminants,
the selective medium contains antimicrobial drugs like vancomycin, colistin,
amphotericin. Forty-eight hours after culture, the organisms can be quickly
MICROBIOLOGY 193
MODULE Nisseriae
Microbiology identified by their apperancce on Gram-stained smear, by oxidase positivity, and

by coagglutination, immunofluorescence staining, or other laboratory test. The
species of bacteria may be determined by rapid carbohydrate utilization tests.
Nucleic Acid Amplification Tests

Several food and drug administration cleared nucleic acid amplification assays
Notes are available for detection of N gonorrhoeae in genitourinary specimens.
Treatment
Penicillin G for inhibition (MIC - 2µg/ml). Pencillinase producing N gonorrhoeae
(PPNG) also have increased in prevalence. Uncomplicated genital or rectal
infections are treated with ceftriaxone 250mg given intramuscularly as a single
dose. Additional therapy with azithromycin 1 gm / doxycycline, orally twice a
day for 7 days, is recommended for the possible concomitant chlamydial
infection.
Fig. 18.2

z Neisseriae are gram-negative cocci occurring in pairs they are non-motile.
Facultative aerobes that are catalase and oxidase positive
z Neisseriae gonorrhoeae (gonococci) and Neisseria meningitidis
(Meningococci) are pathogenic for humans. Some neisseriae are normal
inhabitants of respiratory tract, rarely causing disease
z Neisseriae meningtidis commonly cause of meningitis and septicemia
z Neisseriae gonorrhoeae causes gonorrhea, conjunctivitis, pharyngitis,
proctitis, urethritis and prostatitis.
194 MICROBIOLOGY
Nisseriae MODULE
Microbiology
TERMINAL QUESTIONS
1. Explain laboratory diagnosis of Neisseria
2. Describe the gold standard diagnosis of N. Meningitidis
Notes
1. Negative & Pairs
2. Neisseria gonorrhoeae and Neisseria meningitidis
3. Conjunctivitis
4. Meningitis
MICROBIOLOGY 195
MODULE Corynebacterium
Microbiology
19
Notes
CORYNEBACTERIUM
19.1 INTRODUCTION
Corynebacterium diphtheriae is a pathogenic bacterium that causes diphtheria. It
is also known as the Klebs-Löffler bacillus, because it was discovered in 1884 by
German bacteriologists Edwin Klebs (1834 – 1912) and Friedrich Löffler (1852
– 1915).
OBJECTIVES
z describe the morphological characteristics of the Cornyebacterium diphtheria
z explain the clinical features of diptheria
z discuss the laboratory diagnosis of Corynebacterium diphtheriae
z explain the disease spectrum caused by Corynebacteruim diphtheria
19.1 MORPHOLOGY
Corynebacteria are gram – positive , non- acid fast , nonmotile rods with
irregularly stained segments , and sometimes granules. They frequently shows
club shaped swelling and hence the names corynebacteria( from coryne,
meaning club). The most important member of this genus is C diphtheria sp, the
causative agent of diphtheria.
Corynebacterium diphtheriae
Humans are the sole pathogen reservoir for diphtheria. Infection sources include
infected persons and carriers (rare). The disease is usually transmitted by droplet
196 MICROBIOLOGY
Corynebacterium MODULE
Microbiology
infection, or less frequent indirectly via contaminated objects. It is an acute
and contagious infection characterized by pseudomembranes of dead
epithelial cells, white blood cells, red blood cells, and fibrin that form around
the tonsils and back of the throat. It is an uncommon illness that tends to
occur in unvaccinated individuals, especially school-aged children, those in
developing countries, elderly, neutropenic or immunocompromised patients.
The virulent and toxigenic strains are lysogenic, and produce an exotoxin formed
Notes
by two polypeptide chains, which is itself produced when a bacterium is
transformed by a gene from the ß prophage.
Fig. 19.1
Fig. 19.2: Corynebacterium diphtheriae
MICROBIOLOGY 197
Microbiology Four subspecies are recognized: C. diphtheriae mitis, C. diphtheriae intermedius,

C. diphtheriae gravis, and C. diphtheriae belfanti. The four subspecies differ
slightly in their colonial morphology and biochemical properties such as the
ability to metabolize certain nutrients, but all may be toxigenic (and therefore
cause diphtheria) or non-toxigenic. Unusually, the diphtheria toxin gene is
actually encoded by a bacteriophage which is found in toxigenic strains, not on
the bacterial chromosome itself.
Notes
Clinical Features
z Respiratory: Following an incubation period of 2-4 days, patients typically
report upper respiratory tract symptoms (eg, nasal discharge, sore throat).
The posterior pharynx and tonsillar pillars are most often involved. Onset is
often sudden, with low-grade fevers, malaise, and membrane development
on one or both tonsils, with extension to other parts of the respiratory system.
z Cardiac: The toxic effect in the myocardium characteristically occurs within

1-2 weeks following onset of infection, often when the upper respiratory
tract symptoms are improving. Manifestations are due to arrhythmias and
congestive heart failure (CHF).
z Neurologic: Neurological symptoms can occur immediately or after several

weeks. Bulbar symptoms generally occur within the first 2 weeks after disease
onset and can range from mild symptoms (eg, difficulty swallowing) to
bilateral symmetric paresis of the palatal and ocular muscles. The bulbar
symptoms may remit or progress to paralysis of the proximal and then distal
skeletal muscles over the next 30-90 days. Although recovery can be very
slow, patients generally regain complete neurologic function. Secondary
complications include aspiration from bulbar paralysis and
bronchopneumonia from respiratory muscle dysfunction.
z Skin: Cutaneous infections can occur, often in more tropical climates,

presenting as nonhealing ulcers. A recent surveillance study of Native
Americans presenting to the Indian Health Service clinics in South Dakota
recovered C diphtheriae from 6 (5%) of the 133 patients, 1 of whom had
skin ulcers.
Consists of isolation of the diphtheria bacilli and demonstration of its toxicity.
Samples – two swabs from the lesion are collected under vision
Microscopy – perform gram stain and albert staining
198 MICROBIOLOGY
Gram stain – The bacilli is a slender rod with tendency to clubbing at one or both Microbiology
the ends. The bacilli are pleomorphic. They are nonsporing , noncapsulated and
nonmotile. They are gram positive but tends to decolourised easily. The granules
are composed of polymetaphosphate granules which are more gram positive
from rest of the bacteria.
Albert stain – green colour bacilli are seen with black colour granules.
Morphology – the bacilli is a slender rod with tendency to clubbing at one or both Notes
ends. The bacilli are pleomorphic. They are nonsporing, noncapsulated and
nonmotile. They are gram positive but tends to decolourised easily. The granules
are composed of polymataphosphate granules which are more gram positive
from rest of the bacteria.
Fig. 19.3: Gram staining
Fig. 19.4: Albert staining of Corynebacterium diphtheriae
Cultural Characteristics
Growth is scanty ordinary media. Enrichment with blood, serum or egg is
necessary for good growth. The optimum temperature for growth is 37oc (range
15 - 40oc) and the optimum pH is 7.2. It is an aerobe and a facultative anaerobe.
The usual media employed for the cultivation of the diphtheria bacillus are
Loeffler's serum slope and tellurite blood agar. Diphtheria bacilli grow on
MICROBIOLOGY 199
Microbiology Loeffler's serum slope very rapidly and clolonies can be seen in 6-8 hours, long
before other bacteria grow. Colonies are at first small, circular white opaque
discs but enlarge on continued incubation and may acquire a distinct yellow tint.
Diphtheria bacilli ferment with the production of acid, (but not gas) glucose,
galactose, maltose and dextrin (but not lactose, mannitol or sucrose). Some
strains of virulent diphtheria bacilli have been found to ferment sucrose. It is
Notes necessary to use Hiss's serum water for testig sugar fermentation. Proteolytic
activity is absent. They do not hydrolyse urea or form phosphatase.
Toxin
Virulent strains of diphtheria bacilli produce a very powerful toxin. The
pathogenic effect of the bacilli are due to toxin. Almost all strain of gravis and
intermidius (about 95 – 99 percent) are toxigenic while only about 80 – 85 per
cent of the mitis starins are so.
Diphtheria toxin is a protein. It has two fragments, A and B. Both the fragments
are necessary for toxic effect. .When released by the bacterium , the toxin is
inactive active on fragment A is masked. All the enzymatic activity of the toxin
is present in fragment A. Fragment B is responsible for the binding the toxin to
the cell.
Virulence Test
Virulence test – any isolate of the diphtheria bacilli should be tested for virulence
or toxigenecity for the bacteriological diagnosis to be complete. Virulence
testing may be by vivo or invitro methods, the former by the subcutaneous or
intradermal test and the latter by the precipitation test or the tissue culture test.
Invivo test are done on guinea pig.
Invitro Test
Invitro test- Elek’s gel precipitation test : A rectangular strip of filter paper
impregnated with diphtheria antitoxin (1000 units/ ml) is placed on the surface
of a 20% normal horse serum agar in a petri dish while the medium is still fluid.
When the agar is set, the surface is dried and narrow streaks of the strain are made
at right angle to the filter paper strip. A positive and negative control should be
put up. The plate is incubated at 37°C for 24 – 48 hours. Toxin produced by the
bacterial growth will diffuse in the agar and where it meets the antitoxin of
optimum concentration, will produce a line of precipitation . The presence of
arrow head lines of precipitates indicates that the strain is toxigeneic. No
precipitate will form in the case of nontoxigenic strains.
200 MICROBIOLOGY
Tissue Culture Test Microbiology
Tissue culture test : The toxigenicity of diphtheria bacilli can be demonstrated by

incorporating the strains in the agar overlay of the cell culture monolayers. The
toxin produced diffuses into the cells below and kills them.
Prophylaxis
Diphtheria can be controlled by immunization . Diptheria toxiod is usually
given in children as a trivalent preparation containing tetanus toxoid and Notes
pertussis vaccine also., as a DPT, DPT or triple vaccine
Sensitivity
The bacterium is sensitive to the majority of antibiotics, such as the penicillins,
ampicillin, cephalosporins, quinolones, chloramphenicol, tetracyclines,
cefuroxime and trimethoprim.
Diphtheroides
Corynebacterium resembling C.diphtheriae occurs as normal commensals in the
throat, skin, conjunctiva and other areas. These may sometimes be mistaken for
diphtheria bacilli and are diphtheroids. In general they stain more uniformly than
diphtheria bacilli, possess few or no metachromatic granules and tend to be
arranges in parallel rows (palisade) rather than cuneiform pattern.

1. Which of the following is not the staining method of corynebacterium
(a) Gram stain
(b) Albert stain
(c) Ponders stain
(d) Ziehl Neelsen stain
2. What is selective media for growth of corynebacterium diphtheria
(a) Potassium tellurite blood agar
(b) Loweinstein Jenson medium
(c) Sabourds Dextrose agar
(d) Maconky ‘s agar
3. Corynbacteium are ................. shaped
4. Causative agent of diphetheria is .................
5. Diphtheria is transmitted by ................. infection
6. Culturally the bacteria is .................
MICROBIOLOGY 201
Microbiology

z Corynebacterium is a Gram-positive, rod-shaped bacteria, frequently shows
club shaped swelling and hence the name Coryne meaning club
z Four Subspecies are recognized C.diphtheriae mitis, C.diphtheriae
intermedius, C.diptheriae gravis and C. diphtheriae belfanti.
Notes
z C. diphtheriae sp causes diphtheria, an acute and contagious form which
is transmitted by droplet infection
z Diptheria occurs in unvaccinated individuals especially school-aged children.
z Laboratory diagnosis consists of isolation of diphtheria bacilli and
demonstration of its toxicity
z For Microscopic examination gram staining and Albert staining
z Diphtheria cna be controlled by immunization of DPT vaccine
z Diphtheroides resembles corynebacterium occuring as normal commensals
in throat, skin, conjuctiva
TERMINAL QUESTIONS
1. Clinical features of diphtheria?
2. Laboratory diagnosis of corynebacterium diphtheria?
3. Short note on diphtheroides?er to intext questions
ANSWER TO INTEXT QUESTIONS

1. (d)
2. (a)
3. Club
4. C.diphtheria
5. Droplet
6. Pleomorphic
202 MICROBIOLOGY
Mycobacterium MODULE
Microbiology
20
Notes
MYCOBACTERIUM
20.1 INTRODUCTION
Mycobacterium are slender rods that sometimes show branching filamentous
forms resembling fungal mycelium. In liquid cultures they form a mould-like
pellicle. Hence the name ‘mycobacteria’, meaning fungus like bacteria. They do
not stain readily, but once stained, resist decolourisation with dilute mineral
acids. Hence they are called ‘Acid fast bacilli’. They are aerobic, nonmotile,
noncapsulated and nonsporing.
OBJECTIVES
z describe the morphology of Mycobacterium tuberculosis & M. leprae
z describe the characteristics of Mycobacterium tuberculosis & M. leprae
z explain about pathogenesis of Mycobacterium tuberculosis & M. leprae
z explain the laboratory diagnosis Mycobacterium tuberculosis & M. leprae
The first member of this genus to be identified was Lepra bacillus discovered by
Hansen. Koch (1882) isolated the mammalian tubercle bacillus and proved its
causative role in tuberculosis. In humans tuberculosis is caused by mycobacterium
tuberculosis and also by bovine type called Mycobacterium bovis.
The second human pathogenic mycobacterium is the lepra bacillus causing
Leprosy. The third group of mycobacterium is a mixed group from varied sources
like birds, cold-blooded and warm blooded animals, from skin ulcers, soil, water
and other environmental sources. They are called as atypical mycobacteria. They
are opportunistic pathogens and can cause many types of diseases.
MICROBIOLOGY 203
MODULE Mycobacterium
Microbiology
20.2 MYCOBACTERIUM TUBERCULOSIS
Morphology
M tuberculosis is a straight or slightly curved rod, about 3 X 0.3 µm in size,
occurring singly, in pairs or as small clumps. M bovis is usually straighter,
shorter and stouter.
Tubercle bacilli have been described as Gram positive, even though after
Notes
staining with basic dyes they resist decolourisation by alcohol even without the
effect of iodine. When stained with carbol fuchsin by Ziehl-Neelsen method or
by fluorescent dyes they resist decolorisation by acids such as 20% Sulphuric
acid as well as by alcohols. The unsaponifiable wax (mycolic acid) forms a
semipermeable membrane around the cell that makes it acid fast.
Fig. 20.1: AFB smear
Fig. 20.2: Mycobacterium tuberculosi Acid-Fast stain
Culture characteristics
The bacilli grow and invitro time for generation is 14-15 hours. The optimum
temperature is 37°C and growth does not occur below 25°C or above 40°C.
Optimum pH is 6.4-7.0. M tuberculosis is an obligate aerobe while M bovis is
microaerophilic on primary isolation. M tuberculosis grows luxuriantly in
culture as compared to M bovis which grows sparsely.
204 MICROBIOLOGY
On solid media, dry, rough, raised, irregular colonies with a wrinkled surface are Microbiology
senn and they are creamy white, becoming yellowish coloured on further
incubation. M bovis forms flat, smooth, moist, white colonies that break up
easily.
INTEXT QUESTIONS 20.1 Notes
1. Mycobacteria means ................

2. Mycobacteria are ................ bacilli
3. Presence of ................ around cells makes it acid fast
4. Mycobacteria tuberculosis is an ................ aerobe
Resistance
Mycobacteria are not heat resistant, being killed at 60°C in 15-20 minutes.
Cultures may be killed by exposure to direct sunlight for two hours. But bacilli in
spectrum may remain alive for 20-30 hours. Bacilli are relatively resistant to
chemical disinfectants, surviving exposure to 5% phenol, 15% sulphuric acid,
3% Nitric acid, 5%oxalic acid and 4% sodium hydroxide. They are sensitive to
formaldehyde and glutaraldehyde.
Niacin test: Human tubercle bacilli form niacin when grown on an egg medium.
When 10% cyanogens bromide and 4% aniline in 96% ethanol are added to a
suspension of the culture, a canary yellow colour indicates a positive reaction.
The test is positive with human type and negative with bovine type.
Aryl sulphatase test: This test is positive only with atypical mycobacteria. The
bacilli are grown in a medium containing 0.001 M tripotassium phenolphthalein
disulphate. 2N NaOH is added drop by drop to the culture and pink colour
indicates a positive reaction.
Neutral red test: Virulent strains of tubercle bacilli are able to bind neutral red in
alkaline buffer solution.
Catalase-Peroxidase tests: this is used to differentiate tubercle bacilli from

atypical mycobacteria. Most atypical mycobacteria strains are catalase positive
while tubercle bacilli are weakly positive. Tubercle bacilli are peroxidase
positive but not atypical mycobacteria.
MICROBIOLOGY 205
Microbiology A mixture of equal volumes of 30 volumes of H2O2 and 0.2% catechol in

distilled water is added to 5 ml of test culture and are allowed to stand for few
minutes. Effervescence indicates catalase production and browning indicates
peroxidase activity.
Amidase tests: The ability to split amides has been used to differentiate
mycobacteria. A 0.00165 M solution of the amide is incubated with the bacillary
Notes suspension at 37°C and 0.1 ml MnSO4.4 H2O, 1.0 ml of phenol solution and 0.5
ml hypochlorite solution are added. The tubes are placed in boiling water for 20
minutes. A blue colour indicates a positive test.
Nitrate reduction test: this is positive with M tuberculosis and negative with M
bovis.
Antigenic properties: Antigens have been identified in mycobacteria. Group
specificity is due to polysaccharides and type specificity to protein antigens.
Delayed hypersensitivity develops following an infection of tubercle bacilli to
the bacillary protein. M tuberculosis stains are antigenically homogeneous but is
not useful in diagnosis or in immunity.
Bacteriophage: Mycobacteriophages have been isolated from soil, water and
other environmental sources as well as from lysogenic strains.
There are four phage types A,B,C and a intermediate type between A & B as I,
which is common in india.
Molecular typing: DNA fingerprinting provides a method for differentiating
between strains of tubercle bacilli. Restriction endonuclease treatment yields
nucleic acid fragments of varying lengths and the pattern are strain specific. This
restriction fragment length polymorphism (RFLP) is used in strain typing.
Pathogenesis: Open case of pulmonary tuberculosis is the source of infection,
which is most common in India. One open case may infect 25 contacts. The mode
of infection is by direct inhalation of aerosolized bacilli in droplet nuclei of
expectorated sputum. Coughing, sneezing and speaking releases numerous
droplets as many as 3000 infectious nuclei per cough. Dried bacilli in dust are
much less infectious.
The majority of inhaled bacilli are arrested by natural defenses of the upper
respiratory tract and which reaches the lungs are ingested by alveolar macrophages.
Number and virulence of the infecting bacilli, host factors including genetic
susceptibility, age, immunocompetence, stress, nutrition and coexisting illness
influence the outcome of the infection.
Humans have effective defence against the infection as only a tenth of the
infected develop active tuberculosis. Cell mediated immunity appears to be
206 MICROBIOLOGY
effective, whereas humoral immunity is irrelevant. The key cell is the activated Microbiology
CD4+ helper T cell which develops as Th-1 or Th-2 cells, releasing cytokines
such as interferon γ (gamma) interleukins 1 and 2, toxic necrosis factor α (alpha)
and others exerting different biological effects. Th-1 dependent cytokines
activate macrophages resulting in protective immunity and containment of the
infection. Th-2 cytokines induce delayed type hypersensitivity (DTH), tissue
destruction and progressive disease.
Notes
The essential pathology in tuberculosis is the production in infected tissues of a
characteristic lesion the tubercle, this is an avascular granuloma composed of a
central zone containing giant cells with or without caseation and a peripheral
zone of lymphocytes and fibroblasts.
Tuberculosis may be classified as primary and post primary.

Primary tuberculosis is the initial infection by tubercle bacilli. In endemic
countries like India this usually occurs in young children, the bacilli engulfed by
alveolar macrophages multiply and give rise to a subpleural focus of tuberculous
pneumonia, commonly in upper lobe, the Ghon factor. The hilar lymph nodes are
involved. The Ghon focus together with enlarged hilar lymph node constitutes
primary complex. This occurs about 3-8 weeks from the time of infection and is
associated with the development of tuberculin hypersensitivity. In most of the
cases the lesion heals spontaneously in 2-6 months leaving a calcified nodule and
a few bacilli may survive and remain latent. In children with impaired immunity
or other risk factors they may cause miliary, meningeal or other forms of
disseminated tuberculosis.
The post primary type of tuberculosis is due to reactivation of latent infection or
exogenous reinfection. It affects mostly in the upper lobes of the lungs, the
lesions undergoing necrosis and tissue destruction, leading to cavitation. The
necrotic materials are released through airway, to expectoration of latent sputum,
which is the main source of infection.

1. Niacin test is negative in ................
2. Aryl sulphatase test is positive in ................
3. In molecular typing ................ is used in stain typing
4. Mycobacteria gets transmitted by ................ infection
5. Primary complex constitutes of ................ & ................
MICROBIOLOGY 207
Microbiology Laboratory Diagnosis: Tuberculosis may be demonstrated in the lesion by

microscopy, culture isolation and molecular methods.
Pulmonary Tuberculosis
The sputum is tested for pulmonary tuberculosis. The bacterial shedding in the
sputum is abundant in cases with caseation, but scanty in lesions that do not
communicate with airways. Sputum is best collected in the morning before any
Notes meal. If scanty, a 24-hour sample may be tested and sputum sampling on three
days increases the chances of detection. Laryngeal swabs or bronchial washings
may be collected and in children gastric lavage can be examined.
Microscopy
Direct or concentration smears of sputum are examined. Sputum microscopy is
the most reliable single method in the diagnosis and control of tuberculosis. New
slides should be used for smears and should not be reused as acid fast bacilli may
not always be removed from slides by cleaning.
Smear should be prepared from the thick purulent part of the sputum. Smears are
dried, heat fixed and stained by Ziehl-Neelsen technique. This smear is covered
with strong carbol fuchsin and gently heated to steaming for 5-7 minutes,
without letting the stain boil and become dry. The slide is then washed with water
and decolourised with 20% sulphuric acid till no more stain comes off and then
with 95% ethanol for two minutes. Decolourisation may be carried out as a single
step with acid alcohol. After washing, the smear is counter stained with
Loeffler’s methylene blue, 1% picric acid or 0.2% malachite green for one
minute. Under the oil immersion objective, acid fast bacilli are seen as bright red
rods while the background is blue, yellow or green depending on the counter
stain used. Atleast 10,000 acid fast bacilli should be present per ml of sputum for
them to be readily demonstrable in direct smears. A negative report should not be
given till at least 300 fields have been examined, taking about 10 minutes. A
positive report can be given only if two or more typical bacilli have been seen.
Smears are seen depending on the number of bacilli seen.
No. of AFB Seen in Report
(oil immersion field)
0 300 F AFB not seen
1-2 300 F Doubtful, repeat smear
1-9 100 F 1+
1-9 10 F 2+
1-9 1F 3+
10 or more 1F 4+
208 MICROBIOLOGY
When several smears are to be examined daily, fluorescent microscopy is used. Microbiology
Smears are stained with auramine phenol or auramine rhodamine fluorescent
dyes and when examined under ultraviolet illumination, the bacilli will appear as
bright rods against a dark background.
Concentration methods
(i) Petroff’s method
Notes
This method is widely used. Sputum is incubated with an equal volume of 4%
sodium hydroxide solution at 37°C with frequent shaking till it becomes clear. It
is then centrifuged at 3000 rpm for 20 minutes and the sediment neutralized with
N/10 HCl and used for smear, culture and animal inoculation.
A simpler method like, treating the sputum with an approximately equal volume
of a sterile solution containing 20 g cetrimonium bromide and 40 g of NaOH per
litre of distilled water. The contents are mixed with cotton swab and left to stand
for five minutes. About 0.2 ml of the inoculum is smeared firmly with the swab
over the entire surface of acid buffered medium.
Culture
Culture is a very sensitive diagnostic technique for tubercle bacilli, detecting as
few as 10 to 100 bacilli per ml. The concentrated material is inoculated into
atleast two bottles of IUAT-LJ medium. If the specimen is positive by
microscopy a direct drug sensitivity test may be done. Cultures are examined for
growth after incubation at 37°C for four days, for rapid growing mycobacteria,
fungi and contaminant bacteria and atleast twice weekly thereafter. A negative
report is given if no growth occurs after 8-12 weeks. Any growth seen is smeared
and tested by Ziehl Neelsen staining. For routine purposes, a slow growing, non
pigmented, niacin positive acid fast bacillus is taken as M.tuberculosis.
Confirmation is by biochemical studies
Sensitivity tests
As drug resistance is an important problem in tuberculosis it is desirable to have
sensitivity of isolates tested as an aid to treatment and they are of three types. The
first is absolute concentration method in which a number of media containing
serial concentration of the drugs are inoculated and the minimum inhibitory
concentrations calculated
The second is resistance ratio method in which two sets of media containing
graded concentrations of the drugs are inoculated. One set with the test strain and
other with a standard strain of known sensitivity
The third is proportion method which indicates average sensitivity of the strain.
MICROBIOLOGY 209
Microbiology Allergic test – Mantoux test
0.1ml of Purified Protein Derivative (PPD) containing 5 TU is injected

intradermally on the forearm with a tuberculin syringe causing a wheal. The
injection should not be given subcutaneously but in between the layers of the
skin, intradermally. The site is examined 48-72 hrs later and induration measured
at its widest point transversely. Induration of diameter 10mm or more is
Notes considered positive, 5mm or less is considered negative and 6-9mm equivocal.
A positive tuberculin test indicated hypersensitivity to tuberculoprotein denoting

infection with tubercle bacilli or BCG immunization, recent or past with or
without clinical disease

1. ................ is the most reliable single method in the diagnosis of tuberculosis
2. ................ technique is used in demonstration of tubercle bacilli
3. ................ is the common concentration method in diagnosis of tubercle
bacilli
4. ................, ................ & ................ are the common sensitivity tests in the
diagnosis of tubercle bacilli
5. ................ is the allergic test used in the diagnosis of tuberculosis
6. ................ is injected in allergic test
20.3 MYCOBACTERIUM LEPRAE

Leprosy is a disease recognized since vedic times in India. The person suffering
with leprosy is considered ‘unclean’ and a social outcast. The lepra bacillus was
first observed by Hansen in 1868 and hence it is also called as Hansen’s disease.
Morphology
M leprae is a straight or slightly curved rod, 1-8 X 0.2-0.5 µm in size, showing

considerable morphological variation. It is Gram positive and stains more
readily than tubercle bacillus. It is acid fast, but less so than tubercle bacillus.
Hence 5% sulphuric acid instead of 20% is used for a decolourisation after
staining with carbol fuchsin. In stained smears, live bacilli appear solid and
uniformly stained, while the dead bacilli are fragmented and granular.
210 MICROBIOLOGY
Microbiology
Notes
Fig. 20.3
The bacilli are seen, singly and in groups, intracellularly or lying free outside the
cells. Mostly they appear as agglomerates, the bacteria being bound together by
a lipid-like substance known as ‘globi’.
Resistance:
Lepra bacilli have been found to remain viable in a warm humid environment for
9-16 days and in moist soil for 46 days. They survive exposure to direct sunlight
for 2 hours and ultraviolet light for 30 minutes.

1. Leprosy is caused by ................
2. Leprosy is also called as ................ disease
3. Bacteria being bound with a lipid like substance known as ................
4. Leprosy is a chronic ................ disease of humans
Leprosy
Leprosy is a chronic granulomatous disease of humans primarily involving the
skin, peripheral nerves and nasal mucosa but capable of affecting any tissue or
organ.
The disease may be classified into four types namely Lepromatous, tuberculoid,
dimorphous and indeterminate.
Lepromatous type is seen where the host resistance is low. The bacilli are seen in
large numbers or as globi inside lepra cells or extracellularly. This is known as
‘multibacillary disease’. Superficial nodular lesions (lepromata) develop which
consist of granulation tissue containing a dense collection of vacuolated cells in
different stages of development from mononuclear cells to lepra cells. The
MICROBIOLOGY 211
Microbiology nodules ulcerate, become secondarily infected and cause distortion and mutilation.
Bacilli invade the mucosa of the nose, mouth and upper respiratory tract and are
shed in large numbers in nasal and oral secretions. Cell mediated immunity is
deficient and the lepromin test is negative. Lepromatous type is more infective
than the other types.
Tuberculoid leprosy is seen in patients with high degree of resistance. The skin
lesions are few and sharply demarcated, consisting of macular anesthetic
Notes patches. Neural involvement occurs early leading to deformities of hand and
feet. Bacilli are scanty in the lesions and infectivity is minimal and this is known
as ‘paucibacillary disease’. Cell mediated immunity is adequate and the
lepromin test is positive.
Borderline or dimorphous type refers to lesions possessing characteristics of
both tuberculoid and lepromatous types. It may shift to the lepromatous or
tuberculoid part of the spectrum depending on chemotherapy or alterations in
host resistance.
The indeterminate type is the early unstable tissue reaction which is not
characteristic of either the lepromatous or tuberculoid type.

1. Bacilli seen in large number is known as ................ disease
2. ................ is more infective than other types
3. Neural involvement develops early in ................ leprosy
4. Tuberculoid leprosy is also known as ................
Lepromin test
Lepromin test first described by Mitsuda, is a skin test for delayed hypersensitivity.
The response to the intradermal injection of lepromin is typically biphasic,
consisting of two separate events. The first is the early reaction consists of
erythema and induration developing in 24-48 hours and usually remaining for 3-
5 days. The second and more meaningful is the late reaction starting in 1-2
weeks, reaching a peak in four weeks and gradually subsiding in the next few
weeks. The late reaction is a indication to measure cell mediated immunity
induced by injected lepromin.
The lepromin test is not used to diagnose leprosy, nor does it indicate prior
contact with lepra bacillus. The test is used for following purposes:
z To classify the lesions of leprosy patients. The lepromin test is positive in
tuberculoid, negative in lepromatous and variable in dimorphous and
indeterminate types of disease.
212 MICROBIOLOGY
z To assess the prognosis and response to treatment. A positive reaction Microbiology
indicates good prognosis and a negative reaction indicates bad prognosis.
Conversation to lepromin positivity during treatment is evidence of
improvement
z To assess the resistance of individual to leprosy. It is desirable to recruit only
lepromin positive persons for work in leprosaria as Lepromin-negative
persons are more prone to develop the disease Notes
Bacteriological diagnosis is easy in the lepromatous but difficult in tuberculoid
cases. The diagnosis is of demonstration of acid fast bacilli in the lesions.
Specimens are collected from the nasal mucosa, skin lesions and ear lobules. A
blunt narrow scalpel is introduced into the nose and intestinal septum scraped
sufficiently to remove a piece of mucosa membrane, which is transferred to a
slide and teased out into a uniform smear. Skin is pinched up tight to minimize
bleeding and a cut about 5mm with scalpel. Blood or lymph, is wiped and the
blade is turned transversely to scrape the slides and bottom of the cut so as to
obtain a little tissue pulp which is uniformily smeared on the slide. About 5-6
different areas of the skins should be sampled, including the skin over buttocks,
forehead, chin, cheek and ears. The smears are stained using Ziehi-Neelsen
technique using 5% instead of 20% sulphuric acid for decolourisation
Smears are graded based on the number of bacilli
1-10 bacilli in 100 fields 1+
1-10 bacilli in 10 fields 2+
1-10 bacilli per field 3+
More than 1000 bacilli clamps 6+
The bacteriological Index (BI) is calculated by totaling the number of +s scored
in the smears and divided by the number of smears. Thus, if eight smears
examined have a total of sixteen pluses, the BI will be 2. For calculating this a
minimum of four skin lesions, a nasal swab and both the ear lobes have to be
examined
Detection of antibody against M.Leprae phenolic glycolipid antigen has been
claimed to be a specific diagnostic test. Microscopic demonstration of lepra
bacilli and histology remain the most useful diagnostic procedures
MICROBIOLOGY 213
Microbiology

1. ............... is the skin test used for demonstration of delayed hypersensitivity
2. Bacteriological diagnosis is easy in ............... cases
Notes 3. ............... is used in demonstration of M.leprae bacilli

4. Specific diagnostic test in diagnosis of M.leprae is detection of ...............

z Mycobacteria tuberculosis is an obligatory aerobic, nonmotile, nonsporing,
rod shaped bacterium which strains poorly by the Gram strain because its
cell wall contains abundant amount of lipids. It retains Carbol Fuchsin dye
during attempted decolourisation with acid and alcohol in Ziehl-Neelsen
staining technique. M.tuberculosis is acid and alcohol fast by ZN staining
method. It grows very slowly, taking several weeks to form a visible colony
on enriched culture media.
z M. leprae is a obligate intercellular organism that gains access to skin and
pheripheral nerve tissue. Least severe form is tuberculoid tuberculoid (TT)
and the most severe form is lepromatous lepromatous
TERMINAL QUESTIONS
1. Describe the Laboratory Diagnosis of mycobacteria Tuberculosis and
Leprae.
2. Explain Lepromin and Mantoux test.
20.1
1. Fungus like bacteria
2. Acid fast bacilli
3. Mycolic acid
4. Obligate
214 MICROBIOLOGY
20.2 Microbiology
1. M. bovine
2. Atypical mycobacteria
3. Restriction Fragment Length Polymorphism (RFLP)
4. Droplet
5. Ghon focus & hilar lymph nodes Notes
20.3
1. Sputum microscopy
2. Ziehl-Neelson
3. Petroff’s method
4. Absolute Concentration method, Resistance ratio method & Propotion
method
5. Mantoux test
6. Purified Protein Derivative (PPD)
20.4
1. Mycobacterium leprae
2. Hansen’s
3. Globi
4. Granulomatous
20.5
1. Multibacillary
2. Lepromatous type
3. Tuberculoid
4. Paucibacillary disease
20.6
1. Lepromin test
2. Lepromatous
3. Ziehl-Neelson technique
4. Antibodies
MICROBIOLOGY 215
MODULE Escherichia Coli and Klebsiella
Microbiology
21
Notes
ESCHERICHIA COLI AND
KLEBSIELLA
ESCHERICHIA COLI
21.1 INTRODUCTION
Escherichia coli (commonly abbreviated E. coli) is a Gram-negative, facultative
anaerobic, rod-shaped bacterium that is commonly found in the lower intestine
of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but
some serotypes can cause serious food poisoning in humans, and are occasionally
responsible for product recalls due to food contamination. The harmless strains
are part of the normal flora of the gut, and can benefit their hosts by producing
vitamin K, and by preventing the establishment of pathogenic bacteria within
the intestine.
E. coli and related bacteria constitute gut flora, and fecal – oral transmission is
the major route through which pathogenic strains of the bacterium cause disease.
Cells are able to survive outside the body for a limited amount of time, which
makes them ideal indicator organisms to test environmental samples for fecal
contamination.
The bacterium can be grown easily and inexpensively in a laboratory setting, and
has been intensively investigated for over 60 years. E. coli is the most widely
studied prokaryotic model organism, and an important species in the fields of
biotechnology and microbiology, where it has served as the host organism for
the majority of work with recombinant DNA.
216 MICROBIOLOGY
Escherichia Coli and Klebsiella MODULE
Microbiology
OBJECTIVES
z describe the morphology of Escherichia coli and Klebsiella
z describe the Cultural Characteristics of Escherichia coli and Klebsiella
Notes
z explain pathogenesis of Escherichia coli and Klebsiella
z discuss the virulence determinance
21.2 SCIENTIFIC CLASSIFICATION

Domain: Bacteria
Kingdom: Eubacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Escherichia
21.3 HABITAT
Escherichia coli are common inhabitants of the terminal small intestine and large
intestine of mammals. They are often the most abundant facultative anaerobes
in this environment. They can occasionally be isolated in association with the
intestinal tract of nonmammalian animals and insects. The presence of E. coli
in the environment is usually considered to reflect fecal contamination and not
the ability to replicate freely outside the intestine. There is evidence however
to suggest that E. coli may freely replicate in tropical fresh water (Bermudez and
Hazen, 1988).
21.4 MORPHOLOGY
E. coli is Gram-negative, facultative anaerobic and non-sporulating. Cells are
typically rod-shaped, and are about 2.0 micrometers (μm) long and 0.25-1.0 μm
in diameter, with a cell volume of 0.6–0.7 μm. It can live on a wide variety of
substrates.
Strains that possess flagella are motile. The flagella have a peritrichous
arrangement.
MICROBIOLOGY 217
Microbiology
Escherichia coli or E.coli cells may grow on a solid or in a liquid growth medium
under a laboratory condition. Solid and liquid media may have exactly the same
composition except that the solid medium contains an extra 1.5% agar. Different
E.coli clones may have different properties. Colonies growing on solid media
represent different clones.
Notes
Fig. 21.1
Table 21.1
Temperature 37°C MacConkey Agar Eosin-methylene
for 24 hrs blue Agar
Size in mm 1 1
Shape Circular Circular
Color Pink Metallic sheen
Margin Complete Complete
Elevation Slightly Raised Convex
Opacity Opaque Translucent
Consistency Soft Soft
21.6 BIOCHEMICAL REACTIONS

E. coli uses mixed-acid fermentation in anaerobic conditions, producing lactate,
succinate, ethanol, acetate and carbon dioxide. Since many pathways in mixed-
acid fermentation produce hydrogen gas, these pathways require the levels of
hydrogen to be low, as is the case when E. coli lives together with hydrogen-
consuming organisms, such as methanogens or sulphate-reducing bacteria.
218 MICROBIOLOGY
Table 21.2 Microbiology
Test Reactions
Oxidase –
Urease –
TSI Acid butt, with
gas, acid slant Notes
MR +
VP –
Nitrate +
Citrate –
Indole (TW) +
Gelatin –
Key: + = reaction positive

– = reaction negative
Optimal growth of E. coli occurs at 37°C (98.6°F) but some laboratory strains
can multiply at temperatures of up to 49°C (120°F).
21.7 THERAPEUTIC USE OF NONPATHOGENIC E. COLI

Nonpathogenic Escherichia coli strain Nissle 1917 also known as Mutaflor and
Escherichia coli O83:K24:H31 (known as Colinfant are used as a probiotic
agents in medicine, mainly for the treatment of various gastroenterological
diseases, including inflammatory bowel disease.
21.8 ANTIGENIC AND TOXINS

The worst type of E. coli, known as E. coli O157:H7, causes bloody diarrhea
and can sometimes cause kidney failure and even death. E. coli O157:H7 makes
a toxin called Shiga toxin and is known as a Shiga toxin-producing E. coli
(STEC). There are many other types of STEC, and some can make you just as
sick as E. coli O157:H7.
One severe complication associated with E. coli infection is hemolytic uremic

syndrome (HUS). The infection produces toxic substances that destroy red blood
cells, causing kidney injury. HUS can require intensive care, kidney dialysis, and
transfusions.
MICROBIOLOGY 219
Microbiology
21.9 ROLE IN DISEASES
The commonest infection caused by E. coli is infection of the urinary tract, the
organism normally spreading from the gut to the urinary tract. E. coli is also the
commonest cause of cystitis (infection of the bladder), and in a minority of
patients the infection may spread up the urinary tract to the kidneys, causing
pyelonephritis. Otherwise healthy patients in the community may develop
Notes cystitis, and patients in hospital who have catheters, or tubes, placed in the
urethra and bladder are also at risk. E. coli is also present in the bacteria that
cause intra-abdominal infections following leakage from the gut into the
abdomen, as for example with a ruptured appendix or following traumatic injury
to the abdomen.
E. coli bacteria may also cause infections in the intestine. Diarrhoeal infections
(intestinal) are caused by a group of E. coli known as ‘enterovirulent’ (harmful
to the intestines).
Overspill from the primary infection sites to the bloodstream may cause blood
poisoning (E. coli bacteraemia). In rare instances, E. coli may cause meningitis
in very young children.
21.10 LABORATORY DIAGNOSIS

E. coli infections can be diagnosed by the detection of E. coli in a laboratory
test of your stool, urine, blood or other relevant sample. Infections with some
types of E. coli, e.g. E. coli O157, can be detected by a serum antibody test.
Specimen used in lab for E.coli are
1. Urine
2. Stool
Culture
1. Isolation media- a) nutrient agar, b) MacConkey’s agar c) eosin- methylene
blue agar
Biochemical media
z Glucose phosphate broth
z Motility agar
z TSI slant
z Tryptone water
z Simmon’s citrate agar
220 MICROBIOLOGY
z Christensen’s urea medium Microbiology
z Nitrate broth
z Nutrient gelatin medium
z Sugars: xylose, glucose, mannitol, sucrose, maltose, etc
Reagents
Notes
1. Oxidase reagent
2. Hydrogen peroxide
3. Methyl red
4. Kovac’s reagent
5. Sulfanilic acid reagent
6. O’meara reagent
Procedure
1. Perform gram staining on the given culture organisms.
2. Study motility of the organism.
3. Streak a loop-full of the organism on nutrient Agar, and MacConkey’s agar
4. Keep in the incubator at 35 degree C for 24 hrs.
5. Observe for colony characters from both plates.
6. Inoculate into the various biochemical media from a single colony of the
organisms.
8. Read the result.
Observation
1. Gram staining: gram negative bacilli
2. Motility: sluggishly motile
3. Cultural characters
Treatment of E coli infection

Patients, especially healthy adults, often require no treatment for E. coli
O157:H7 since many infections are self–limited. Moreover, if required can be
treated according to antibiotic sensitivity pattern.
MICROBIOLOGY 221
Microbiology

1. E.coli are Gram ................. rods
2. Culturally E.coli are .................
3. E. Coli required ................. in the media
Notes
4. E. Coli is a consistent inhabitant of the ................. tract
21.11 KLEBSIELLA AEROGENS

Klebsiella pneumoniae can be found as a commensal in the mouth and upper
respiratory tract, it is also found in moist environments, particularly in the
intestinal tract of humans and animals. These are also found in plants, water and
soil.
Fig. 21.2
21.12 CLINICAL SIGNIFICANCE

Klebsiella causes pneumonia, urinary infections, septicemia and other pyogenic
infections. Sometimes it also causes diarrhea. K.pneumoniae is generally
associated with lower respiratory tract infections and middle ear infections,
K.aerogenes is generally associated with wounds and urinary tract infections.
Requirements
1. Cultural suspension
2. Isolation media - (a) nutrient agar, (b) MacConkey’s agar (c) eosin-
methylene blue agar
222 MICROBIOLOGY
Biochemical media Microbiology
z Glucose phosphate broth

z Motility agar
z TSI slant
z Tryptone water
z Simmon’s citrate agar
z Christensen’s urea medium Notes
z Nitrate broth
z Nutrient gelatin medium
z Sugars:- xylose, glucose, mannitol, sucrose, maltose, etc
Reagents
1. Oxidase reagent
2. Hydrogen peroxide
3. Methyl red
4. Kovac’s reagent
5. Sulfanilic acid reagent
6. O’meara reagent
Procedure
1. Perform gram staining on the given culture organisms.
2. Study motility of the organism.
3. Streak a loop-full of the organism on nutrient Agar, and MacConkey’s agar
5. Observe for colony characters from both plates.
6. Inoculate into the various biochemical media from a single colony of the
organisms.
8. Read the result.
Specimen
Specimen received in the laboratory are
1. Urine
2. Sputum
3. Ear swabs
4. Wound swabs
MICROBIOLOGY 223
Microbiology Observation
Organism- klebsiella aerogenes
1. Gram staining : gram negative bacilli
2. Motility : non-motile
3. Cultural characters
Notes
Table 21.3
Temperature 37°C Mac Conkey Agar Eosin-methylene
for 24 hrs blue Agar
Size in mm 3-4 3-4
Shape round round
Color Pink pink
Margin Complete Complete
Elevation Slightly Raised slightly raised
Opacity Translucent Translucent
Consistency Mucoid Mucoid
Table 21.4
Test Organism K.aerogenes Organism K.pneumoniae
Reactions Reaction
Oxidase – –
Urease + + slow
TSI acid slant, with gas, acid slant, with gas,
acid butt acid butt
MR – +
VP + –
Citrate + +
Indole (TW) – –
Gelatin – –
Nitrate + (delayed) + (delayed)
224 MICROBIOLOGY
Microbiology

1. Enterotoxigenic E.coli (ETEC) causes diarrhea among .............. & ..............
2. Dysentry like diarrhea with fever are caused by ..............
3. Haemorrhagic colitis caused by Enterohemorrhagic E.Coli may lead to
Notes
..............
4. Klebsilla are .............. shaped and gram .............. bacteria
5. Pathogenesis of klebsiella is determined by ..............
6. Klebsiella commonly causes .............., .............. & ..............

z Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped
bacterium that is commonly found in the lower intestine of warm-blooded
organisms (endotherms).
z E. coli and related bacteria constitute gut flora, and fecal–oral transmission
is the major route through which pathogenic strains of the bacterium cause
disease.
z E. coli are common inhabitants of the terminal small intestine and large
intestine of mammals. They are often the most abundant facultative
anaerobes in this environment
z E.coli cells may grow on a solid or in a liquid growth medium under a
laboratory condition. Solid and liquid media may have exactly the same
composition except that the solid medium contains an extra 1.5% agar
z E. coli uses mixed-acid fermentation in anaerobic conditions, producing
lactate, succinate, ethanol, acetate and carbon dioxide
z Optimal growth of E. coli occurs at 37°C (98.6°F) but some laboratory
strains can multiply at temperatures of up to 49°C (120°F).
z The commonest infection caused by E. coli is infection of the urinary tract,
the organism normally spreading from the gut to the urinary tract
z E. coli infections can be diagnosed by the detection of E. coli in a laboratory
test of your stool, urine, blood or other relevant sample
MICROBIOLOGY 225
Microbiology z Klebsiella pneumoniae can be found as a commensal in the mouth and upper
respiratory tract, it is also found in moist environments, particularly in the
intestinal tract of humans and animals. These are also found in plants, water
and soil.
z Klebsiella causes pneumonia, urinary infections, septicemia and other
pyogenic infections. Sometimes it also causes diarrhea
Notes z Biochemically they are Oxidase negative, Urease positive
TERMINAL QUESTIONS
1. Describe the morphology & cultural characteristic E.coli.
2. Describe the virulence determinants of pathogenic E.coli
3. Describe the intestinal diseases caused by E.coli
4. List the cultural characteristics of Klebsiella
21.1
1. Negative
2. Facultative anaerobes
3. glucose
4. intestinal
21.2
1. Infants & travellers
2. Enteroinvasice E.coli
3. Haemolytic uremic syndrome
4. Rod, negative
5. Polysaccharide capsule
6. Community acquired pneumonia, urinary tract infection & Nosocomial
infection
226 MICROBIOLOGY
Citrobacter, Edwardsiella, Enterobacter, and Serratia MODULE
Microbiology
22
Notes
CITROBACTER, EDWARDSIELLA,
ENTEROBACTER AND
SERRATIA
22.1 INTRODUCTION
These are members of Enterobacteriaceae. They are gram negative bacilli, motile
with peritrichous flagella, non-sporing, non-acid fast. They are oxidase negative,
catalase positive and reduce nitrates to nitrites. They are considered as
environmental contaminants and normally isolated from soil, water and faeces of
man and animals.
OBJECTIVE
z describe characteristics of Citrobacter, Edwardsiella, Enterobacter, and
Serratia
z describe pathogenicity of Citrobacter, Edwardsiella, Enterobacter, and
Serratia.
z differentiate between Citrobacter, Edwardsiella, Enterobacter, and Serratia.
22.2 CITROBACTER
It belongs to tribe Citrobacteriaceae of Enterobacteriaceae family. Members of
this genus are motile, grow well on ordinary media producing smooth, convex,
non pigmented 2-3 mm colonies. On MacConkey agar they form pale to pink
coloured colonies due to fermentation of lactose which can be late. Capsule may
be present. Genus has three species: C.freundii, C.koseri, and C.amalonaticus.
They are indole positive except C.freundii, MR positive, VP negative, Citrate
MICROBIOLOGY 227
MODULE Citrobacter, Edwardsiella, Enterobacter, and Serratia
Microbiology
positive, urease weakly positive, H2S is produced by C.freundii. Mannitol
fermentation is always positive. Lactose fermentation may or may not be
positive but they always produce β-galactosidase (ONPG positive).
Notes
Fig. 22.1
22.2.1 Pathogenecity
They are normal commensals of human gastrointestinal tract. Pathogenecity can
be due to endotoxins, O (somatic) and H (flagellar) antigen, capsular antigen,
adhesion proteins produced by bacteria. They share O Ag with Salmonella and if
isolated in faecal samples, they can be mistaken as Salmonella. They can cause
UTI, infection of gall bladder, middle ear. C.koseri may occasionally cause
neonatal meningitis.
22.3 EDWARDSIELLA
It belongs to tribe Edwardsielleae. Edwardsiella tarda is the only recognised
human pathogen. It is non-capsulated, motile, produce hydrogen sulphide in KI
medium. On MacConkey agar they form pale coloured colonies which can
become pink on further incubation due to fermentation of lactose which can be
late. Term tarda refers to slow or weak fermentation of sugars by this organism.
Only glucose and maltose are fermented. It is indole positive, utilise citrate,
urease negative.
Fig. 22.2
228 MICROBIOLOGY
22.3.1 Pathogenicity Microbiology
Normal habitat is intestine of cold blooded animals and fresh water. It is mainly
pathogenic to water animals. It causes occasional infection in humans. Its
pathogenic role is uncertain but it has been isolated from wound, urine, blood
and CSF.
22.4 ENTEROBACTER Notes

It belongs to tribe Klebsielleae. Members of this genus are motile, capsulated,
form pink coloured mucoid colonies on MacConkey agar. It is MR negative, VP
positive, indole negative, citrate positive. It does not produce H2S. It has 12
species of which E.aerogenes and E.cloacae are most commonly encountered in
clinical specimen. E.aerogenes is urease negative while E.cloacae is urease
positive.
22.4.1 Pathogenicity
Both the species are widely distributed in water, sewage, soil and on vegetables.
They are associated with opportunistic infection including UTI, respiratory tract
infection and cutaneous wounds. They may occasionally cause meningitis and
septicaemia. They are an important cause of hospital acquire infections.
22.5 SERRATIA
It belongs to tribe Klebsielleae. They are motile, gram negative coccobacilli.
They may form capsule. Several species have been described of which
S.liquifaciens, S.rubidaea and S.marcescens are of clinical significance.
S.marcescens is the most frequently encountered species. It forms smooth,
convex colonies with crenated edges. It form red coloured pigment (prodigiosin)
which is insoluble in water and does not diffuse into the media. Therefore
colonies are red to pink in colour. Pigment is soluble in alcohol, ether, acetone
and chloroform. Pigment is best formed at 15-20°C however, growth is poor at
this temperature.
Fig. 22.3 Serratia marcescens
MICROBIOLOGY 229
MODULE Citrobacter, Edwardsiella, Enterobacter, and Serratia
Microbiology Serratia species are ONPG positive but fail to ferment lactose. Form pale
coloured colonies on MacConkey agar. It is indole, MR and urease negative,
citrate and VP positive. It does not form H2S.
22.5.1 Pathogenicity
It is basically a saprophyte, found in water, soil and food. It can cause nosocomial
Notes infection mainly in newborns and patients receiving immunosuppressive therapy
which are increasing in frequency now days. It is associated with meningitis,
endocarditis, septicaemia, peritonitis and respiratory tract infections. Multidrug
resistance is common making it serious pathogens in hospital setting.
Table 22.1: Characteristics of Citrobacter, Edwardsiella,

Enterobacter and Serratia.
Characteristics Citrobacter Edwarsiella Enterobacter Serratia

Habitat Human gut Intestine of cold Water, soil, Saprophyte, water
blooded animals sewage and soil
Colonies on MCA Pale to pink Pale pink pale

Motility + + + +
Capsule ± _ + +
ONPG + + + +
Lactose ± +(slow) + _
Indole ± + _ _
MR + + _ _
VP _ _ + +
Citrate + + + +
Urease _ _ + _
H2S production + + _ _

1. Citrobacter are gram ................ bacilli
2. Enterobacteria are enviromental contamiants commonly present on ................,
................ & ................
3. Citrobacter are mannitol ................ bacilli
4. Serrratia causes nosocomial infection in ................ and patients receiving
................ therapy.
230 MICROBIOLOGY
Microbiology

z They are important members of enterobacteriaceae. They occur mainly as
either normal commensal of GIT or as saprophyte in the environment.
Infections caused by this genus are nowdays increasing in frequency. They
are often multidrug resistant and difficult to treat. They can be easily
differentiated from each other on the basis of colony morphology and Notes
biochemicals. Citrobacter is often mistaken as Salmonella. Enterobacter
colonies do look like that of Klebsiella but it can be differentiate from it
by its motility.
TERMINAL QUESTIONS
1. Describe the characteristics of genus Edwardsiella?
2. Write pathogenesis of Citrobacter?
3. Describe the colonies and morphology of Enterobacter?
4. Name the pigment produced by S.marsecens?
22.1
1. Negative
2. Water, soil, faeces
3. Fermenting
4. Newborns & immunosuppresive
MICROBIOLOGY 231
MODULE Salmonella
Microbiology
23
Notes
SALMONELLA
23.1 INTRODUCTION
Salmonella consists of bacilli leading to Enteric fever, Gastroenteritis, Speticemia
etc. The important member of the genus is Salmonella typhi, which causes
Typhoid fever.
OBJECTIVES
z describe the morphology of Salmonella
z discuss the cultural characteristics of Salmonella
z explain the biochemical reactions of Salmonella
z demonstrate the Widal reaction.
Salmonella are of two groups;
(i) Enteric fever group consisting of typhoid & Paratyphoid bacilli exclusively
or primary human parasites
(ii) Food poisoning group, which are animal parasite but may infect humans
causing Gastrointestinal infections
Morphology
Salmonellae are gram negative rods. They are motile with peritrichate flagella
except for S. gallinarum pullorum
232 MICROBIOLOGY
Salmonella MODULE
Microbiology
Notes
Fig. 23.1
Cultural Characteristics
Salmonellae are aerobic and facultatively anaerobic bacteria growing readily on
simple media over a range of pH 6-8 & temperature 15-41°C with optimum
temperature of 37°C. Colonies are large, circular and smooth on MacConkey and
Deoxycholate citrate media, colonies are colourless due to absence of lactose
fermentation. Selenite F and Tetrathionate broth are commonly employed as
enrichment media.
Biochemical reaction
Salmonellae ferment glucose, mannitol and maltose forming acid and gas.
Whereas S. typhi is an aerogenic i.e. it does not form fermentation of sugars like
glucose etc. Lactose, Sucrose and Salicin are not fermented. Indole is not
produced. They are MR positive, VP negative and citrate positive.
Resistance
The bacilli are killed at 55oC in one hour or at 60oC in 15 minutes. Boiling or
chlorination of water and pasteurization of milk destroy the bacilli. In polluted
water it may survive for weeks and in ice for months.
Antigenic Structure
Salmonellae possess the antigens and based on which they are classified as
(i) flagella antigen H,
(ii) Somatic antigen O and
(iii) surface antigen Vi
H antigen
This antigen present on flagella is heat labile protein. It is destroyed by boiling or
by treatment with alcohols but not by formaldehyde.
MICROBIOLOGY 233
MODULE Salmonella
Microbiology O antigen
O antigen is a Phospholipid-protein-polysaccharide complex which forms an
integral part of the cell wall. It is identical with endotoxin. This is unaffected by
boiling, alcohol or weak acids
Notes
1. Salmonellae are gram ................ rods
2. Culturally salmonellae are facultative ................
3. ................ & ................ broth are commonly used as enrichment media for
salmonellae
4. Flagella antigen is ................ & somatic antigen is ................
5. ................ antigen is unaffected by boiling, alcohol & weak acids
23.2 CLASSIFICATION AND NOMENCLATURE

Classification within the genus is on antigenic characterisation based on
Kauffman-White scheme and this depends on identification by agglutination of
the O and H antigens of the strains. Salmonellae are classified into serological
groups based on the presence of distinctive O antigen factors and designated as
1, 2, 3 etc
Biochemically Kauffman proposed Salmonellae classification as
Subgenus I: Largest and medically most important group causing human and
animal infections
Subgenus II: Species isolated from reptiles.
Subgenus III: Species isolated from reptiles and human beings
Subgenus IV: These are rarely encountered.
Pathogenecity
Salmonellae cause the following clinical syndrome in human beings
1. Enteric fever
2. Gastroenteritis or food poisoning
3. Septicemia with or without local suppurative lesions
234 MICROBIOLOGY
Salmonella MODULE
I. Enteric Fever Microbiology
This includes typhoid fever caused by S.typhi and paratyphoid fever caused by S.
Paratyphi A,B,C. the infection is acquired by ingestion of contaminated food, on
reaching the gut the bacilli attach to microvilli of the ileal mucosa and penetrate
submucosa. They are phagocytosed by polymorphs and macrophages. Their
ability to resist intercellular killing and to multiply within the cells is a measure
of their virulence. They enter the mesenteric lymph nodes, where they multiply
and via thoracic duct, enter the blood stream causing bacteremia. Notes
Fig. 23.2
As bile is a good culture medium for the bacillus it multiplies abundantly in the
gall bladder and is discharged continuously into the intestine where it involves
the Peyer’s Patches and lymphoid follicles of the ileum, which ulcerate and may
lead to intestinal perforation & haemorrhage as complication. The incubation
period is usually 7-14 days but may range from 3-56 days.
Bacteriological diagnosis of enteric fever consists of isolation of the bacilli and
demonstration of antibodies in serum. A positive blood culture is diagnostic;
demonstration of antibodies is not conclusive of current infection. A third
method is the demonstration of typhoid bacilli in blood or urine.
Blood culture
Bacteremia occurs early in the disease and blood cultures are positive mostly in
the first week of fever. About 5-10ml of blood is collected and inoculated into
culture bottle containing 50-100ml of 0.5 percent bile broth. After incubation
overnight at 37oC, the bile broth is subcultured on MacConkey agar, pale non-
lactose fermenting colonies that may appear on this medium are picked up for
biochemical tests and motility. Salmonellae are motile, indole and urease
negative and ferment glucose, mannitol and maltose but not lactose or sucrose.
MICROBIOLOGY 235
MODULE Salmonella
Microbiology The typhoid bacillus will be anaerogenic, while the paratyphoid bacilli will form
and gas from sugars. Identification of the isolate is by slide agglutination. A
loopful of the growth from an agar slope is emulsified in two drops of saline on
a slide. One emulsion acts as a control to show that the strain is not auto
agglutinable.
If Salmonellae are not obtained from first subculture from bile broth, subcultures
should be repeated every other day till growth is obtained. Cultures should be
Notes declared negative only after incubation for ten days.
Feces culture
Salmonellae are shed in feces throughout the course of disease and even in
convalescence, with varying frequency. A positive fecal culture, however may
occur in carriers as well as in patients. The use of enrichment and selective media
and repeated sampling increase the rate of isolation.
Fecal samples are plated directly on MacConkey, DCA and Wilson-Blair media.
On MacConkey and DCA it appears as pale colonies. On Wilson-Blair medium
S typhi forms large black colonies. S paratyphi A produces green colonies on this
medium.
Urine culture
Salmonellae are shed in urine irregularly and infrequently. Hence urine culture is
less useful than culture of blood or feces. Cultures are generally positive in
second and third weeks.
Widal reaction
This is a test for measurement of H and O agglutinins for typhoid and
paratyphoid bacilli in the patient’s sera. Two types are generally used for the test-
a narrow tube with a conical bottom (Dreyer’s agglutination tube) for H
agglutination and short round-bottomed tube (Felix tube) for O agglutination.
Equal volumes (0.4 ml) of serial dilutions of the serum and the H and O antigens
are mixed in Dreyer’s and Felix agglutination tubes and incubated in a water bath
at 37°C overnight. Control tubes containing the antigen and normal saline are set
to check for autoagglutination. The agglutination titres of the serum are read. H
agglutination leads to the formation of loose, cotton woolly clumps, while O
agglutination is seen as a disc-like pattern at the bottom of the tube.
Salmonellae gastroenteritis
Salmonellae gastroenteritis or food poisoning is zoonotic disease, the source of
infection being animal products and may be caused by any Salmonellae except
S typhi.
236 MICROBIOLOGY
Salmonella MODULE
Human infection results from ingestion of contaminated food and most common Microbiology
source of food poisoning are poultry, meat, milk products. Salmonellae can enter
through the shell if eggs are left on contaminated chicken feed or feces and grow
inside.
Laboratory diagnosis is made by isolating the Salmonellae from feces and from
the article of food which confirms the diagnosis.
Notes
Salmonellae Septicemia
S choleraesuis in particular, may cause septicemic disease with focal suppurative
lesions such as osteomyelitis, deep abscesses, endocarditis, pneumonia and
meningitis.

1. Enteric fever is acquired by ingestion of ................
2. ................ & ................ are complication of Enteric Fever
3. ................ is a good culture medium for salmonellae
4. ................ tube is used for H agglutination
5. ................ tube is used for O agglutination

z Salmonella belong to the family Enterobacteriaceae
z Salmonella are gram-negative, facultatively anaerobic, motile rods that are
catalase positive and oxidase negative.
z Salmonella species are responsible for enteric fever spread only from human
to human. Water contaminated with feces is a common source.
z The bacilli invade mucos cells in small intestine, transported through
lymphatics and reach blood stream.
z For diagnosis, samples of feces or blood are plated on solid medium, such
as MacConkey agar and Salmonellae-Shiella agar which yields gram-
negative bacilli with flagella.
z The organisms are identified by biochemical characteristics and by slide
agglutination tests using reference antibody to O, H, vi. antigens.
MICROBIOLOGY 237
MODULE Salmonella
Microbiology
TERMINAL QUESTIONS
1. Describe the cultural characteristics of salmonellae
2. Explain the laboratory diagnosis of enteric fever by Blood culture
3. Describe Widal reaction
Notes 4. Write about the prevention of Salmonella infection
23.1
1. Negative
2. Aerobes
3. Selenite f & tetrathionate
4. H & O
5. O
23.2
1. Contaminated food
2. Intestinal perforation & Haemorrhage
3. Bile broth
4. Felix
238 MICROBIOLOGY
Shigella MODULE
Microbiology
24
Notes
SHIGELLA
24.1 INTRODUCTION
Bacillary dysentery is caused by genus Shigella, named after Shiga who isolated
them.
OBJECTIVES
z describe the characteristics of Shigella species
z classify Shigella species
z describe the laboratory diagnosis of shigellosis
24.2 MORPHOLOGY
Shigellae are short, Gram-negative rods. They are non-motile, non sporing and
non capsulated
They are aerobes and facultative anaerobes, with growth temperature range of
10-40°C and optima of 37°C and pH 7.4. They grow on ordinary media.
Deoxycholate citrate Agar (DCA) is a useful selective medium. Growth is
inhibited on Wilson and Blair’s bismuth sulphite medium
Resistance
Shigella are not specially resistant. They are killed at 56°C in one hour and by 1%
phenol in 30 minutes. In ice they last for 1-6 months. They remain viable in moist
environment, in faeces they die within few hours due acidity produced by growth
of coliforms.
MICROBIOLOGY 239
MODULE Shigella
Microbiology Biochemical reactions

Shigella are MR positive and reduce nitrates to nitrites. Catalase is produced,
except in Sh.dysenteriae type I. glucose is fermented with the production of acid,
without gas. Fermentation of mannitol is of importance in classification & based
on these shigella is divided as mannitol fermenting and non-fermenting species.
Notes
Fig. 24.1

1. Shigellae are Gram .............. rods
2. Culturally shigellae are facultative ..............
3. .............. agar is used as a selective medium
4. Shigella is divided as .............. & .............. species
24.3 CLASSIFICATION
Shigella are classified into four species based on combination of biochemical
and serological characteristics
Sh.dysenteriae (subgroup A)
This is mannitol nonfermenting bacilli consisting of ten serotypes. It is indole
negative and is always catalase negative. Sh.dysenteriae type 1 forms a toxin.
Three types of toxic activity have been demonstrated
(i) Neurotoxicity
(ii) Entereotoxicity
(iii) Cytotoxicity
240 MICROBIOLOGY
Shigella MODULE
Sh. dysenteriae type 2 forms indole and ferments sorbitol and rhannose Microbiology
Sh. Flexneri (subgroup B)

These are mannitol fermenting species, which are biochemically heterogeneous
and antigenically complex.
Sh. Boydii (subgroup C)
Notes
This group consists of dysentery bacilli and named after Boyd who described
this
Sh. Sonnei (Subgroup D)
This was described by Sonne, ferments lactose and sucrose late. It is indole
negative, this causes mildest form of bacillary dysentery.
Pathogenecity
Shigellae cause bacillary dysentery. Infection occurs by ingestion. The minimum
infective dose is as low as 10-100 bacilli as they can survive gastric acidity.
Human beings are the only natural hosts for Shigella
Bacillary dysentery has short incubation period 1-7 days usually 48 hours. The
onset and clinical course are variable and are largely determined by the virulence
of the strain. The clinical features are frequent passage of loose, scanty feces
contacting blood and mucus with abdominal cramps and tenesmus. Complications
include arthiritis, toxic neuritis, conjunctivitis, parotitis and intussusceptions,
Hemolytic Uremic Syndrome may also occur. Shigellosis includes a whole
spectrum of disease caused by shigellae.
Subgroup A B C D
Species Sh.dysenteriae Sh.flexneri Sh.boydii Sh.sonnei
Mannitol - A A A
Lactose - - - A late
Sucrose - - - A late
Dulcitol - - D -
Indole d d d -
Ornithine decarboxylase - - - +
A – acid, d – variable
MICROBIOLOGY 241
MODULE Shigella
Microbiology Laboratory diagnosis

Diagnosis may be made by isolating bacilli from feces. Fresh feces should be
inoculated without any delay or transported in medium such as Sachs’ buffered
glycerol saline, pH 7.0 – 7.4. For innoculation it is best to use mucus containing
feces. MacConkey and DCA plates are inoculated. After overnight incubation
at 37°C, the plates are inspected for nonlactose fermenting colonies, which are
tested for motility and biochemical reactions.
Notes

1. Shigella causes ..............
2. Natural host of shigella are ..............
3. Incubation period of Bacillary dysentery is ..............
4. .............. is the spectrum of disease caused by shigellae

z Shigella is a genus of the family Enterobacteriacae, which are rod shaped
bacteria that are nonmotile facultatively anaerobic usually catalase positive
and oxidase negative and Gram negative bacteria.
TERMINAL QUESTIONS
1. Describe the cultural characteristics of shigella
2. Classify shigella and explain any two species in detail
3. Describe the laboratory diagnosis of shigella
24.1
1. Negative
2. Anaerobes
242 MICROBIOLOGY
Shigella MODULE
3. Deoxycholate Citrate Microbiology
4. Mannitol fermenting & mannitol non-fermenting
24.2
1. Bacillary dysentery
2. Human beings
Notes
3. 1-7 Days
4. Shigellosis
MICROBIOLOGY 243
MODULE Proteus and Providencia
Microbiology
25
Notes
PROTEUS AND PROVIDENCIA
25.1 INTRODUCTION
The genera Proteus and Providencia belong to the tribe Proteae of the family
Enterobacteriaeceae. The members of both these genera are gram negative,
motile bacilli, aerobes and facultative anaerobes and can grow on basic media.
A characteristic feature which distinguishes tribe Proteae from other members
of Enterobactereaceae is the presence of the enzyme phenylalanine-deaminase
which converts phenylalanine to phenylpyruvic acid (PPA reaction).They also
produce a powerful urease enzyme which rapidly hydrolyses urea to ammonia.
OBJECTIVES
z define the tribe Proteae and distinguish it from the other members of
Enterobacteriaceae.
z differentiate among the various members of this tribe with the help of
various biochemical reactions.
z discuss the pathogenecity, laboratory diagnosis and treatment of Proteus and
Providencia.
25.2 GENUS PROTEUS

They are gram-negative bacilli, 1-3 µm long and 0.6 µm wide. They are non-
capsulated and are actively motile by peritrichous flagella. The name ‘Proteus’
refers to their pleomorphism, after the Greek God Proteus who could assume
any shape. Four species: Proteus mirabilis, P vulgaris, P penneri and P
244 MICROBIOLOGY
Proteus and Providencia MODULE
myxofaciens are recognized. Proteus mirabilis, P. vulgaris are widely recognised Microbiology
as human pathogens.
25.3 CULTURE CHARACTERISTICS

These can grow on ordinary media like nutrient agar with a characteristic fishy
or seminal odour. On MacConkey and Teepol lactose agar, lactose non-
fermenting pale colonies, around 2-3 mm in size are formed. On non-inhibtory Notes
solid media such as blood and nutrient agar Proteus mirabilis and P vulgaris
show characteristic swarming growth in the form of a uniform film, which
spreads over the whole surface of the plate. In young swarming cultures, many
of the bacteria are long, curved and filamentous, sometimes reaching upto 80
µm in length. When two different strains of swarming proteus mirabilis encouter
one another on an agar plate, swarming ceases and a visibile line of demarcation
forms. This is known as the Dienes phenomenon.
Swarming inhibitory methods: Swarming of Proteus can be prevented by
z Increasing the concentration of agar from 1-2% to 6%.
z Incorporation of sodium azide, boric acid, or chloral hydrate.
z Introducing growth inhibitors like sulphonamides.
z On Teepol Lactose agar by Teepol(surface active agent)
z On MacConkey agar or DCA by presence of bile salts.
z On CLED agar by the absence of electrolytes.
In liquid medium (peptone water, nutrient broth), Proteus produces uniform
turbidity with a slight powdery deposit and an ammonical odour.
Fig. 25. 1: Swarming of Proteus
MICROBIOLOGY 245
Microbiology
Notes
Fig. 25. 2: Dienes phenomenon

Like all other members of the family Enterobacteriaceae, all the species of genus
Proteus are catalase positive, oxidase negative, reduce nitrates to nitrites and
show fermentative reaction on Hugh Leifson’s of media. All members of the
tribe Proteeae are PPA positive and, hydrolyse urea to ammonia which
differentiates them from other Enterobacteriaeceae.
Table 25.1: Biochemical Reactions of Tribe Proteeae
Biochemical Proteus Proteus Providencia Providencia Providencia
reaction mirabilis vulgaris stuartii rettgeri alcalifaciens
Indole - + + + +
Methyl red + + + + +
Voges Proskauer - - - - -
Citrate+/- - + + +
Urease+ + +/- + -
H2S production + + - - -
Gas from glucose + + - - +
25.5 ANTIGENIC STRUCTURE

The bacilli possess thermostable ‘O’ antigen (somatic) and thermolabile ‘H’
(flagellar) antigen. Weil and Felix observed that certain non-motile strains of P
vulgaris, called the ‘X’ strains, were agglutinated by sera from patients with
typhus fever. This heterophile agglutination due to the sharing of carbohydrate
antigen by certain strains of Proteus and Rickettsia forms the basis of the Weil
Felix reaction and is used for diagnosing rickettsial infections. Three non-motile
Proteus strains OX-2 and OX-19 of P vulgaris and OX-K of P mirabilis are used
in the agglutination test.
25.6 TYPING METHODS

z Serotyping
z Phage typing
246 MICROBIOLOGY
z Dienes typing Microbiology
z Bacteriocin(proticin) typing
Dienes phenomenon: This method forms the basis of typing swarming strains
of Proteus for local epidemiological studies. Different cultures are inoculated
as discrete spots on the same plate and allowed to swarm towards one another.
A line of complete or partially inhibited growth is formed where cultures of
different strains meet; no line is formed between culture of the same strain. Notes
25.7 PATHOGENECITY
Proteus species are saprophytic and widely distributed in nature. They also occur
as commensals in the intestine. They are opportunistic pathogens and may cause
many types of infections such as:
z Urinary tract infections (UTI) with predilection for upper UTI. It produces
urease which liberates ammonia from urea. The alkaline conditions lead to
the precipitation of phosphates and the formation of calculi in the urinary
tract.
z Pyogenic lesions
z Wound infections
z Bed sores
z Otitis media
z Meningitis
z Septicaemia
z Osteomyelitis

(i) Specimen:
z UTI- midstream urine
z Wound/ abscesses, osteomyelitis, otitis media: pus
z Meningitis: CSF
z Septicaemia – blood culture
(ii) Culture: Clinical specimens should be cultured on MacConkey agar/
Teepol lactose agar, 6% blood agar and in case of urine on CLED (Cysteine
lactose electrolyte deficient agar). Culture media are incubated at 37ºC
for 18-24 hours. Pale coloured Non-Lactase Fermenting (NLF) colonies
are seen on MacConkey agar. Identification is done by standard biochemical
reactions mentioned above.
MICROBIOLOGY 247
Microbiology
(iii) Antibiotic susceptibility: Proteus are resistant to many of the common
antibiotics, except P mirabilis which is sensitive to ampicillin and
cephalosporins but nitrofurantoin is not effective.
25.9 GENUS PROVIDENCIA

Like Proteus, strains of Providencia are Non-lactase fermenting (NLF), methyl
Notes red and PPA positive bacilli which are motile by peritrichous flagella. However,
they do not swarm on solid media. They can often be recognized by their ‘fruity’
smell. Three important pathogenic species include Prov alcalifaciens, Prov
rettgeri and Prov stuartii. It has been suggested that Prov alcalifaciens causes
diarrhea, Prov rettgeri and Prov stuartii have been associated with hospital-
acquired urinary-tract, wound and other infections. Providencia are very
resistant to antibiotics, particularly Prov stuartii which is also resistant to
disinfectants, making it a major pathogen in burn units.
1. Proteus are Gram .............. bacilli

2. Name proteus refers to ..............
3. Proteus are catalase .............. and oxidase ..............
4. .............. phenomenon is the basis of typing swarming strains of proteins.
z This unit summarizes for you the features of tribe Proteae which include
the genera Proteus and Providencia. All the members of the tribe Proteae
are gram negative, non-capsulated, lactose non-fermenting, motile bacilli
with a characteristic swarming growth of genus Proteus on blood and
nutrient agar. The distinguishing test for the tribe is PPA. They are mainly
saprophytes, but may lead to various infections, particularly UTI with
predilection to the upper urinary tract and the risk of formation of phosphate
stones.
248 MICROBIOLOGY
Microbiology
TERMINAL QUESTIONS
1. What are the swarming inhibitory factors?
2. Discuss the Weil-Flix reaction.
3. What is dienes phenomenon?
Notes
4. What are the biochimical reactions of Proteus
25.1
1. Negative
2. Pleomorphism
3. Positive, Negative
4. Dienes
MICROBIOLOGY 249
MODULE Yersinia
Microbiology
26
Notes
YERSINIA
26.1 INTRODUCTION
Genus Yersinia belongs to tribe Yersinieae of the family Enterobacteriaceae.
Yersinia are Gram-negative rod shaped bacteria and are facultative anaerobes.
Important human pathogens are Yersinia pestis, Y. enterocolitica and Y.
pseudotuberculosis.
OBJECTIVES
z differentiate characteristics between various species of Yesinia.
z describe diseases caused by Yersinia species.
z discuss the laboratory diagnosis of plague.
26.2 YERSINIA PESTIS

Yersinia Pestis: Causative agent of plague.
26.2.1 General characterstics

Gram negative bacilli or coccobacilli, with rounded ends, convex or parallel
sides. Non motile.
Capsule present when grown at 37ºC. Bipolar staining (safety pin appearance)
with methylene blue or giemsa stain.
250 MICROBIOLOGY
Yersinia MODULE
Microbiology
Notes
Fig. 26.1: Bipolar straining (safety pin appearance)
Pleomorphic when grown in unfavourable conditions (nutrient agar with 3%

sodium chloride).
Grow on ordinary media. Colonies are dark brown on blood agar because of
absorption of haemin. On Maconkey agar NLF colonies are produced. Optimum
temperature 27ºC
Liquid media- granular deposit and surface pellicle. Shows stalactite growth if a
drop of sterile oil/gheebroth is allowed to float on broth and the medium is not
disturbed growth hangs down from oil/ghee into the liduid medium which looks
like stalactites.
Catalase: Positive
Oxidase, Urease, and Indole: Negative
26.2.2 Antigen, toxins and other virulence factors

z Fraction-1 or F1: heat labile protein envelope antigen ⇒ antiphagocytic
z V and W antigens: always produced together ⇒inhibits phagocytosis and
intracellular killing,
z Pesticin 1 (bacteriocin), fibrinolysin ,coagulase : inhibits strains of Y.
enterocolitica and Y. Pseudotuberculosis and E.coli
z Plague toxin (endotoxin -LPS and Murinetoxin)
z Ability to synthesize purine

1. Yersinia is gram ................ bacilli.
2. Yersinia are catalase ................
3. ................ appearance is seen with methylene blue stain.
4. Yersinia are culturally facultative ................
MICROBIOLOGY 251
MODULE Yersinia
Microbiology 26.2.3. Pathogenesis

Causative agent of plague. It is a zoonotic disease. Rodents are the natural
reservoirs. It is transmitted through a bite of an infected rat flea (Xenopsylla
species), but can also be transmitted by air (especially during pandemics of the
disease).
Rat fleas become infected after taking blood meals from septicemic animals. Y.
Notes pestis grows in the midgut and eventually blocks the proventriculus, starving the
flea for blood. The insects attempt to feed more often but end up giving back
infected blood into the wound. Rat flea can’t fly. It jumps to a height of <2 feet,
usually biting on the legs of humans.
Fig. 26.2: Rat flea
Plague can occur in 3 forms:

(a) Bubonic plague: Incubation period of 2–6 days. General malaise, fever,
headache and chills occur suddenly at the end of the incubation period.
Swelling of lymph nodes resulting in buboes, the classic sign of bubonic
plague. The inguinal nodes are most frequently affected (“boubon” is
Greek for “groin.”)
Fig. 26.3: Bubonic plague showing bubos on wrist.
252 MICROBIOLOGY
Yersinia MODULE
(b) Pneumonic plague: Spread person to person by droplet infection as the Microbiology
bacilli spread through the lungs producing haemorrhagic pneumonia. The.
bloody mucoid sputum that is coughed out contain enormous number of
bacilli Fever, chills, cough, chest pain, dyspnea, hemoptysis, hypotension
and shock.This type is highly infectious.
(c) Septicemic plague: End stage of bubonic or pneumonic plague but may
occur primarily also. Hypotension, fever, hepato-splenomegaly, delirium,
Notes
seizures in children and shock. Symptoms of bubonic or pneumonic plague
are not always present. Patient may die before any symptoms appear.
Also called as ‘Black death’.
26.2.4 Laboratory diagnosis

Specimen
z For pneumonic plague Sputum /Bronchial wash/ tracheal aspirate,
z For septicemic plague - blood.
z For bubonic plague - Aspirate or biopsy of bubo.
Microscopy: Exudates/ sputum/ other specimens can be stained by gram stain
and methylene blue to look for characterstic safetypin morphology.
Culture: Exudates/other specimen cultured on blood agar. Perform biochemical
testing for identification of the isolate. Blood culture can be done in septicemic
cases.
z GNB, non motile, NLF
z Catalase +ve, oxidase –ve,
z Ferments glucose , maltose, sucrose, and mannitol anaerogenically.
z MR +ve,
z Indole, VP, urease, and Citrate –ve,
z Gelatin is not liquefied.
Animal pathogenicity: Guinea pigs or white rats.Diagnosis in rats: rat is
immersed in disinfectant like 3% Lysol and necropsy is performed to look for
following characterstic features-
z Enlarged lymph nodes especially cervical.
z Subcutaneous injection
z Pleural effusion
MICROBIOLOGY 253
MODULE Yersinia
Microbiology z Granular congested liver

z Enlarged spleen with some white areas
z Microscopy and culture of heart blood, spleen and liver also done.
26.2.5. Prevention
Control of rat flea and rats using pesticides and rat poison.
Notes
Vaccines:
1. Killed vaccine containing 2000 million bacteria/ ml. Protects for about 6
months after 3 doses.This is used in India.
2. Live vaccine using avirulent strains of Y. Pestis is no longer recomended.
26.2.6. Treatment
Aminoglycosides such as streptomycin and gentamicin, tetracyclines and
fluoroquinolones can be given.
26.3 YERSINIA ENTEROCOLITICA

Causative agent of gastroenteritis, mesenteric lymphadenitis and septicaemia.
Laboratory diagnosis is by isolation of organism from blood, lymph node, feces,
food or soil or by serology using tube agglutination test. Cold enrichment often
helps.
The organism is motile at 25°C but non motile at 37°C.
26.4 YERSINIA PSEUDOTUBERCULOSIS

Causative agent of pseudotuberculosis (a zoonosis).
Causes mesenteric lymphadenitis and erythema nodosum especially in young
males. Diagnosis is by serology using tube agglutination test (for antibody
detection).
This organism is motile at 25°C but non motile at 37°C and hydrolyses urea and
has relatively poor growth on MaConkey agar.

1. Yersinia causes ................... in human.
2. ................... are the natural reservoirs of yersinia.
3. Septicemic plague is also called as ...................
4. ................... agglutination test is used to diagnosis serology.
254 MICROBIOLOGY
Yersinia MODULE
Microbiology

z This chapter deals with various Yersinia species i.e Y.pestis, Y. enterocolitica
and Y. pseudotuberculosis their pathogenesis and laboratory diagnosis. The
oraganisms are gram negative bacilli, some of them showing pleomorphism
or involution forms. Bipolar staining is characterstic with methylene blue
Notes
stain. Y. enterocolitica and Y. pseudotuberculosis are motile at 22°C but non
motile at 37°C while Y.pestis is non motile.
TERMINAL QUESTIONS
1. Name the causative agent of plague and its mode of transmission.
2. Describe morphological characterstics of Y. pestis.
3. How Y. pestis can be identified in the laboratory.
4. How is Y.pestis different biochemically from Y.enterocolitica?

26.1
1. Negative
2. Positive
3. Safety pin
4. Anaerobes
26.2
1. Plague
2. Rodents
3. Black death
4. Tube
MICROBIOLOGY 255
MODULE Vibrio and Related Organism
Microbiology
27
Notes
VIBRIO AND RELATED
ORGANISM
27.1 INTRODUCTION
Vibrios are Gram-negative, rigid, curved rods that are actively motile by means
of a polar flagellum. The name ‘Vibrio’ is derived from the characteristic
vibratory motility (from vibrare, meaning to vibrate). They are asporogenous and
noncapsulated. Vibrios are present in marine environments and surface waters
worldwide. The most important member of the genus is Vibrio cholerae, the
causative agent of cholera. It was first isolated by Koch (1883) from cholera
patients in Egypt, though it has been observed earlier by Pacini (1884) and
others.
Fig. 27.1: Vibrio Cholera
OBJECTIVES
z describe the salient characteristics of Vibrio cholerae.
256 MICROBIOLOGY
Vibrio and Related Organism MODULE
z describe the pathogenesis, clinical presentation, diagnosis, management and Microbiology
prevention of cholera.
z list the features of other pathogenic vibrios, such as Vibrio parahaemolyticus
and Vibrio vulnificus.
27.2 VIBRIO CHOLERAE

Morphology : The cholera vibrio is a short, curved, cylindrical rod, about 1.5
× 0.2-0.4 mm in size, with rounded or slightly pointed ends. The cell is typically Notes
comma shaped (hence the old name V comma) but the curvature is often lost
on subculture. S-shaped or spiral form may be seen due to two or more cells lying
end to end. Pleomorphism is frequent in old cultures. In stained films of mucus
flakes from acute cholera cases, the vibrios are seen arranged in parallel rows,
described by Koch as the ‘fish in stream’ appearance. It is actively motile, with
a single sheathed polar flagellum. The motility is of the darting type, and when
acute cholera stool or a young culture is examined under the microscope, the
actively motile vibrios suggest a ‘swarm of gnats’. The vibrios stain readily with
aniline dyes and are Gram negative and non-acid fast.
Culture Characteristics
The cholera vibrio is strongly aerobic, growth being scanty and slow anaerobically.
It grows within a temperature range of 16-40oC (optimum 37 oC), Growth is
better in an alkaline medium, the range of pH being 6.4-9.6 (optimum 8.2). NaCl
(0.5-1%) is required for optimal growth though high concentration (6% and
above) are inhibitory.
It grows well on ordinary media. On MacConkey’s agar, the colonies are
colourless at first but become reddish on prolonged incubation due to the late
fermentation of lactose. On blood agar, colonies are initially surrounded by a
zone of greening, which later become clear due to hemodigestion. In peptone
water, growth occurs in about six hours as a fine surface pellicle, which on
shaking breaks up into membranous pieces.
A number of special media have been employed for the cultivation of cholera
vibrios. They may be classified as follows :
Fig. 27.2: Microscopic Slide of Vibrio Cholera
MICROBIOLOGY 257
Microbiology Holding or transport media

1. Venkatraman – Ramakrishnan (VR) medium : In this medium vibrios do
not multiply but remain viable for several weeks.
2. Cary-Blair medium : It is a suitable transport medium for Salmonella and
Shigella as well as for vibrios.
Notes Enrichment media

1. Alkaline peptone water at a pH of 8.6;
2. Monsur’s taurocholate tellurite peptone water at ph 9.2.
Both these are good transport as well as enrichment media.
Plating media
1. Alkaline bile salt agar (BSA) at pH 8.2 : The colonies after overnight
growth, colonies are moist, translucent, round discs, about 1-2mm in
diameter; with a bluish tinge in transmitted light.
2. TCBS medium : This medium, containing thiosulfate, citrate, bile salts and
sucrose, is available commercially and is very widely used at present.
Cholera vibrios produce large yellow convex colonies which may become
green on continued incubation.
Biochemical reactions : Carbohydrate metabolism is fermentative, producing
acide, but no gas. Cholera vibrios ferment glucose, mannitol, maltose, mannose
and sucrose but not inositol, arabinose or lactose, though lactose may be split
very slowly. Indole is formed and nitrates are reduced to nitrites. These two
properties contribute to the ‘cholera red reaction’ which is tested by adding a
few drops of concentrated sulphuric acid to a 24-hour peptone water culture.
With cholera vibrios, a reddish pink colour develops due to the formation of
nitroso-indole, Catalase and oxidase testes are positive. Methyl red and urease
tests are negative. Vibrios decarboxylate lysine and ornithine but not utilize
arginine.
Resistance : Cholera vibrios are susceptible to heat, drying and acids, but resist
high alkalinity. They are destroyed at 55°C in 15 minutes. Dried on linen or
thread, they survive for 1-3 days but die in about three hours on cover slips.
Survival in water is influenced by its pH temperature, salinity, presence of
organic pollution and other facts. In general, the E1 Tor vibrio survives longer
than the classical cholera vibrio. In the laboratory, vibrios survive for months
in sterile sea water, and this has been suggested as a method for the survival of
vibrios in nature. They survive in clean tap water for thirty days. In untreated
night soil, they may survive for several days. Vibrios are susceptible to common
disinfectants.
258 MICROBIOLOGY
On fruits, they survive for 1-5 days at room temperature and for a week in the Microbiology
refrigerator. In general, food materials left at room temperature do not act as an
important source of infection for longer than a day or two but those stored in
the cold may harbour vibrios for more than two weeks.
They are killed in a few minutes in the gastric juice of normal acidity but they
may survive for 24 hours in achlorhydic gastric juice.
Classification Notes
1. Serological Classification
2. According to Growth requirement
1. Serological Classification : VIBRIO are classified according to biochemical

properties & H antigen into Group A and Group B.
(i) Group A : This group includes vibrio cholarae and have similar
biochemical properties and Common H (flagellar) Antigen. Group A
is again classified based on somatic O antigen into O1 & Non-O1
O1 (agglutinable vibrio): These vibrios agglutinate with O1 anitserum.
(ii) Non-O1 (non-agglutinable vibrio) : This are not agglutinated by O1
antiserum. They are currently upto O-139.
O1 are classified into Biotypes-CLASSICAL and ELTOR and these
are further classified into serotypes- OGAWA, INABA, HIKOJIMA
Subgroup O1 can further be classified into two groups as follows
Classical Eltor
Hemolysis _ +
Voges Prosker test _ +
Chick erythrocyte agglutination _ +
Polymyxin B Sensitivity + _
Group IV phage susceptibility + _
Group V phage susceptibility _ +
2. According to growth requirement

Genus Vibrio includes 33 species the important are :
1.1 Non halophillic : It includes following & they are able to grow in
media without salts.
V. cholerae
MICROBIOLOGY 259
Microbiology 1.2 Halophillic : They are unable to grow in media without salts – have
a high requirement of NaCl.
V. Parahemolyticus is responsible for food poisoning
V. alginoltyticus is responsible for diarrheal disease
V. vulnificus is responsible for diarrheal disease
Notes

1. What is the causative agent of Cholera ?
2. Vibrio shows which type of motily ?
3. Which is the widely used medium for Cholera ?
4. Group A Vibrios has which common antigen ?
5. What is the requirement halophilic species ?
27.3 PATHOGENECITY OF VIBRIO CHOLERAE
Cholera
Disease caused by Vibrio Cholerae
Introduction
Cholera is an acute diarrheal disease caused by V cholerae. In its most severe
form, cholera is a dramatic and terrifying illness in which profuse painless
watery diarrhea and copious effortless vomiting may lead to hypovolemic shock
and death in less than 24 hours. In treated cases, the disease may last 4-6 days,
during which period the patient may pass a total volume a liquid stool equal to
twice his body weight.
Fig. 27.3: Pathogeneis Disease
260 MICROBIOLOGY
Clinical Feature of Cholera Microbiology
z All the clinical features of severe cholera result from this massive loss of
fluid and electrolytes.
z The cholera stool is typically a colourless watery fluid with flecks of mucus,
said to resemble water in which rice has been washed (hence called ‘rice
water stools).
Notes
z The clinical severity of cholera varies widely, from the rapidly fatal disease
to a transient asymptomatic colonization of the intestine by the vibrios.
z The incidence of mild and asymptomatic infections is more with EI Tor
vibrios than with the classical cholera vibrios.
z The incubation period varies from less than 24 hours to about five days.
The clinical illness may begin slowly with mild diarrhea and vomiting in
1-3 days or abruptly with sudden massive diarrhea.
Pathogenesis
z Vibrio Cholerae enters through orally contaminated food & water.
z If gastic acidity is less then they will be destroyed otherwise they passes
to intestine attaches the mucus membrance breach the epithelial cell &
attach to produce toxin.
Cholera Toxin
z It is very similarly to heat labile toxin of E.coli in structural, biochemical,
biological & antigenic properties but is more potent.
z It’s production is determined by filamentous phage integrated with bacterial
chromosome.
Mode of action
z Cholera Toxin has 2 sub units Sub-Unit A and Sub-Unit B
z Sub-Unit B binds to GM1 ganglioside on gut epithelial cells.
z This binding activates A subunit & it divides into A1 & A2.
z A1 activates adenly cyclase wich converts ATP to CAMP.
z CAMP increases the outflow of electrolytes & water to gut lemen & causes
diarrhea.
z Sub-Unit A2 act as binding agent for A1 to B
MICROBIOLOGY 261
Microbiology
Aim
z To demonstrate Vibrio Cholerae & identify the species.
Specimen
Notes 1. Stool
z Collected in acute stage of the disease before administration of Antibiotics.
z Collected by introducing lubricated catheter into rectum or directly in screw
capped bottle.
2. Rectal Swab
z Very useful in convalescent carrier in which there is no watery diarrhea.
z Transport Media for Cholera are as follows If chances of delay occur then
use V-R medium, Bile peptone transport medium, Monsur’s medium, Carry
Blair medium, Autoclaved sea water
Methods
The Methods for demonstration of the disease are as follows:
1. Direct Demonstration of bacilli
1.1 Microscopy :For rapid diagnosis , the characteristic darting motility
of vibrios and its inhibition by antiserum can be demonstrated
hanging drop method using cholera stool from acute cases or more
reliably after enrichment for 6 hours.
3. Culture
z Media
Enrichment media – Alkaline peptone water
For the plating medias are – Bile Salt Agar, & TCBS
Fig. 27.4: Colonies of V Cholerae on TCBS
262 MICROBIOLOGY
Microbiology
z Method
If the laboratory facilities are available then the specimen which is collected is
a directly inoculated on plating media otherwise it transport by holding media
then inoculated on enrichment media for 6-8 hrs. then subculture on plating
media after colonies appear the slide agglutination test with O1 antiserum is
done after the species is biochemically identified.
Notes
4. Biochemical Reactions
z V Cholera is identified from other by Biochemical reaction as follows it
shows Oxidate positive, Catalese Positive, Indole Positive, V.P. Positive test
& Citrate Positive but it shows Urease & Methyl red test negative.
5. For isolation of carries

z Essentially the same technique is required only more than one cycle of
enrichment is required.
z As the excretion of bacilli is intermittent repeated stool examination is
required.
Prophylaxis
The prevention of cholera requires essentially general measures such as
provision of protected water supply and improvement of environmental
sanitation. An ideal cholera vaccine is yet to be found.
Other Vibrio
z Vibrio Mimicus
So named because it closely resembles cholera vibrios in biochemical
features, V mimicus can be differentiated by its failure to ferment sucrose.
z Halophilic Vibrios
Vibrios that have a high requirement of sodium chloride are known as
halophilic vibrios.
z Vibrio Parahaemolyticus
V parahaemolyticus is an enteropathogenic halophillic vibrio. It is causative
agent of an outbreak of food poisoning due to sea fish. Gastronteritis due
to his vibrio has since been identified in several countries and it is now
considered an important cause of food poisoning throughout the world. It
resembles the cholera vibrio, except that it is capsulated, shows bipolar
staining and has a tendency to pleomorphism, especially when grown on
3% salt sagar and in old cultures. It grows only in media containing NaCl.
MICROBIOLOGY 263
Microbiology z Vibrio Alginolyticus

This halophillic vibrio resembles V parahaemolyticus in many respects and
was formerly considered a biotype of the latter. It has a higher salt tolerance,
is VP positive and ferments sucrose.
z Vibrio Vulnificus
V vulnificus, previously known as L+ vibrio or Beneckea vulnifica, is a
Notes marine vibrio of medical importance. It is VP negative and ferments lactose
but not sucrose. It has a salt tolerance of less than 8 per cent. It causes two
types of illness. The first is wound infection following contact of open wounds
with sea water. The second type occurs in compromised hosts particulars
those with liver disease. Following ingestion of the vibrio, usually in oysters,
it penetrates the gut mucosa without causing gastrointestinal manifestations
and enters the bloodstream, rapidly lading to septicemia with high mortality.
z Aeromonas and Pleasiomonas
Aeormonas hydrophila, originally isolated from frogs, in which it causes the

‘red leg disease’, has been reported from many cases of diarrhea and from
some pyogenic lesions in human beings. Plesiomonas shilgelloides also has
been reported from diarrheal disease.

1. How long is the incubation period of V Cholerae ?
2. V Cholarae toxin is similar to which toxin ?
3. Toxin activates which enzyme in Gut Lumen ?
4. Before administration of antibiotics which specimen is collected for
laboratory diagnosis ?
5. In Convalescent carrier which specimen is commonly collection ?
6. Which is the most common plating media for Vibrio ?

z Members of the genus Vibrio are curved, gram negative rod-shaped bacteria
which exhibit darting motility, are facultative anaerobes, positive by the
catalase and oxidase tests. Vibrios are found in aquatic environments. In
this genus, Vibrio Cholerae is the most important cause of human disease,
264 MICROBIOLOGY
but Vibrio parahaemolyticus and Vibrio Vulnificus are also sometimes Microbiology
implicated in human infections.
Virulent V cholerae organisms:
z Live in salt water attached to algae, copepods or shells of crustaceans; if
conditions become unfavourable, they become dormant and unculturable.
Drinking contaminated water or vegetables washed with contaminated
water can lead to epidemics in humans. Cycles of transmission are Notes
perpetuated when bacteria are shed into water sources by fecal contamination.
z Possess O1 or O-139 somatic antigens, and O1 isolates are serotyped as
Inaba, Ogawa or Hokujima and as two biotypes, classical and El Tor.
z Cholera causes a voluminous watery diarrhea that is life-threatening
because the patient becomes markedly dehydrated. After ingestion, the
bacteria gain access to the epithelium of the small intestine, where they
become attached and then secrete cholera toxin. Secretion of the toxin
results in marked efflux of water and ions into the lumen of the intestine,
which manifests as diarrhea and dehydration.
z For diagnosis of cholera, clinical signs in areas of endemicity or during
epidemics are highly suggestive. Vibrios grow on culture media designed
for enteric organisms, with added sodium chloride where appropriate. Feces
samples are plated on thiosulphate-dtrate-bile softs-sucrose (TCBS) agar to
obtain yellow colonies or BSA to get translucent colonies; Gram stain of
these colonies reveals curved Gram-negative rods. In epidemics, feces can
be examined for darting motility, which will be reduced by adding specific
antiserum.
z Cholera can be prevented by proper treatment of drinking water. Cholera
is treated with rehydration therapy (ORS) and, in severe cases, with
tetracycline or doxycycline to shorten the course of the disease. Antibody
to cholera toxin greatly reduces the severity of the disease.
z Vibrio parahaemofyticus is halophilic (has an exceptionally high requirement
for sodium chloride) and is found in prawns and other seafood. It also
produces an enterotoxin but its effect is much milder than that of cholera.
Other vibrios (Vibrio vulnificus) occasionally cause human disease, including
traumatic wound infections and, rarely, eye infections.
TERMINAL QUESTIONS
1. What is the Morphology of Vibrio Cholerae ?
2. What are the holding or transport media for Vibrio Cholerae ?
MICROBIOLOGY 265
Microbiology 3. Describe the serological classification of Vibrio Cholerae ?

4. What are the differences between Classical and EL TOR Vibrios ?
5. What is the mechanism of action of Cholera Toxin ?
6. What are the biochemical reactions for demonstration Vibrio Cholerae ?
Notes
27.1
1. V. Cholerae
2. Darting Motility
3. Thiosulphate Citrate Bile Salt Medium(TCBS) and Bile salt agar (BSA).
4. H Antigen
5. They grow only on high salt containing medias
27.2
1. Incubation period is 24 hrs. to 5 days
2. Hear labile, toxin of E.Coli
3. They activates adenyl cyclase
4. Stool Sample and Rectal swab
5. By double enrichment
6. Thiosulphate Citrate Bile Salt Medium (TCBS) and Bile salt agar (BSA).
266 MICROBIOLOGY
Pseudomonas MODULE
Microbiology
28
Notes
PSEUDOMONAS
28.1 INTRODUCTION
Pseudomonas is a bacteria mostly saprophytic in nature, is found in soil, water
and other moist environment. It has emerged as an important cause of Health
Care Associated and Opportunistic Infections. Most of the clinical isolates of
Pseudomonas are resistant to many antibiotics. Pseudomonas aeruginosa is also
a pathogen of plants. Pseudomonas is a strict aerobe, motile Gram negative
bacteria and belongs to the order Pseudomonadales, family- Pseudomonadaceae
and Genus Pseudomonas. The family comprises of about eight groups and 191
species, the type species is Pseudomonas aeruginosa. Walter Migula vaguely
described these Gram negative motile organisms in 1890 and named as
Pseudomonas. Recently, it has been postulated that Pseudomonas may be the
common nucleator of ice crystals in clouds, so the organism is important to
the formation of snow and rain around the world.
OBJECTIVES
z classify Pseudomonas;
z state the morphological characteristics of Pseudomonas;
z enumerate the biochemical characteristics;
z explain the mechanism of virulence, pathogenicity;
z describe how the biofilm is formed;
z enumerate the infections caused by Pseudomonas;
z culture and identify Pseudomonas from specimen;
z carry out antibiotic susceptibility and interpret the results.
MICROBIOLOGY 267
MODULE Pseudomonas
Microbiology
28.2 HISTORY
As already explained that it was sometimes in 1890-1900 that this Gram negative
bacteria was described. A scientist by the name of Migula gave the Genus name
of Pseudomonas in 1894 to these bacteria. Pseudomonas is comprised of two
Greek words-Pseudo meaning false and Monas meaning unit, so literally the
term means “false unit”. However Pseudomonas is a real bacteria so we really
do not know the basis of the nomenclature by Walter Migula. The bacteria now
Notes has many groups and species. Recently the Pseudomonas whole genome has
been sequenced and the analysis of 16S rRNA has lead to the exclusion of many
groups and inclusion of many groups to the Genus pseudomonas.
28.3 CLASSIFICATION
The classification of Pseudomonas is given below:
z Class: Gamma Proteobacteria
z Order: Pseudomonadales
z Family: Pseudomonadaceae
z Genus: Pseudomonas
z Eight groups: P. aeruginosa ; P. chlororaphis; P. fluorescens; P.
pertucinogena; P. putida; P. stutzeri; P. syringae; P. incertae sedis.
z The family has 191 valid species. These include bacteria which are
saprophytic, free living, and human, animal and plant pathogens. The type
species is Pseudomonas aeruginosa.
28.4 MORPHOLOGY
Pseudomonas is rod shaped, slender (0.5 to 0.8 µm by 1.5 to 3.0 µm) Gram
negative organism, motile by polar flagella, sometimes more than two flagella
may be present. Some strains of Pseudomonas particularly those isolated from
cases of cystic fibrosis are very mucoid and have kind of pseudo capsule
(glycocalyx) made of polysaccharides. The glycocalyx protects pseudomonas
from host defense.
Fig. 28.1: Gram staining of pseudomonas: Gram negative,

rod shaped bacteria seen
268 MICROBIOLOGY
Pseudomonas MODULE
Microbiology
Pseudomonas is a strict (obligate) aerobe, but sometimes it can grow anaerobically
if nitrates (NO3 act as respiratory electron acceptor) are present in the medium.
Pseudomonas can grow at wide ranges of temperature; the optimum temperature
is 37° C. It can grow on ordinary media like nutrient agar and grows almost on
all the culture media used routinely in the bacteriology lab. Pseudomonas has
been seen to grow in distilled water, also. Notes
Fig. 28.2: Pseudomonas aeruginosa on nutrient agar showing greenish colouration

due to production of Pyoverdin pigment.
Fig. 28. 3: Growth of P. aeruginosa showing bluish green colouration due to production
of Pyocyanin, produced by some strains of Pseudomonas
Pseudomonas produces large, opaque, flat colonies with irregular margins and
distinctively fruity odour colonies. The colour of growth will depend upon the
type of pigments (enumerated below) produced by the organism. The isolates
from water and soil produce small round colonies. The isolates from clinical
specimens like respiratory, urine, etc. may produce mucoid colonies. The
bacteria which form mucoid colonies are more virulent compared to others.
MICROBIOLOGY 269
MODULE Pseudomonas
Microbiology The pigments produced by Pseudomonas are:

z The fluorescent pigment pyoverdin (greenish yellow)
z The blue pigment pyocyanin (bluish green)
z Pyorubin (red)
z Pyomelanin (brown)
Notes

1. Pseudomonas is an ..................
2. Pseudomonas is Gram .................. bacteria.
3. Shape of Pseudomonas is ..................
4. Pseudomonas grow on .................. media.
28.6 BIOCHEMICAL CHARACTERISTICS

Pseudomonas has oxidative metabolism. Since organism is non fermentative the
acid is not produced from peptone water sugars.
The important biochemical characteristics of Pseudomonas include:
z Oxidase test positive;
z Catalase test positive
z Nitrates are reduced to nitrites;
z Arginine dihydrolase test positive;
z Glucose is utilized oxidatively ⇒ Oxidative reaction in of media
z Indole, Methyl red (MR), Vogues Prauskar (VP) and H2S production test
are negative.
Commonest screening diagnostic biochemical test used in lab is the oxidase test.
Fig. 28.4: Oxidative reaction of Pseudomonas in Hugh and Leifson’s of media
270 MICROBIOLOGY
Pseudomonas MODULE
Microbiology
Notes
Fig. 28.5: Nitrate Test
Fig. 28.6: Arginine dihydrolase test
28.7 VIRULENCE AND PATHOGENICITY

Pseudomonas can infect any tissue, any organ system in an immune-compromised
host. Pseudomonas usually cannot infect normal host. So, you see as compromised
hosts are found in hospitals, Pseudomonas has emerged as a common cause of
health care associated or nosocomial or hospital associated infections. In
addition Pseudomonas produces many different organ system infections in the
humans. These are described below.
P. aeruginosa produces exotoxin A which is a virulence factor. This exotoxin
inactivates ADP ribosylate eukaryotic elongation factor 2 (EF 2) and thus
interferes with the synthesis of protein resulting in death of the cell. P.
aeruginosa also produces an exoenzyme “Exo U” which damages the cell
membrane leading to lysis of the membrane and cell death.
MICROBIOLOGY 271
MODULE Pseudomonas
Microbiology It is reported that low levels of phosphate in human intestines activate the
symbiotic Pseudomonas to produce lethal toxins inside the intestinal tract which
may severely damage or kill the host.
The most important risk factor for Pseudomonas infections is break down of host
defense due to disease or other factors. Pseudomonas is both invasive and
toxinogenic. Infection involves the following three steps:
Notes z bacterial attachment and colonization;
z local invasion;
z disseminated systemic disease.
28.7.1 Bacterial attachment and Colonization

Pseudomonas infection may be endogenous (may be from intestines) or may be
acquired from outside (exogenous). Individuals outside the hospital may be
colonized with Pseudomonas at different sites (0-24%).The adhesins are the pili
of P aeruginosa with which bacteria adhere to specific galactose or mannose
or sialic acid receptors on the mucosal epithelial cells of the upper respiratory
tract and others. Production of protease enzyme by bacteria breaks down the
fibronectin and exposes the pilus specific receptors on the epithelial cell surface.
Tissue injury caused by viral infection and other phenomenon facilitates
colonization by Pseudomonas (Opportunistic colonization).
Pseudomonas can also colonize by formation of biofilm which we will discuss
later. Pseudomonas pili, mucoid polysaccharide, probably surface-bound
exoenzyme S and possibly other cell surface adhesins help Pseudomonas to
colonize.
28.7.2 Bacterial Invasion

P. aeruginosa produces enzymes and toxins that break down barrier to enter and
damage host cells, resist phagocytosis and host immune defenses. The
polysaccharide slime and sort of false capsule produced by Pseudomonas
effectively protects cells from opsonization by antibodies, complement deposition,
and phagocyte engulfment. Elastase and alkaline protease protease destroy the
ground substance of the cornea and other supporting structures composed of
fibrin and elastin resulting in invasion and injury. Pseudomonas aeruginosa
produces three other soluble proteins involved in invasion: a cytotoxin (mw 25
kDa) and two hemolysins. The cytotoxin is a pore-forming protein. It was
originally named leukocidin because of its effect on neutrophils, but it appears
to be cytotoxic for most eucaryotic cells. Of the two hemolysins, one is a
phospholipase and the other is a lecithinase. They appear to act synergistically
272 MICROBIOLOGY
Pseudomonas MODULE
to break down lipids and lecithin. The cytotoxin and hemolysins contribute to Microbiology
invasion through their cytotoxic effects on neutrophils, lymphocytes and other
eucaryotic cells. Pyocyanin the pigmen, impairs the normal function of human
nasal cilia and disrupts the respiratory epithelium. So, you see that Pseudomonas
has a varied armamentarium consisting of pilli, enzymes, capsule and pigments
which help it to establish in human tissues and produce harmful effects.
28.7.3 Bacterial dissemination: Notes
Pseudomonas can invade the blood stream from initial site of infection and
through blood is disseminated to different organs. The factors which help
bacteria to invade as described above help to invade the organs, tissues wherever
the bacteria reach. Bacterial endotoxin during septicemia, may cause fever,
hypotension, and intravascular coagulation.
The virulence/pathogenicity armamentarium of Pseudomonas includes:
Adhesins
Pili (N-methyl-phenylalanine pili)
Polysaccharide capsule (glycocalyx)
Slime
Invasins
Elastase
Alkaline protease
Hemolysins (phospholipase and lecithinase)
Cytotoxin (leukocidin)
Pyocyanin
Toxins
Exoenzyme S
Exotoxin A
Lipopolysaccharide (LPS)
Antiphagocytic elements
False capsules, slime layer
Lipo polysaccharide
Biofilm formation
MICROBIOLOGY 273
MODULE Pseudomonas
Microbiology
28.8 BIOFILM FORMATION
Like some other bacteria Pseudomonas forms biofilm which serves as a safe
haven for the bacteria. Biofilm is formed on quorum sensing. When a critical
number of bacteria are reached bacterial cells communicate with each other by
forming some molecules. Quorum sensing results in expression of genes which
help bacteria to adapt to the environment, multiply, release virulence factors and
Notes produce enzymes, exotoxins, slime, galactocalyx, etc. This is the biofilm, in
which are embedded colonies of bacteria. These bacteria are protected from host
defense and antibiotics. Bacteria from this biofilm can invade, enter blood
stream and cause septiceamia. Biofilms of P aeruginosa result in chronic
opportunistic infections.

1. Virulence factor of Pseudomonas is ..................
2. Pseudomonas produce .................. and .................. which resists phagocytosis.
3. Pseudomonas invade by .................. and ..................
4. Pseudomonas colonies by the formation of ..................
28.8 INFECTIONS AND DISEASES CAUSED BY

PSEUDOMONAS
Pseudomonas aeruginosa is the most common cause of infection of burn injuries
and otitis externa (infection of outer ear). As stated earlier, Pseudomonas can
infect any tissue/organ system in the immunocompromised host. The disease
produced will depend on the organ system infected. Pseudomonas colonizes
medical devices, forms biofilms and causes chronic opportunistic infections.
Pseudomonas can be present as commensal in healthy hosts and does not cause
any disease.
The diseases caused by Pseudomonas include:
z Respiratory infections: Pneumonia in neutropenic cancer patients

undergoing chemotherapy; diffuse broncho pneumonia, infection in cystic
fibrosis patients;
z Bacteremia and septicemia: Pseudomonas causes hospital acquired Gram-
negative bacteremias in immunocompromised patient and in severe burns.
274 MICROBIOLOGY
Pseudomonas MODULE
Pseudomonas accounts for 25% of hospital acquired BSI caused by Gram Microbiology
negative bacilli.
z Ear infections: Pseudomonas usually causes otitis externa and”swimmer’s
ear”.
z Central nervous system infections: Pseudomonas can invade meninges
from near by structures like external ear /paranasal sinuses after invasive
procedure or trauma to the head. Notes
z Urinary tract infection: Usually causes hospital-acquired UTI related to
urinary tract catheterization, instrumentation or surgery. Pseudomonas
aeruginosa is the third leading cause(12%) of all hospital-acquired UTIs.
z Endocarditis: Pseudomonas bactreamia may result in infection of damaged
heart valves and prosthetic heart valves.
z Bone and joint infections: Pseudomonas bactreamia may result in infection
of bones and joints by direct inocculation. May cause chronic contiguous
osteomyelitis from direct inoculation of bone. Pseudomonas also causes
osteochondritis after puncture wounds of the foot.
z Gastrointestinal infections: Any part of the gastrointestinal system can be
infected by Pseudomonas in immunocompromised host. The diseases
produced include diarrhoea, gastroenteritis, perirectal infections. Sometimes
Pseudomonas can produce necrotizing enterocolitis. It is an important cause
of antibiotic associated diarrhea.
z Skin and soft tissue infections, including wound infections, pyoderma
and dermatitis: Any part of skin and soft tissue compromised by trauma,
burn injury, bad hygiene may be infected by Pseudomonas. The infections
caused include folliculitis, acne vulgaris and abscesses.
28.9 DRUG RESISTANCE

Pseudomonas is heat sensitive is killed at 55° C in one hour. Pseudomonas is
inherently resistant to many antibiotics, common disinfectants and can merrily
grow in bottles of antiseptic solutions. The resistance to antibiotics is by
different mechanisms like multi drug efflux pumps; antibiotic resistance
chromosomal genes (mexAB, mexXY, etc.) and the low permeability of the
bacterial false capsule in biofilms.Pseudomonas also acquires drug resistance
by mutations which may be spontaneous and also drug induced.Pseudomonas
is sensitive to aminoglycosides (amikacin and gentamicin); cephalosporins
(ceftazidime and cefotaxime); fluoroquinolones (ciprofloxacin, pefloxacin) and
penicillins like piperacillin, ticarcillin) and colistin. Localized infections can be
treated with topical colistin, polymyxin B.
MICROBIOLOGY 275
MODULE Pseudomonas
Microbiology
28.10 LABORATORY DIGNOSIS OF INFECTIONS
CAUSED BY PSEUDOMONAS
z Collect the appropriate sample. This will be according to the tissue/system
affected. Specimen can be pus, urine, blood, CSF, tissue, etc.;
z Carry out the Gram staining- Gram negative bacilli and pus cells will be
seen;
Notes
z Culture the specimen on Blood agar and MacConkey agar plates. Incubate
overnight at 37° C;
z Examine the bacterial growth : Examine type of colonies, pigment production,
odour and do oxidase test from MacConkey agar. Pale colonies on
MaConkeyAgar, fruity odour, pigment (greenish, brownish) and oxidase
positive test means the growth is probably Pseudomonas;
z Confirm by OF test and Arginine dihydrolase test
z Carry out the antibiotic susceptibility testing by Kirby Bauer disc diffusion
method. Read the result-measure the inhibition zones and label as sensitive
, resistant or intermediate sensitivity taking into account the zone size as
has been explained to you in the chapter on antibiotic susceptibility testing.

1. Pseudomonas causes .............. and .............. infection
2. Pseudomonas are .............. sensitive and resistant to .............., ..............
3. Pseudomonas are cultured on .............. and ..............
4. Pseudomonas are ..............

z Pseudomonas is a bacteria mostly saprophytic in nature, is found in soil,
water and other moist environment. It is an important plant pathogen also.
It has emerged as an important cause of Health Care Associated and
Opportunistic Infections.
z Pseudomonas has eight groups: P. aeruginosa ; P. chlororaphis; P.
fluorescens; P. pertucinogena; P. putida; P. stutzeri; P. syringae; P.incertae
sedis and The family has 191 valid species.
276 MICROBIOLOGY
Pseudomonas MODULE
z Pseudomonas is rod shaped, slender (0.5 to 0.8 µm by 1.5 to 3.0 µm) Gram Microbiology
negative organism, motile by polar flagella.
z Pseudomonas is a strict (obligate) aerobe, can grow at wide ranges of
temperature; the optimum temperature is 37° C. It can grow on ordinary
media like nutrient agar and others and even indistilled water.
z Pseudomonas aeruginosa on nutrient agar showing greenish yellow
colouration due to production of Pyoverdin pigment.
Notes
z The pigments produced by Pseudomonas are:
z The fluorescent pigment pyoverdin (greenish yellow)
z The blue pigment pyocyanin (bluish green)
z Pyorubin (red)
z Pseudomonas has oxidative metabolism.
z Since organism is non fermentative the acid is not produced from peptone
water sugars.
z The important biochemical characteristics include: Oxidase test positive
and Catalase test positive, no fermentation of sugars in peptone water.
z Pseudomonas can infect any tissue, any organ system in an immune-
compromised host. Pseudomonas usually cannot infect normal host.
z Pseudomonas has emerged as a common cause of health care associated
or nosocomial or hospital associated infections.
z The most important risk factor for Pseudomonas infections is break down
of host defense due to disease or other factors. Pseudomonas is both
invasive and toxinogenic.
z Pseudomonas forms biofilm which serves as a safe haven for the bacteria.
z Pseudomonas causes a large number of infections affecting almost any part
of body in immunocompromised host. These include: Respiratory infections,
Bacteremia and septicemia, hospital acquired Gram-negative infection,
otitis externa and”swimmer’s ear”, Central nervous system infections,
hospital-acquired UTI related to urinary tract catheterization, instrumentation
or surgery, Endocarditis, Bone and joint infections, Pseudomonas also
causes skin and soft tissue infections, including wound infections, and is
an important cause of infection in burns patients.
z To detect and identify Pseudomonas from specimen in the lab: Collect the
appropriate sample; make smear and do Gram staining; culture the specimen
on Blood agar and MacConkey agar plates; examine the bacterial growth;
and do oxidase test from MacConkey agar.
z Pale colonies on Mac Agar, fruity odour, pigment (greenish, brownish) and
oxidase positive test means the growth is Pseudomonas; carry out the
antibiotic susceptibility testing.
MICROBIOLOGY 277
MODULE Pseudomonas
Microbiology
TERMINAL QUESTIONS
1. Briefly classify Pseudomonas.
2. What are the important morphological, cultural and biochemical
characteristics of Pseudomonas aeruginosa.
Notes 3. Enumerate the important enzymes and toxins produced by Pseudomonas
which help the bacteria to evade the host defense and produce disease.
4. Briefly describe how biofilm is formed and how it helps Pseudomonas?
5. Enumerate the infections and diseases caused by Pseudomonas.
6. Is Pseudomonas a sensitive organism? If not give reasons for resistance.
7. Briefly describe the steps for identification of Pseuodomonas from pus
collected from burn case.
28.1
1. Aerobe
2. Negative
3. Rod
4. Nutrient
28.2
1. Exotoxin
2. Enzyme, toxin
3. Cytotoxin, hemolysins
4. Biofilm
28.3
1. Septicemia, Opportunistic
2. Heat, antibiotic, disinfectants
3. Blood agar, MacConkey agar
4. Aerobes
278 MICROBIOLOGY
Haemophilus MODULE
Microbiology
29
Notes
HAEMOPHILUS
29.1 INTRODUCTION
The genus Haemophilus contains small, nonmotile, nonsporing, oxidase positive,
pleomorphic, gram negative bacilli that are parasitic on human beings or
animals. Haemophilus means blood loving organisms.
OBJECTIVES
After reading this lesson you will be able to:
z describe the morphology of Haemophilus Influenzae
z discuss the cultural characteristics
z describe the pathogenesis of Haemophilus influenza

z explain the laboratory diagnosis
29.2 MORPHOLOGY
H. influenzae is a small gram negative, nonmotile, nonsporing bacillus
exhibiting pleomorphism. In sputum it usually occurs as clusters of coccobacillary
forms. The bacilli are relatively difficult to stain. Staining for 5-15 minutes with
Leoffler’s methylene blue or dilute carbol fuchsin gives good results.
Fig. 29.1
MICROBIOLOGY 279
MODULE Haemophilus
Microbiology
The bacillus has fastidious growth requirement. Growth factors namely X & V,
present in blood are essential for growth. It is aerobic but grows anaerobically
also. The optimum temperature is 37°C and does not grow below 20°C. when
staphylococcus aureus is streaked across a plate of blood agar on which a
specimen containing H influenza has been inoculated after overnight incubation,
Notes the colonies of H. influenzae will be large & well developed alongside the streak
of staphylococcus and smaller farther away. This phenomenon is called
satellitism and demonstrates the dependence of H. influenzae on v factor which
is high near staphylococcal growth. This is a routine test in clinical bacteriology
for identification of H. influenzae
When blood agar is heated to 80-90°C, or boiled for a few minutes, the V factor
is released from within erythrocytes & hence these media are superior to blood
agar for growing H. influenzae. Clear transparent media may be prepared by
boiling & filtering a mixture of blood & nutrient broth or by adding a peptic agar
is best for primary isolation of H. influenzae and gives a copious growth.
Capsulated strains produce translucent colonies with a distinctive iridescence on
Levinthal’s agar.

1. H. influenzae is small gram ................. bacilli
2. H. influenzae exhibits ................. characteristic
3. Hemophilus means ................. organism
4. ................. staining is commonly used in identification of Haemophilus
5. ................. phenomenon is seen in Haemophillus
6. Streaks of ................. enhances the growth of heamophillus

Glucose and xylose are fermented with acid production but not lactose, sucrose
and mannitol. Catalase and oxidase reactions are positive. Nitrates are reduced
to nitrites.
29.5 RESISTANCE
H. influenzae is a delicate bacterium, destroyed by heating at 55°C for 30
minutes and refrigeration at 0-4°C, drying and disinfectants. In culture, the cells
280 MICROBIOLOGY
Haemophilus MODULE
die within two or three days due to autolysis. Cultures may be preserved for about Microbiology
a month on chocolate agar slopes in screw capped bottles.
29.6 ANTIGENIC PROPERTIES

Capsular polysaccharide, outer membrane protein and lipooligosaccharide are
the major surface antigens. The major antigenic determinant of capsulated
strains is the capsular polysaccharide based on which H. influenzae strains have Notes
been classified with six capsular types a to f. Capsular typing is of medical
importance as major acute invasive infections belong to type b. The type capsular
polysaccharide has a unique chemical structure, containing the pentose sugars
ribose and ribitol instead of the hexoses and hexosamines as in the other five
serotypes. H. Influenzae strains lacking a capsule cannot be typed and are called
nontypable strains. Next to Hib, the nontypable strains are the most relevant in
clinical infections.
Fig. 29.2: Haemophilus influenzae

1. ..............., ............... & ............... are the major surface antigens of haemophilus
2. Common infections caused by invasive haemophilus are ..............., ...............
& ...............
3. Common infections caused by non invasive haemophilus are ...............,
............... & ...............
4. Capsulated stains caused ............... infections
MICROBIOLOGY 281
MODULE Haemophilus
Microbiology
29.7 PATHOGENECITY
H. influenzae is an exclusively human pathogen. Diseases due to H. influenzae
are invasive and noninvasive. In invasive the bacillus acts as a primary pathogen,
causing acute invasive infections. The bacilli spread through blood, being
protected from phagocytes by their capsule. Haemophillus meningitis is the most
important infection in this group, and others are laryngoepiglottitis, conjunctivitis,
Notes Bacteremia, pneumonia, arthritis, endocarditis and pericarditis. These are most
commonly seen in children and mostly capsulated and type b antigen strain.
In noninvasive the bacillus spreads by local invasion along mucosal surfaces and
causes secondary or superadded infections, usually of the respiratory tract. These
are otitis media, sinusitis and exacerbations of chronic bronchitis and
bronchiectasis. These are usually seen in adults and are often caused by
noncapsulated strains.

In meningitis, presence of pleomorphic in CSF, and Gram-negative bacilli that
do not stain well are suspicion of H. influenzae infection. The capsular
polysaccharide antigen may be present in the CSF in meningitis and in urine in
systemic infection. Its demonstration by latex particle agglutination or CIE is
useful in diagnosis.
For isolation, CSF should be plated promptly on blood agar or chocolate agar
and incubated in an environment of 5-10 percent of CO2 and high humidity. The
specimen should not be refrigerated before inoculation as the bacillus is very
sensitive to low temperatures. A strain of staphylococcus should be streaked
across the plate. After overnight incubation at 37°C, small opaque colonies
appear that show satellitism. Iridescence may be demonstrated on Levinthal’s
medium. Typing may be done if antisera are available.

z Haemophilus means blood loving organisms.
z Haemophilus are non-motile, non-sporing, oxidase positive, pleomorphic,
gram negative bacilli
z Haemophilus can be Stained with Leoffler’s methylene blue or dilute carbol
fuchsin
z Growth factors namely X & V, present in blood are essential for growth
z They demonstrate satellitism
282 MICROBIOLOGY
Haemophilus MODULE
Microbiology
TERMINAL QUESTIONS
1. What is the morphology of haemophilus
2. What are the cultural characteristics of haemophilus
3. Describe the laboratory diagnosis of haemophilus
Notes
29.1
1. Negative
2. Pleomorphism
3. Blood loving
4. Leoffler’s methylene blue
5. Satellitism
6. Staphylococcus aureus
29.2
1. Capsular polysaccharide, outer membrane protein and lipooligosaccharide
2. Haemophillus meningitis, laryngoepiglottitis, conjunctivitis, Bacteremia
3. Otitis media, sinusitis and exacerbations of chronic bronchitis and
bronchiectasis
4. Invasive infections
MICROBIOLOGY 283
MODULE Bordetella
Microbiology
30
Notes
BORDETELLA
30.1 INTRODUCTION
The genus Bordetella are small Gram negative, non-motile, coccobacilli. This
genus contains three species - Bordetella pertussis, B. parapertussis, B.
bronchiseptica. B. pertussisis associated with classical whooping cough or
pertussis and is the most fastidious of the three species. B. parapertussis causes
milder form of pertussis. In contrast to Haemophilus influenzae they do not need
X and V factors.
OBJECTIVES
z describe the characteristics of Bordetella.
z describe the diseases produced by it.

z explain the laboratory diagnosis.
30.2 MORPHOLOGY
Bordetella was identified by Bordet and Gengou. As mentioned earlier B.
pertussis is a small ovoid Gram negative coccobacilli 1-1.5 μm by 0.3?m. It is
non-motile and non-sporing. When isolated from a clinical sample it is
capsulated but rapidly loses it on subculture. In smears from culture, the bacilli
are arranged in loose clumps with clear spaces in between giving a thumb print
like pattern.
30.3 CULTURE CHARACTERISTICS

Bordetella are aerobes and facultative anerobes. They grow best at 35-36oC.
Bordetella are sensitive to various inhibitory substances. Bordet and Gengou
284 MICROBIOLOGY
Bordetella MODULE
developed a special media for growing Bordetella which is called Bordet Microbiology
Gengou medium. It consists of glycerine potato blood agar. A higher concentration
of blood is added not to provide additional nutritive factors but to neutralize
inhibitory substances. The plates are incubated for 48-72 hours as the growth
is slow. The colonies are small, dome shaped, opaque, greyish, refractile and
glistening resembling the appearance of bisected pearls or mercury drops.They
are surrounded with hazy haemolysis. Regan and Lowe is a selective medium
comprising of charcoal, cephalexin, amphotericin along with horse blood. Notes

Bordetella pertussis is oxidase and catalase positive. Biochemically it is inert.
30.4.1 Antigenic Structure

While diphtheria and tetanus are singe toxin diseases, bordetella is a multitoxin
disease. It produces eight different antigenically defined virulence factors.
z Agglutinogens
z Lipopolysaccharide
z Heat labile toxin
z Tracheal cytotoxin
z Pertusssis toxin
z Adenylatecyclase
z Filamentous haemagglutinin
z Haemolysin
Fig. 30.1
z Pertactin
Pertussis toxin, adenylatecyclase and filamentous hemagglutininare the major
virulence factors which also exhibit excellent immunogenicity.

1. Bordetella are Gram ................ bacilli.
2. Culturally Bordetella are facultative ................
3. Bordetella are grown in ................ media.
4. Bordetella are oxidase and catalase ................
MICROBIOLOGY 285
MODULE Bordetella
Microbiology
30.5 PATHOGENESIS
Like diphtheria, measles and chicken pox, pertussis is a childhood disease. B.
pertussis causes whooping cough in 95%, B. parapertussis in 5% and B.
bronchiseptica in 0.1% cases. The route of infection is respiratory. It is amongst
the most contagious diseases. Incubation period is 1-2weeks. The disease can
be divided into three stages-catarrhal, paroxysmal and convalescent. The
Notes infectivity is maximal during the incubation period and the catarrhal stage.
Unfortunately the symptoms are non-specific and Bordetella pertussis cannot be
diagnosed at this stage when treatment is most effective if given in this phase.
Untreated the disease continues for 6-8 weeks. During the paroxysmal stage the
violent paroxysm or spasm of continuous coughing leaves the lungs depleted of
oxygen. This is followed by a long inrush of air in the near empty lungs in the
inspiratory stage with the characteristic whoop. At this stage the patient may
even become cyanosed and vomit. The eyes may bulge and drooling from mouth
can occur. During convalescent stage, the frequency and severity of coughing
slowly decrease.
Specimen collection
Cough plate method, nasopharyngeal aspirate, pernasal swab, West’s post-
pharyngeal swab are the methods used for collection of specimen. The swab
should not be of cotton. It should be either of calcium alginate or dacron on a
nichrome wire.
Cough plate method: Culture plate is held 10-15 cm in front of the patient’s
mouth while coughing.
Pernasal swab: Here a flexible swab is passed gently along the floor of the nasal
cavity till resistance is felt. It is left there for 30 seconds and then withdrawn.
This method collects specimen from the roof of the pharyngeal wall.
Microscopy
Smears may be made from respiratory specimen and stained by Gram’s stain or
preferably by fluorescent antibody technique.
Culture
The samples are plated on Bordet Gengou medium or Regan and Lowe’s
medium and incubated for 48-72 hours. Colonies are identified by biochemical
tests and slide agglutination.
286 MICROBIOLOGY
Bordetella MODULE
Serological investigations Microbiology
Four fold rise in titre of antibodies is diagnostic. It can be demonstrated by

agglutination, complement fixation and immunofluorescent test.
30.7 TREATMENT
Erythromycin is the drug of choice. Other drugs which can be used are
tetracycline, chloramphenicol and ampicillin.
Notes
30.8 PROPHYLAXIS
Vaccination by whole cell killed vaccine in the triple vaccine combination (DPT)
is very effective. Three intramuscular doses at 4-6 month intervals starting at 6
weeks of age is the recommended schedule. The booster dose should be given at
15-18 months of age. B. pertussis also acts as an adjuvant for the tetanus and
diphtheria toxoids enhancing the immunogenicity of the vaccines. Now an
acellular vaccine comprising of pertussis toxin, pertactin and filamentous
hemagglutinin is also being used. It has fewer side effects but is costlier.
30.9 BORDETELLA PARAPERTUSSIS

It is non fastidious and grows on nutrient agar. Colonies are pigmented. Urease
is positive. Whooping cough in 5% cases is attributed to it.
30.10 BORDETELLA BRONCHISEPTICA

In contrast to the other two species, it is motile with peritrichous flagella. It also
grows on nutrient agar. It reduces nitrate to nitrite, hydrolyses urea and is oxidase
and catalase positive. It is known to cause pertussis in 0.1% cases.

1. Bordetella causes ................ in children.
2. The route of infection of pertussis is ................
3. Swab used for specimen collection contains ................ or ................
4. Staining technique preferred for identification by ................

z Bordetella are Gram negative, ovoid shaped coccobacilli. They are aerobes,
non-motile and catalase and oxidase positive.
z Bordetella pertussis is the most important species.
MICROBIOLOGY 287
MODULE Bordetella
Microbiology z When isolated from a clinical sample it is capsulated but rapidly loses it
on subculture.
z In smears from culture, the bacilli are arranged in a thumb print like pattern.
z Bordetella can be cultured on Bordet Gengou medium and has the
appearance of bisected pearls or mercury drops
z Pertussis is a childhood disease, with route of infection as respiratory.
Notes z Cough plate method, nasopharyngeal aspirate, pernasal swab, West’s post-
pharyngeal swab are the methods used for specimen collection.
z Erythromycin is the drug of choice.
z Vaccination by whole cell killed vaccine in the triple vaccine combination
(DPT) is very effective.
TERMINAL QUESTIONS
1. What are the species of Bordetella?
2. What are the stages of Pertussis?
3. What are the virulence factors of B. pertussis?
4. How should a sample from a case of suspected pertussis be collected?
5. Describe the laboratory diagnosis of whooping cough?
6. Write in brief about the vaccines for pertussis?
30.1
1. Negative
2. Anaerobes
3. Bordet Gengou
4. Positive
30.2
1. Whooping cough
2. Respiratory
3. Calcium alginate, Dacron
4. Fluorescent antibody
288 MICROBIOLOGY
Spirochaetes MODULE
Microbiology
31
Notes
SPIROCHAETES
31.1 INTRODUCTION
Elongated, motile, flexible bacteria twisted spirally along the long axis are
termed ‘spirochetes’ (from Speira, meaning coil and chaite, meaning hair). It has
two families: (1) Spirochaetaceae in which the spirochaetes are anaerobic,
facultative anaerobic or microaerophilic and not hooked. This family includes
the genera Treponema and Borrelia and (2) Leptospiraceae which is a family
of hooked and obligate aerobic spirochaetes. The genus Leptospira is included
in this family.
Structurally, the spirochetes have gram-negative-type cell wall composed of an
outer membrane, a peptidoglycan layer and a cytoplasmic membrane. However,
they are more complex than other bacteria. A characteristic feature is the
presence of varying number of endoflagella. These endoflagella impart the shape
and the following types of motions to the Spirochaetes:
z Flexion and extension.
z Cork-screw like rotatory movement along the long axis.
z Translatory motion i.e. from one site to another.
OBJECTIVES

z discuss the characteristics of Spirochaetes.
z differentiate between the various groups of Spirochaetes.
z discuss the pathogenecity, laboratory diagnosis of Syphillis.
z discuss the features of Borrelia and Leptospira.
MICROBIOLOGY 289
MODULE Spirochaetes
Microbiology
31.2 GENUS TREPONEMA
Treponemes are relatively short slender spirochetes with fine spirals and pointed
or rounded ends. Treponemes cause the following diseases in humans:
z Venereal syphilis caused by T pallidum.
z Endemic syphilis caused by T endemicum.
Notes z Yaws caused by T pertenue.
z Pinta caused by T carateum.
Treponema pallidum: It is thin, delicate, spiral filament 6-14 µm by 0.2 µm,
with pointed and tapering ends. It has 6-12 coils which are comparatively small,
sharp and regular. T pallidum cannot be seen under the light microscope. Its
morphology and motility can be seen under the dark ground or phase contrast
microscope. It cannot be stained by ordinary bacterial stains, but can be stained
by silver impregnation methods. Fontana’s method is used for staining films and
Levaditi’s method for tissue sections. On prolonged Giemsa staining they stain
pale pink.
31.2.1 Culture
Pathogenic treponemes cannot be grown in artificial culture media but are
maintained by subculture in susceptible animals e.g. Nichol’s strain has been
maintained in rabbit testes for several decades. Cultivable treponemes such as T
phagedenis (Reiter’s strain - widely used as the antigen in group specific
treponemal tests for the diagnosis of syphillis) and T refringens (Noguchi’s
strain) are non-pathogenic.
31.2.2 Diseases
Treponema pallidum leads to syphilis which is acquired by sexual contact. Other
modes of transmission includes blood borne infections, congenital(from mother
to child) and occupational. Incubation period ranges from 10 to 90 days. The
clinical manifestations fall into three stages- primary, secondary and tertiary.
The primary stage is characterized by a hard, circumscribed, chancre, which is

usually genital. Secondary syphilis sets in 1-3 months after the primary lesion
heals. Roseolar or popular skin rashes, mucous patches in the oropharynx and
condylomata at the muco-cutaneous junctions are the characteristic lesions. This
may be followed by natural cure or in some cases by manifestations of tertiary
syphilis. These include cardiovascular lesions including aneurysms, chronic
granulomata (gummata) and meningovascular manifestations.
290 MICROBIOLOGY
Spirochaetes MODULE
31.2.3 Laboratory Diagnosis Microbiology
Laboratory diagnosis consists of demonstration of the spirochetes under the

microscope and of antibodies in serum or CSF.
(i) Microscopy
(a) Dark ground microscopy: Diagnosis by microscopy is applicable in primary
and secondary stages and in cases of congenital syphilis with superficial Notes
lesions.Specimens should be collected with utmost care as the lesions are
highly infectious. T pallidum is identified by its slender spiral structure and
slow movement in dark ground illumination. Smears can be stained by silver
impregnation method and visualized under light microscope.
(b) Direct fluorescent-antibody staining for T pallidum (DFA-Tp): The smear
to be tested is stained with fluorescein-labelled pathogen specific monoclonal
antibody. The treponemes appear distinct, sharply outlined and have apple-
green fluorescence.
(ii) Serological tests
These are divided into non-treponemal tests (non-specific/standard test for

syphilis) and treponemal tests.
(a) Non-treponemal tests/ standard tests for syphilis(STS): These tests are used
as screening tests.\ Reagin antibodies are detected by cardiolipin antigen.
Cardiolipin antigen is an alcoholic extract of beef heart tissue to which
lecithin and cholesterol are added. Non-treponemal tests include
z Venereal Diseases Research Laboratory (VDRL)
z Rapid Plasma Reagin test(RPR)
z Toluidine Blue Unheated Serum Test(TRUST)
z Wassermann complement fixation test
z Kahn tube flocculation test
The Wassermann reaction and the kahn test are now replaced by the VDRL test.
VDRL is the most widely used simple and rapid test. It is performed as a slide
flocculation test, in which the inactivated patient serum is mixed with a freshly
prepared suspension of cardiolipin-cholesterol-lecithin antigen on a glass slide.
The result is read under low power objective of microscope. Formation of
visible clumps or floccules is taken as positive reaction. In case of negative
result, the antigen particles are seen as evenly dispersed small fusiform needles.
The test is performed both as qualitative and quantitative assay. VDRL test can
be used for testing CSF also but not plasma. The major disadvantages of the
VDRL test are the need to prepare fresh antigen each day and to use microscope
to read the results.
MICROBIOLOGY 291
MODULE Spirochaetes
Microbiology Most laboratories now use RPR test which employs a stabilized VDRL carbon
antigen, which can be stored for upto 6 months at 4-10%C. This test does not
require heat inactivation of patient’s serum and can be read by naked eye.
TRUST is similar to RPR with an added advantage of storage at room
temperature of 26-31%C.
The major disadvantage of standard test for syphilis is the biological false
Notes positive reactions(BFP), because of sharing of cardiolipin antigen of T pallidum
and mammalian tissues.
BFP are defined as positive reactions obtained in cardiolipin tests, with negative
results in specific treponemal tests, in the absence of past or present treponemal
infections. They represent non-treponemal cardiolipin antibody responses. They
can be classified as acute or chronic. Acute BFP reactions last for a few weeks
or months and are associated with acute infections, injuries or inflammatory
conditions. Chronic BFP reactions persist for more than 6 months and are seen
in:
z SLE and other collagen diseases
z Leprosy
z Malaria
z Relapsing fever
z Infectious mononucleosis
z Hepatitis
z Tropical eosinophilia
(b) Treponemal tests for syphilis:
z Tests using cultivable treponemes: RPCF (Reiter protein complement fixation
test)
z Tests using pathogenic treponemes:
(i) Treponema pallidum immobilization test
(ii) Treponema pallidum agglutination tes
(iii) Treponema pallidum immune adherence (TPIA) test
(iv) Fluorescent treponemal antibody test (FTA)
(v) Treponema pallidum haemagglutination test (TPHA)
(vi) Enzyme immunoassay (EIA)
31.2.4 Treatment
Penicillin-G intravenous is the drug of choice.
Non-venereal Treponematoses: Occur in communities with poor standards of
hygiene. Ususally transmitted by direct body to body contact.
292 MICROBIOLOGY
Spirochaetes MODULE
Microbiology
Non-venereal Endemic Syphilis Yaws Pinta
treponematosis
Treponema species T pallidum subsp T pallidum subsp T pallidum subsp

endemicum pertenue carateum
Endemic regions Middle east, Tropical areas of Central and South

Zimbabwe, eastern Asia, Africa and America
Europe, India America, India Notes
Synonyms Bejel, njovera, Frambesia, pian, Carate
Sibbens parangi
Transmission Direct contact Direct contact, flies Direct contact

may act as
mechanical vectors
Primary lesion Usually not seen. Extragenital papule Extragenital papule

May be present on which enlarges and which does not
nipples of mother breaks down to ulcerate but develops
infected by their form an ulcerating into a lichenoid or
children granuloma psoriaform patch
Secondary and Similar to syphilis Similar to syphilis Hyper and hypo

tertiary lesions pigmented patches
Laboratory diagnosis Similar to syphilis. Similar to syphilis. Similar to syphilis.

and treatment
31.3 BORRELIA
Borrelia are large, motile, refractile spirochetes with irregular, wide, open coils.
They are usually 5-30 µm long and 0.3-0.7 µm wide. They are motile and are
readily stained by ordinary stains and are gram negative. A number of species,
although fastidious, can be cultured. These are transmitted to vertebrate hosts by
haematophagous arthropods. Important pathogenic species of Borrelia include:
B recurrentis (relapsing fever), B burdorferi (lyme disease) and B vincenti
(Vincent’s angina).
(a) B recurrentis: Relapsing fever is characterized by the occurrence of one or
more relapses after the subsidence of primary febrile paroxysm. It is of two
types; epidemic or louse borne, caused by B recurrentis and endemic or tick-
borne relapsing fever caused by a number of species like B duttoni, B hermsii,
B parkeri.
Borrelia causing relapsing fever can be seen by light microscopy in
preparations stained by aniline dyes such as Wright or Geimsa. These can be
demonstrated in the peripheral blood by direct stain. In fresh blood, they are
actively motile with forward, backward and corkscrew like motions.
MICROBIOLOGY 293
MODULE Spirochaetes
Microbiology Diagnosis is by direct examination of wet films by dark ground or phase

contrast microscopy or staining blood films with giemsa or gram stain using
dilute carbol fuchsin as counter stain. Culture is too difficult and serology is
unreliable. Tetracyclines, chloramphenicol, penicillin and erythromycin are
effective.
(b) Borrelia burgdorferi: These are flexible, helical and gram-negative. It is
microaerophilic and can be cultured on BSK (barbour stoenner kelly)
Notes medium. It leads to Lymes disease. Transmission is by ixodid ticks. After an
incubation period of 3-30 days , the patient develops a localized infection in
the form of an expanding annular skin lesion (erythema migrans). This is
followed by second stage of disseminated infection, wherein the patient
develops fever, headache, arthralgia and lymphadenopathy. Third stage is of
persistent infection, with chronic arthritis, polyneuropathy, encephalopathy
and acrodermatitis. Diagnosis is by direct examination of skin lesions, blood
or CSF by giemsa or gram stain using carbol fuschin as counterstain.
Darkground or fluorescent microscopy can also be done. Serological tests
like ELISA and immunofluorescence have been described, confirmation is
by immunoblotting. Doxycycline, amoxicillin and cefuroxime are useful
for treatment.
(c) Borrelia vincenti: Borrelia vincenti is a motile spirochete, with 3-8 coils of
variable size. It is easily stained with dilute carbol fuschin and is gram
negative. It can also be stained by methyl violet, giemsa and leishman stains.
It is an obligate anaerobe and can be cultured in sealed tubes containing
digest broth enriched with ascetic fluid. In a symbiotic association with
Leptotricha buccalis, B vincenti leads to ulcerative gingivostomatitis or
oropharyngitis (vincent’s angina). For diagnosis, smears are made from the
ulcerative lesions and are stained with dilute carbol fuschin.
31.4 LEPTOSPIRA
Leptospires are actively motile, delicate spirochetes, possessing a large number
of fine and tightly coiled spirals and hooked ends like umbrella handles. They
cannot be seen under the light microscope. They stain poorly with aniline dyes.
They may be stained with Giemsa stain. Better results are obtained by silver
impregnation methods. The genus Leptospira is classified into two species: L
interrogans containing pathogenic leptospires and L biflexa containing saprophytic
leptospires found predominantly in surface waters. Within each species are
serogroups, which are further classified into serotypes (serovars).
Leptospires are obligate aerobes. Several liquid and semi-solid media, such as
Korthof’s, Stuart’s and Fletcher’s media have been described. Semisynthetic
media such as EMJH (Ellinghausen, McCullough, Johnson and Harris) are now
commonly used.
294 MICROBIOLOGY
Spirochaetes MODULE
L interrogans causes a zoonotic disease, transmitted to humans by direct or Microbiology
indirect contact with water contaminated by urine of carrier animals. Leptospirosis
can be in the form of a mild febrile illness or sometimes the patient may land up
into severe illness with jaundice and albuminuria, known as weil’s disease.
Diagnosis of Leptopirosis can be done by direct microscopic examination of
blood and urine. Serological tests used for diagnosis can be genus specific
(complement fixation test, haemagglutination test, ELISA, etc) or serotype Notes
specific (microscopic and macroscopic agglutination test). Leptopires are
sensitive to penicillin, tetracycline and erythromycin.
(a) (b) (c)

(a) = Borrelia (b) = Spirochete (c) = Leptospira

1. ................. is the most widely used simple and rapid test for syphilis.
2. Syphilis is acquired by ................. contact.
3. Vincent’s angina is caused by .................
4. B burgdorferi is cultured in .................
5. Leptospira are obligate .................
6. Leptospira causes severe form of illness known as .................
7. Match the following:
1. Veneral syphilis (a) T endemicum
2. Endemic syphilis (b) T carateum
3. Pinta (c) T pallidum
4. Yaws (d) T pertenue
MICROBIOLOGY 295
MODULE Spirochaetes
Microbiology

z Spirochetes are elongated, motile, flexible bacteria twisted spirally along
the long axis.
z They are divided into two families: Spirochaetaceae in which the spirochaetes
Notes are anaerobic, facultative anaerobic or microaerophilic and not hooked.
z This family includes the genera Treponema and Borrelia and Leptospiraceae
which are obligate aerobes and have hooked ends.This include the genera
Leptospira.
z Spirochetes have characteristic motility with flexion, extension and cork
screw like movement.
z Treponema pallidum causes a venereal disease syphilis.
z Direct examination by dark ground microscopy can be done for the
diagnosis of syphilis.
z For serology various non-treponemal and treponemal tests are available.
z The non-treponemal tests are also known as non-specific or standard test
for syphilis. These are used as screening tests and include VDRL, RPR,
TRUST.
z The treponemal tests are more specific. These include TPI (T pallidum
immobilization test), TPA (T pallidum agglutination test), TPIA (T pallidum
immune adherence test), FTA (fluorescent treponemal antibody test), FTA-
ABS (fluorescent treponemal antibody-absorption test) and TPHA (T
pallidum haemagglutination assay).
z The non-venereal species of Treponema include T pallidum subsp endemicum,
T pallidum subsp pertenue and T pallidum subsp carateum which causes
endemic syphilis, yaws and pinta respectively. They occur in communities
with poor standards of hygiene and are ususally transmitted by direct body
to body contact.
z Borrelia are large, motile, refractile spirochetes with irregular, wide, open
coils. They are motile and are readily stained by ordinary stains and are gram
negative. A number of species, although fastidious, can be cultured. These
are transmitted to vertebrate hosts by haematophagous arthropods. Important
pathogenic species of Borrelia include: B recurrentis (relapsing fever), B
burdorferi (lyme disease) and B vincenti (Vincent’s angina).
z Leptospires are actively motile, delicate spirochetes, possessing a large
number of fine and tightly coiled spirals and hooked ends like umbrella
handles. They cannot be seen under the light microscope. Leptospires are
296 MICROBIOLOGY
Spirochaetes MODULE
obligate aerobes and can be cultured in artificial media. L interrogans causes Microbiology
a zoonotic disease, which is transmitted by rodents. It can be manifestated
as a febrile illness or may be in the form of weil’s disease with jaundice
and albuminuria.
TERMINAL QUESTIONS Notes
1. What are the characteristics of spirochetes?

2. Describe the Standard tests for syphilis.
3. Discuss the laboratory diagnosis of syphilis.
4. Discuss the pathogenecity of Borrelia.
5. Write a short note on relapsing fever.
6. Write briefly about vincent’s angina.
7. Write a short note on Leptospirosis.
8. Differentiate between Treponema, Borrelia and Leptopsira.

31.1
1. VDRL
2. Sexual
3. Borrelia Vincenti
4. Barbour stoenner Kelly
5. Aerobes
6. Weil’s disease
7. 1. (c)
2. (a)
3. (d)
4. (b)
MICROBIOLOGY 297
MODULE Rickettsiaceae
Microbiology
32
Notes
RICKETTSIACEAE
32.1 INTRODUCTION
Rickettsiae are small, pleomorphic, gram negative bacilli that multiply by binary
fission. They are fastidious bacteria that are obligate intracellular parasites. They
require an arthropod vector as part of their natural cycle and are transmitted to
man by blood sucking arthropods. They possess both DNA and RNA. They
possess a cell wall made of peptidoglycan.They are non motile and non
capsulated.They reproduce by binary fission and are susceptible to antibacterial
agents. However they are not visible by light microscopy. Family Rickettsiaceae
contains three genera: Rickettsia, Orientia, and Ehrlichia-Coxiella burnetti
which causes Q fever and Rochalimaea Quintana causing trench fever are no
longer included as the former is not transmitted by arthropod vector and the latter
is not an obligate intra cellular parasite.It was named after Howard Taylor
Rickett who died of typhus fever contracted while working on this organism.
OBJECTIVES
z enumerate members of family Rickettsiaceae.
z describe characteristics of rickettsiae.
z describe cultivation of rickettsiae.
z describe pathogenesis and disease caused by rickettsiae.
32.2 GENUS RICKETTSIAE

Genus consist of two groups on the basis of disease caused by them
298 MICROBIOLOGY
Rickettsiaceae MODULE
z Typhus fever group Microbiology
z Spotted fever group
Morphology
They are pleomorphic coccobacilli 0.3-0.6µm × 0.8-2µm in size. They possess
trilaminar cytoplasmic membrane and cell wall as seen by electron microscopy.
They are gram negative though do not take stain well. They stain deep red with Notes
Machiavello and Gimenez while bluish purple with Giemsa and Castaneda stain.
Cultivation
z They are obligate intracellular parasites. They cannot be grown on cell free
media. They generally grow in cytoplasm of infected cell but spotted fever
rickettsiae grow in nucleus as well.
z Optimum temperature for growth is 32-35°C.
z They can be cultivated in yolk sac of 5-6 days old embryonated egg.
z They can grow well on HeLa, Hep-2, mouse fibroblast, Detriot 6 and other
continuous cell lines.
z Mice and guinea pig can be used for primary isolation of rickettsiae from
clinical samples.
Antigenic structure
Rickettsiae possess 3 types of antigens
1. Group specific soluble antigen:- It is present on surface of organism and

is protein in nature.
2. Species specific antigen:- It is adherent to the cell and act as adhesin for
host cell
3. An alkali stable polysaccharide:- Found in some rickettsiae and in some non
motile strains of Proteus (OX 19, OX 2, OXK). This sharing of antigens
forms the basis for Weil- Felix reaction used in diagnosis of rickettsial
infections. In this test agglutinins are detected against these Proteus strains.
Pathogenesis
Man acquire infection by bite or faeces of an infected arthropod vector. On entry
into the human body they become localised chiefly in the vascular epithelium
leading to thrombus formation.
MICROBIOLOGY 299
Microbiology

1. Rickettsiae are Gram ................ bacilli.
2. Rickettsiae are obligate ................ parasites.
3. Rickettsiae are transmitted to man by ................
Notes
4. Rickettsiae are ................ coccobacilli.
32.2.1 Typhus fever group

This consist of-
(a) Epidemic (classical) typhus

(b) Brill-Zinsser disease (recrudescent infection)
(c) Endemic typhus
Epidemic Typhus
It is reported from all parts of the world but common in Russia and eastern
Europe. In India the endemic spot is Kashmir. Humans are the only natural
vertebrate hosts. Its causative agent is R. prowazekii. Human body louse
(Pediculus humanus corporis) and head louse (Pediculus humanus capitis) act
as vector. Incubation period is 10-14 days. Clinical features include: high fever,
chills, severe headache and vomiting. On 4-7 days a characteristic maculopapular
rash appear which start from trunk and spread over limbs but sparing face, palm
and soles. Patient become stuporous and delirious in 2nd and 3rd week. The case
fatality is about 40%.
Brill-Zinsser disease (recrudescent infection)

Rickettsiae may remain latent in the lymphoid tissue for years. Such latent
infection may be reactivated leading to recrudescent typhus. It is milder illness
with shorter duration (ˆ2weeks) and lower case fatality. Skin rashes are also
mild. Disease itself is not importance but patient is infectious for louse and an
outbreak can occur.
Endemic typhus
Clinical disease is similar but milder than epidemic typhus and case fatality is
1%. It is caused by R. typhi. Its reservoir is rat and are transmitted by flea
(Xenopsylla cheopsis). Rat flea acquire infection by feeding on infected rat.
300 MICROBIOLOGY
Natural cycle is rat-flea-rat and man are the dead end host. They acquire infection Microbiology
by the bite of infected fleas when their saliva of feces is rubbed in.
R. typhi can be differentiated from R. prowazekii by following test
z Neil-Mooser or tunica reaction :- when male guinea pig is inoculated

intraperitoneally with blood of patient infected with R. typhi they develop
fever and scrotal swelling. The testis cannot be pushed back into abdomen
Notes
due to adhesions. This reaction is negative in case of R. prowazekii.
z IFA
z ELISA
z PCR based DNA tests.
32.2.2 Spotted fever group

This group include:
Table 32.1: Members of spotted fever group, their vectors and reservoir.
Members Disease Vector Vertebrate reservoir
R. rickettsii Rocky Mountain spotted fever Tick Rabbit, dog, rodents

R. siberica Siberian tick typhus Tick Wild animals, cattle
R. conori Indian tick typhus Tick Rodents
R. japonica Oriental spotted fever Tick -
R. akari Rickettsial pox Gamacid mite Mouse
Main vector of spotted fever group are ticks except for R. akari which is
transmitted by mites. Eschar frequently develops at the site of bite except in case
of rocky mountain spotted fever.
Rocky Mountain spotted fever: It is the most serious type of infection,
transmitted by tick (Dermacenter andersoni). Incubation period is 1 week.
Clinical features include fever, headache, vomiting, diarrhoea, photophobia and
cough. Maculopapular rashes appears on 4th day which starts from wrist, ankles,
palms and soles. Rashes may become petechial and in grave cases they may
become hemorrhagic. Patient may lend up into hypotensive shock, hemiplagia
and die within 5 days of onset of symptoms. Mortality varies from 6- 70%.
Rickettsial Pox: Mildest form of rickettsial disease, self-limited, non-fatal,
vesicular exanthema. Also known as varicelliform rickettsiosis. Vector is mite
and reservoir is domestic mouse. Clinically vesicle appear at site of bite and
enlargement of lymphnodes occur followed by fever, headache, malaise within
3-10 days. Illness last for 10-14 days.
MICROBIOLOGY 301
Microbiology
32.3 SCRUB TYPHUS
It is caused by Orientia tsutsugamushi. Migratory birds may act as reservoir and
transporter of disease agent. Vector is trombiculid mites. Man are the accidental
host and may acquire infection when they trespass into mite islands and aare
bitten by their larvae (chiggers).
After 1- 3 weeks of bite, patient develops severe headache, fever, conjunctivitis,
Notes deafness and eschar at site of bite. After 1 week of fever, maculopapular rash
appears which starts from trunk. Mortality varies from 10- 60%.
32.4 LABORATORY DIAGNOSIS OF RICKETTSIAL

INFECTIONS
Samples which can be processed for diagnosis are
z Blood clots
z Serum
z Biopsy specimen of rashes
z Impression smear from organs of infected animals
z Endolymph of ticks
Diagnosis is carried out by
(a) Isolation of rickettsiae in lab animals, fertile hen’s egg and cell cultures
(b) Direct detection of organism and their antigen in clinical samples
(c) Serology
32.4.1 Isolation of Rickettsiae

Blood clots ground in skimmed milk or BHI broth is inoculated intraperitoneally
in male guinea pig or mice. Animal will be observed for 3-4 weeks.
Table 32.2: Reaction in guinea pig produced by different rickettsial agents.
Agent Reaction in Guinea pig
R. rickettsii Develop fever, scrotal necrosis and may even die

R. typhi Animal develops fever and tunica reaction
R. akari Animal develops fever and tunica reaction
R. prowazekii Animal develops fever without tunica reaction
O. tsutsugamushi Mice is preferred. Animal develops ascitis
302 MICROBIOLOGY
Smears from peritoneum, tunica and spleen of infected animals are stained by Microbiology
Giemsa or Gimenez method to demonstrate rickettsiae. They can also be grown
in yolk sac of embryonated hen’s eggs and cell culture.
32.4.2 Direct detection of organism and their antigen

Aggregates of the organism or their antigen in biopsy specimen from rashes and
liver, impression smears from organs of infected animals may be demonstrated Notes
by
z Giemsa staining
z Macciavello staining
z Gimenez staining
z Direct immunoflourescence
z Indirect immunoflourescence
z PCR
z Sequence Amplification

1. Epidemic typhus is caused by ................
2. Rickettsiae may remain latent in ................ tissue of human
3. R.akari causing spotted fever is transmitted by ................
4. The reservoir of Rickettsiae pox is ................
5. Scrub typhus is caused by ................
32.4.3 Serology
(a) Non-specific reaction:- Weil- Felix Reaction ( see table. 3).
(b) Specific:- using rickettsial antigen
z CFT with purified antigen
z Immunofluorescence on microdots of purified rickettsial antigen
z ELISA with particulate or extracted antigen
z Latex agglutination
MICROBIOLOGY 303
Microbiology Table 32.3 Weil- Felix Reaction in diagnosis of rickettsial diseases
Disease Agglutination with Proteus strains

OX19 OX2 OXK
Epidemic typhus ++++ ± -
Brill-Zinsser disease ± - -
Notes
Endemic typhus +++ ± -
Spotted fever group ++ ++ -
Scrub typhus - - +++
Rickettsial pox - - -
Treatment: Tetracyclines and chloramphenicol can be given to treat rickettsial

infections.
32.5 GENUS EHRLICHIA

They are small, gram negative, obligate intracellular bacteria. They have affinity
towards blood cells and their vacuoles inside phagocytic cells are known as
morula. They are tick borne. Leucopenia, thrombocytopenia and raised liver
enzymes are seen in patients. Three species have been reported to infect humans.
(a) E. sennetsu
(b) E. chaffeensis
(c) E. phagocytophila
z Demonstration of bluish intracytoplasmic inclusion (morula) in Geimsa
stained blood smears.
z Demonstration of specific antibodies by immunofluorescence using cell
cultures.
z PCR
Treatment: Doxycycline is recommended for treatment of ehrlichioses.
32.6 COXIELLA BURNETII (Q FEVER)- NO LONGER

INCLUDED IN GENUS RICKETTSIAE
Q fever is a worldwide zoonosis and is caused by Coxiella burnetii. It is also an
obligate intracellular pathogen. It is pleomorphic coccobacilli 0.2µm- 0.4µm ×
0.4- 1.0µm. It is Gram negative but better stained with Gimenez and other
rickettsial stains. It can survive holder method (63°C for 30 min) of pasteurization
304 MICROBIOLOGY
of milk but flash method is effective. It can be inactivated by 2% formaldehyde, Microbiology
1% Lysol and 5% H2O2.
Pathogenesis
It is transmitted among cattle, sheep and poultry by ticks. They can be shed in
milk of infected animals and products of conception. They can contaminate the
environment during parturition. Humans can get infection through
Notes
z Consumption of infected milk
z Handling of infected wool, meat and other animal products
z Contaminated soil, straw and clothing
Coxiella may enter through abraded skin, inhalation, ingestion. Incubation
period is 2-4 weeks. Patient develop fever, chill, headache, pneumonia,
endocarditis and meningoencephalitis. Spontaneous recovery is usual.
Coxiella can be isolated from blood, sputum and other clinical samples but is not
recommended due to serious risk of infection to laboratory personnel. Culture
can be done in guinea pig, mice and embryonated hen’s eggs. Cell cultures can
also be used. Diagnosis mainly relies upon the demonstration of antibodies by
CFT and indirect immunofluorescence assay. PCR can be done.
Treatment: Doxycycine may be given for treatment. In serious cases prolonged
treatment with tetracycline, chloramphenicol may be required.
32.7 GENUS BARTONELLA

It belongs to family Bartonellaceae. They are Gram negative bacilli. This genus
include
32.7.1 Bartonella bacilliformis

It is pleomorphic, Gram negative, motile coccobacillus. They are strict aerobes.
They can grow on semi-solid nutrient agar containing rabbit serum and
haemoglobin but growth is slow. It causes Oraya fever (Carrion’s disease) which
is transmitted by sandfly. Incubation period is 3 weeks to 3months. Clinical
features include fever, severe headache followed by severe anaemia. Organism is
found inside erythrocytes of infected patients. Hepato-splenomegaly can be
present. Case fatality is 40%. A late sequel in survivors is verruga peruana.
Diagnosis is done by demonstration of organism in blood smears by Giemsa
staining and by culture. Penicillin, streptomycin and tetracycline can be used for
treatment of Oraya fever and verruga peruana.
MICROBIOLOGY 305
Microbiology 32.7.2 Bartonella Quintana

It is small, gram negative bacillus. It does not possess flagella. it may show
twitching movement caused by fimbriae. Growth is slow on rabbit or sheep
blood agar when incubated in 5% CO2. There is no animal reservoir of this
disease. It causes trench fever. Disease is spread by body louse. Infection is
acquired when infected feaces are scratched into the skin. Incubation period is
Notes 14- 30 days. Clinical features include headache, fever, severe pain in legs and
back and roseolar rash on chest, abdomen and back. Recovery is frequent but
relapses can occur.
Diagnosis is done by allowing healthy lice on patient blood and the organism
may be detected in gut of lice. Culture can be done on rabbit or sheep blood agar.
It can be detected in tissues by PCR.
32.7.3 Bartonella henselae

It is small, slightly curved, Gram negative bacillus. It also displays twitching
motility. It can be grown on chocolate agar, Columbia agar with 5% sheep or
rabbit blood and BHI agar supplemented with blood. Growth occurs in 5-15
days. It causes cat- scratch disease. Infection is acquired by contact, scratch or
bite of infected cat. Clinical features include fever and lymphadenopathy. Cat
scratch disease is self limiting and requires no treatment. Endocarditis can occur.
In AIDS patient, it can lead to bacillary angiomatosis and bacillary peliosis.
Diagnosis is done by demonstration of organism in section of lymph node

biopsies stained with Warthin- Starry silver impregnation stain. It can be gown
on chocolate and Columbia agar.

1. Serologically ................... method is used in diagnosis of rickettsial disease.
2. Ehrlichia infections are transmitted by ...................
3. Q fever is caused by ...................
4. Baronella bacilliform causes ...................
5. Bartonella bacilliform is transmitted by ...................
6. Bartonella Quinteana causes ................... fever
7. Trench fever is spread by ...................
306 MICROBIOLOGY
Microbiology

z Family Rickettsiaceae contains small coocobacillary organisms. They cause
typhus fever and other related diseases. They are true intracellular parasites.
They cannot be grown on artificial media. They are arthropod borne. Among
the typhus fever group RMSF is the most serious form of disease while the
Notes
rickettsial pox is the mildest form of disease. Ehrlichia have affinity for
blood cell of patient. Coxiella causes Q fever. Bartonella can be grown on
blood and chocolate agar. They are gram negative but are better visualised
by special staining.
TERMINAL QUESTIONS
1. Describe characteristics of family Rickettsiaceae?
2. Describe pathogenesis of rickettsial diseases?
3. Describe Weil- Felix reaction?
4. Describe Neil Mooser reaction?
5. Differentiate between epidemic and endemic typhus?
6. Write short note on Brill- Zinsser disease?
32.1
1. Negative
2. Intracellular
3. Anthropods
4. pleomorphic
32.2
1. R.prowazekii
2. Lymphoid
3. Mites
MICROBIOLOGY 307
Microbiology 4. Domestic mouse

5. Orientia tsutsugamushi
32.3
1. Weilfelix reaction
2. Ticks
Notes 3. Coxiella burnetii
4. Oraya fever
5. Sandfly
6. Trench
7. Body louse
308 MICROBIOLOGY
Chlamydia MODULE
Microbiology
33
Notes
CHLAMYDIA
33.1 INTRODUCTION
Chlamydiae are obligate, aerobic, intracellular parasites of eukaryotic cells.
They are small Gram-negative coccoid or rod shaped, non-motile bacteria.
Chlamydiae exhibit characteristics intermediate between bacteria and viruses.
They are widespread in the natural world, being parasites of people, animals and
birds with tropism for squamous epithelial cells and macrophages of the
respiratory and gastrointestinal tract.
They are recognized as bacteria as
z They have both DNA and RNA.
z They have cell wall (that resembles that of GNB) and ribosomes
z Replicate by binary fission
z Susceptible to antibiotics
OBJECTIVES
z distinguish characteristics of Chlamydia.
z describe the diseases produced by it.
z explain the laboratory diagnosis.
33.2 CLASSIFICATION
Classification of Chlamydiae is as follows:
Order Chlamydiales
MICROBIOLOGY 309
MODULE Chlamydia
Microbiology Family Chlamydiaceae

Genus Chlamydia,Chlamydophila
Species There are three species C. trachomatis, Chlamydophila psittaci,
Chlamydophila pneumoniaae
C. trachomatis has two biovars; TRIC and LGV.
Notes
33.3 CELL STRUCTURE
Chlamydiae have a cytoplasmic membrane and an outer membrane similar to
Gram-negative bacteria but lack a peptidoglycan cell wall. Chlamydiae cannot
synthesize their own ATP and require intracellular abode to remain viable.
Chlamydiae exist in two forms: the elementary body and the reticulate body.
Both of them play a pivotal part in the life cycle of chlamydia. Although Gram
negative, Chlamydiae stain better with Castaneda, Machiavello or Gimenez
stains.
33.4 ELEMENTARY BODY (EB)

The elementary body is the dispersal form, which is analogous to a spore. This
dispersal form is about 0.3 µm or 200-300 nm in diameter. It is the extracellular
infective form. It induces its own endocytosis upon exposure to target cells.
33.5 RETICULATE BODY (RB)

Reticulate body is the intracellular, multiplicative form. It represents the non-
infectious growing form.
33.6 LIFE CYCLE

The life cycle of Chlamydia trachomatis consists of two stages: elementary body
and reticulate body. Upon endocytosis into the host cell EB prevents
phagolysosomal fusion enabling intracellular survival of the bacteria. Once
inside the endosome, the elementary body transforms into the larger reticulate
body (500 – 1000 nm) as a result of the glycogen that is produced. The reticulate
body is the reproductive form. It divides through binary fission at approximately
2-3 hours per generation. It contains no cell wall and is detected as an inclusion
in the cell arranged as a mantle around the nucleus. The inclusion bodies are
basophilic. They can also be stained by Lugol’s iodine because of the presence
of glycogen matrix. After division, the reticulate body transforms back to the
elementary form and is released by the cell by exocytosis. One phagolysosome
usually produces 100-1000 elementary bodies. The entire process takes 24 – 48
hours. The EB may infect new cells and the cycle continues.
310 MICROBIOLOGY
Chlamydia MODULE
Microbiology
33.7 ANTIGENIC STRUCTURE
Chlamydia antigens consist of 3 groups: genus-specific antigen, species-
specific protein antigen, serotype-specific. The sero type-specific antigens are
located on MOMP and on the basis of this chlamydiae have been divided into
many serovars or serotypes.
33.8 CULTURE Notes

Chlamydiae can be isolated by the following methods:
(a) Animal inoculation: Mice can be inoculated through intranasal,
intraperitoneal or intracerebral route. Mice die within 10 days. Smears
made from lung, spleen, brain or peritoneal exudate demonstrate elementary
bodies.
(b) Egg inoculation: Organisms can be isolated by egg yolk inoculation of
the specimen. Impression smears can be stained by Giemsa or Gimenez.
(c) Tissue culture: McCoy cells treated with cycloheximide are the most
commonly used cell lines. Irradiated or metabolically inhibited cell lines
can also be used for isolation of chlamydia. Inclusion bodies can be
visualized by staining the cell lines.
33.9 DISEASES PRODUCED BY CHLAMYDIA

(a) Ocular infections: C. trachomatis serotype A,B,Ba,C- is the leading cause
of preventable blindness (caused by a chlamydia infection called trachoma)
in the world. Other diseases produced are inclusion conjunctivitis (serotype
D to K) and ophthalmia neonatorum.
(b) Genital infections: C. trachomatis is also the leading cause of sexually
transmitted disease worldwide. It is associated with non- gonococcal
urethritis and lymphogranuloma venereum (serotype L1, L2, L3). C.
trachomatis is one of the major causes of pelvic inflammatory disease
(PID) and infertility in women.
(c) Respiratory infections: C. pneumoniae causes pneumonia. C. psittaci
causes psittacosis.

Specimen collection: Specimen should be collected by scraping the mucosa.
Discharge should not be collected. Depending on the site of infection, ocular,
urethral, cervical, sputum, respiratory secretions can be collected. In suspected
Psittacosis, blood and sputum are collected for microscopy and culture and
serum for serology.
MICROBIOLOGY 311
MODULE Chlamydia
Microbiology Direct detection of antigen: Antigen detection is a rapid method of diagnosing

chlamydial infection.
1. Light Microscopy: Inclusion bodies of C. trachomatis can be detected by
staining with Lugol’s iodine. Iodine can be used because inclusion bodies
contain a glycogen matrix. Giemsa, Castaneda, Machiavello and Giminez
methods are better and can be used to stain ocular, cervical or urethral
specimen.
Notes 2. Immunofluoresence: Direct fluorescent antibody test detects major outer
membrane proteins. It is now considered by many the test of choice for
diagnosis.
ELISA: Antigen and antibodies can be detected by ELISA. Antigen detection

is more specific than antibody detection.
Isolation: Mice, fertilized hen’s egg and tissue cultures can be used for isolation
of chlamydia. The clinical specimen can be inoculated into the yolk sac of 6 to
8 day old eggs. Irradiated or cycloheximide treated McCoy cell culture is the
preferred isolation method.
Molecular tools: Polymerase chain reaction, ligase chain reaction can be used
for detection of chlamydia
33.11 TREATMENT
Sulphonamides and tetracycline are the drugs of choice. Single dose azithromycin
is the drug of choice for non-gonoccocal urethritis.

1. Chlamydiae are ............... parasites
2. Chlamydiae are gram ............... cocci
3. ............... body enables intracellular survival of the bacteria
4. ............... body is the reproductive form
5. C.trahomatis causes ............... & ...............
312 MICROBIOLOGY
Chlamydia MODULE
Microbiology

z Chlamydiae are obligate, aerobic, intracellular parasites of eukaryotic cells.
z They are small Gram-negative coccoid or rod shaped, non-motile bacteria.
z Chlamydiae has elementary body which is similar to a spore.
z Life cycle of Chlamydia trachomatis consists of two stages namely Notes
elementary and reticulate body.
z Chlamydiae can be isolated by animal inoculation, egg inoculation & tissue
culture
z Chlamydia trachomatis causes preventable blindness, conjunctivitis &
ophthalmia neonatorum, non- gonococcal urethritis and lymphogranuloma
venereum, inflammatory disease, infertility in women.
z Inclusion bodies of C. trachomatis can be detected by staining with Lugol's
iodine
z Giemsa, Castaneda, Machiavello methods are used to stain ocular, cervical
or urethral specimen.
z Direct fluorescent antibody is test of choice for diagnosis.
TERMINAL EXERCISES
1. Describe the life cycle of chlamydia.
2. Name the various staining techniques used for chlamydia.
3. Enumerate the diseases caused by Chlamydia trachomatis.
4. How can we cultivate chlamydia?
5. What are the rapid diagnostic methods for detecting chlamydia?

33.1
1. Intracellular
2. Negative
3. Elementary
4. Reticulate
5. Blindness & lymphogranuloma venerum
MICROBIOLOGY 313
MODULE Mycoplasma and L-Forms
Microbiology
34
Notes
MYCOPLASMA AND L-FORMS
34.1 INTRODUCTION
Mycoplasma species are the smallest free-living organisms. These organisms are
unique among prokaryotes in that they lack a cell wall, hence lack fixed shape
or size and also lack Gram stain reaction and their lack of susceptibility to beta-
lactams. Because of their plasticity, they can pass through bacterial filters of
450nm pore size and have often been mistaken for viruses. Mycoplasmal
organisms are usually associated with mucosal surfaces of respiratory and
urogenital tracts. They rarely penetrate the submucosa, except in the case of
immunosuppression or instrumentation, when they may invade the bloodstream
and disseminate.
Species most commonly associated with infections are Mycoplasma pneumoniae,

Mycoplasma hominis, Mycoplasma genitalium, and Ureaplasma species.
Fig. 34.1: Fried egg colonies of Mycoplasma
Culture: They can be cultivated on fluid (PPLO broth) or solid media (PPLO
agar) enriched with 20% horse or human serum and yeast extract and addition
of antibiotics as selective agents. Colonies appear after incubation for 2-6 days
and are about 10-600 µm in size with a typical “fried egg” appearance. Colonies
314 MICROBIOLOGY
Mycoplasma and L-Forms MODULE
may be seen with a hand lens but are best studied after staining by Dienes Microbiology
method. For this, a block of agar containing the colony is cut and placed on a
slide. It is covered with a cover slip on which an alcoholic solution of methylene
blue and azure has been dried. Colonies cannot be picked with loops; instead
subculture is carried out by cutting a agar block with colonies and rubbing it on
fresh plates In the liquid medium no turbidity is appreciated.
Notes
Fig. 34.2: Dienes stain
OBJECTIVES
z describe the structure of Mycoplasm & L.form
z explain the pathogenesis of Mycoplasm & L. Form
34.2 PATHOPHYSIOLOGY
M pneumoniae causes community-acquired atypical pneumonia, tracheobronchitis
or bronchiolitis. Pneumonia develops in only 5-10% of persons who are infected.
Acute pharyngitis may also occur.
After inhalation of respiratory aerosols, the organism attaches to host epithelial
cells in the respiratory tract. It produces adhesions and other accessory proteins
which mediate attachment, followed by induction of ciliostasis, local inflammation
and tissue destruction that may be mediated by liberation of hydrogen peroxide.
Recently, M pneumoniae has been shown to produce an exotoxin: community-
acquired respiratory disease toxin (CARDS) that is also believed to play a major
role in the damage to the respiratory epithelium. The organism replicates
intracellularly, which contribute to chronicity of illness and difficult eradication.
Spread of infection throughout households is common. the incubation period is
2-3 weeks.
MICROBIOLOGY 315
Microbiology Antimicrobials
Oral erythromycin or one of the newer macrolides such as azithromycin or
clarithromycin have long been the Drug of Choice for mycoplasmal respiratory
tract infections. Tetracycline and its analogues are also active. Fluoroquinolones
such as levofloxacin or moxifloxacin exhibit bactericidal antimycoplasmal
activity but are generally less potent in vitro than macrolides. As would be
Notes predicted by the lack of a cell wall, none of the beta-lactams is effective against
M pneumonia.
In addition to the administration of antimicrobials for the management of M

pneumoniae infections, other measures (eg, cough suppressants, antipyretics,
analgesics) should be administered as needed to relieve other systemic
symptoms.

1. Mycoplasma lack ...............
2. Mycolasma commonly causes ...............
3. Mycoplasma produces ............... & ............... which mediate attachment
4. M.pneumoniae produce ............... toxin
34.3 L-FORM BACTERIA

L-form bacteria, also known as L-phase bacteria, L-phase variants, and cell wall-
deficient (CWD) bacteria, are strains of bacteria that lack cell walls.[1] They
were first isolated in 1935 by Emmy Klieneberger-Nobel, who named them “L-
forms” after the Lister Institute in London where she was working
Two types of L-forms are distinguished: unstable L-forms, spheroplasts that are
capable of dividing, but can revert to the original morphology, and stable L-
forms, L-forms that are unable to revert to the original bacteria.
L-forms can be generated in the laboratory from many bacterial species that
usually have cell walls, such as Bacillus subtilis or Escherichia coli. This is done
by inhibiting peptidoglycan synthesis with antibiotics or treating the cells with
lysozyme, an enzyme that digests cell walls
Some of the species of L-form bacteria that have been implicated in chronic
disease include Bacillus anthracis, Treponema pallidum, Mycobacterium
tuberculosis, Helicobacter pylori, Rickettsia prowazekii, and Borrelia burgdorgeri.
316 MICROBIOLOGY
Mycoplasma and L-Forms MODULE
Although L-forms can develop from Gram-positive as well as from Gram- Microbiology
negative bacteria, in a Gram stain test, the L-forms always colour Gram-
negative, due to the lack of a cell wall.

1. L-form are ............... deficient bacteria
2. L-forms that are unable to revert to the original bacteria are ...............
3. L-froms can be generated in laboratory from bacterial species that have

...............
4. Due to the lack of cell wall l-forms ............... on staining

z Mycoplasma species are the smallest free-living organisms
z These organisms lack a cell wall and hence lack Gram stain reaction
z Mycoplasmal organisms are habitant of mucosal surfaces of respiratory and
urogenital tracts & cause infections is immunosuppressed patients.
z M pneumoniae causes community-acquired atypical pneumonia,
tracheobronchitis or bronchiolitis
z L-form bacteria, also known as L-phase bacteria, L-phase variants, and cell
wall-deficient bacteria.
z L-form bacteria are two types namely unstable L-forms, which can revert
to the original morphology.
z Stable L-forms, L-forms that are unable to revert to the original bacteria.
34.1
1. Cell wall
MICROBIOLOGY 317
Microbiology 2. Atypical pneumonia

3. Adhesions & proteins
4. Community acquired respiratory disease
34.2
1. Cell wall
Notes
2. Stable l-froms
3. Cell wall
4. Gram negative
318 MICROBIOLOGY
Spore Forming Anaerobes MODULE
Microbiology
35
Notes
SPORE FORMING ANAEROBES
35.1 INTRODUCTION
Spore-forming bacteria produce a unique resting cell called an endospore. They
are Gram-positive and usually rod-shaped, but there are exceptions. Bacteria are
a large group of microscopic, unicellular organisms that exist either independently
or as parasites. Some bacteria are capable of forming spores around themselves,
which allow the organism to survive in hostile environmental conditions.
Bacterial spores are made of a tough outer layer of keratin that is resistant to
chemicals, staining and heat. The spore allows the bacterium to remain dormant
for years, protecting it from various traumas, including temperature differences,
absence of air, water and nutrients.
The two medically-important genera are Bacillus, the members of which are
aerobic spore formers in the soils, and Clostridium, whose species are anaerobic
spore formers of soils, sediments and the intestinal tracts of animals.
Some spore formers are pathogens of animals, usually due to the production of
powerful toxins. Bacillus anthracis causes anthrax, a disease of domestic
animals (cattle, sheep, etc.), which may be transmitted to humans. Bacillus
cereus causes food poisoning. Clostridium botulimum causes botulism, a form
of food poisoning, and Clostridium tetani is the agent of tetanus. Clostridium
perfringens causes food poisoning, anaerobic wound infections and gas gangrene,
and Clostridium difficile causes a severe form of colitis called pseudo-
membranous colitis. Whenever the spore-formers act as pathogens, it is not
uncommon or surprising that their spores are somehow involved in transmission
or survival of the organism between hosts.
OBJECTIVES
After reading this chapter, the student will be able to
z explain why bacteria produce spores
MICROBIOLOGY 319
MODULE Spore Forming Anaerobes
Microbiology
z identify various types of spore forming bacteria
z describe the structure of Spores
z explain Sporulation Cycle
35.2 STRUTURE OF SPORES

It consists of following layers
Notes
Exosporium - A thin delicate covering made of protein.
Spore coats - Composed of layers of spore specific proteins.
Cortex - Composed of loosely linked peptidoglycan and contains dipicolinic
acid (DPA), which is particular to all bacterial endospores. The DPA cross links
with calcium ions embedded in the spore coat. This cross linkage greatly
contributes to the extreme resistance capabilities of the endospores because it
creates a highly impenetrable barrier. The calcium DPA cross linkages compose
10% of the dry weight of the endospores.
Core - The core contains the usual cell wall and, cytoplasmic membrane,
nucleoid, and cytoplasm. The core only has 10-30% of the water content of
vegetative cells; therefore the core cytoplasm is in a gel state. The low water
content contributes to the endospores success in dry environments. However, the
low water concentration and gel cytoplasm contributes to the inactivity of
cytoplasmic enzymes. The core cytoplasm is also one unit lower in pH than the
vegetative cell, thus conferring acidic environment survival. SASPs, small acid
soluble spore proteins, are formed during sporulation and bind to DNA in the
core. SASPs protect the DNA from UV light, desiccation, and dry heat. SASPs
also serve as a carbon energy source during germination, the process of
converting a spore back to a vegetative cell.
Fig. 35.1: Endospore
320 MICROBIOLOGY
Microbiology
Sporulation Cycle: You may have read elsewhere in this site that bacteria
sometimes form protective spores to help them survive through tough times.
Some other kinds of microbes do, too. Here’s how that transformation takes
place.
First of all, you might think of a bacterial spore roughly as a mummified
bacterium. The spore has a hard protective coating that encases the key parts of
the bacterium – think of this coating as the sarcophagus that protects a mummy. Notes
The spore also has layers of protective membranes, sort of like the wrappings
around a mummy. Within these membranes and the hard coating, the dormant
bacterium is able to survive for weeks, even years, through drought, heat and
even radiation. When conditions become more favorable again – when there’s
more water or more food available – the bacterium “comes to life” again,
transforming from a spore back to a cell. Some bacterial spores have possibly
been revived after they lay underground for more than 250 million years!
Ok, so how do bacteria turn themselves into spores? First, the bacterium senses
that its home or habitat is turning bad: food is becoming scarce or water is
disappearing or the temperature is rising to uncomfortable levels. So it makes
a copy of its chromosome, the string of DNA that carries all its genes.
Fig. 35.2: ASM Digital image collection merkel
Then, the rubbery cell membrane that surrounds the bacterial cell fluid begins
pinching inward around this chromosome copy, until there’s a little cell within
the larger bacterial cell. This little cell is called the “daughter cell” and the bigger,
original one, what starts out as the “vegetative cell” in this illustration, is now
called the “mother cell.” Next, the membrane of the mother cell surrounds and
swallows up the smaller cell, so that now two membrane layers surround the
daughter cell. Between these two membranes a thick wall forms made out of
stuff called peptidoglycan, the same stuff found in bacteria’s rigid cell walls.
Finally, a tough outer coating made up of a bunch of proteins forms around all
this, closing off the entire daughter cell, which is now a spore. As the mother
cell withers away or gets blasted by all kinds of environmental damage, the spore
lies dormant, enduring it all, just waiting for things to get better. Not all bacteria
MICROBIOLOGY 321
Microbiology can form spores. But several types that live in the soil can. Bacteria in the
Bacillus and Clostridium groups are spore-formers. Their spores are called
endospores.
Another group of bacteria called Methylosinus produces spores called exospores.
The difference between endospores and exospores is mainly in how they form.
Endospores form inside the original bacterial cell, as described above. Exospores
Notes form outside by growing or budding out from one end of the cell. Exospores also
don’t have all the same building blocks as endospores, but they’re similarly
durable.
Fig. 35.3: Sporulation Cycle

1. Spore forming bacteria produce unique cell called ....................
2. Bacillus groups produce .................... spores
3. Methylosinus produces .................... spores
4. Endospores forms .................... the bacterial cell
5. Exospores forms .................... the bacterial cell.
322 MICROBIOLOGY
Microbiology
35.3 DEMONSTRATION OF SPORES
Endospores can be examined with both light and electron microscopes. Because
spores are impermeable to most stains, they often are seen as colourless areas
in bacteria treated with Methylene blue and other simple stains; special spore
stains are used to make them clearly visible. Spore position in the mother cell
or sporangium frequently differs among species, making it of considerable value
in identification. Spores may be centrally located, close to one end (subterminal), Notes
or definitely terminal. Sometimes a spore is so large that it swells the
sporangium.
Electron micrographs show that endospore structure is complex. The spore often
is surrounded by a thin, delicate covering called the exosporium .A spore coat
lies beneath the exosporium, is composed of several protein layers, and may be
fairly thick. It is impermeable and responsible for the spore’s resistance to
chemicals. The cortex, which may occupy as much as half the spore volume,
rests beneath the spore coat. It is made of a peptidoglycan that is less cross-linked
than that in vegetative cells. The spore cell wall (or core wall) is inside the cortex
and surrounds the protoplast or core. The core has the normal cell structures such
as ribosomes and a nucleoid, but is metabolically inactive.
Bacterial Endospore: Endospores or spores are highly resistant dormant
structure produced by number of Gram negative bacteria. Eg. Sporasacrium spp,
Bacillus spp, Clostridium spp. Bacteria normally grow, matured, & reproduce
by somatic cells. When there is nutrient depletion or environmental stress (heat,
UV radiation, chemical disinfection desiccation), spore former bacteria begin
spore formation. After return of suitable environmental spores produce vegetative
cell. Some endospores remain viable for 100 yrs. Since spores often survive
boiling for an hour or more therefore, autoclave must be used to sterilize any
bacteria.
Bacillus: Bacillus is a specific genus of rod-shaped bacteria that are capable of
forming spores. They are sporulating, aerobic and ubiquitous in nature. Bacillus
is a fairly large group with many members, including Bacillus cereus, Bacillus
clausii and Bacillus halo denitrificans. Bacillus spores, also called endospores,
are resistant to harsh chemical and physical conditions. This makes the bacteria
able to withstand disinfectants, radiation, desiccation and heat. Bacillus are a
common cause of food and medical contamination and are often difficult to
eliminate.
Sporolactobacillus: Sporolactobacillus is a group of anaerobic, rod-shaped,
spore forming bacteria that include Sporolactobacillus dextrus, Sporolactobacillus
inulinus, Sporolactobacillus laevis, Sporolactobacillus terrae and Sporolacto-
bacillus vineae. Sporolactobacillus are also known as lactic-acid bacteria for
MICROBIOLOGY 323
Microbiology
they are capable of producing the acid from fructose, sucrose, raffinose,
mannose, inulin and sorbitol. Sporolactobacillus are found in the soil and often
in chicken feed. According to “Fundamentals of Food Microbiology,” the spores
formed by Sporolactobacillus are less resistant to heat than those formed by the
Bacillus genus.
Notes
Fig. 35.4: Bacillus anthracis
Sporosarcina: Sporosarcina are a group of round-shaped (cocci) aerobic

bacteria that include Sporosarcina aquimarina, Sporosarcina globispora,
Sporosarcina halophila, Sporosarcina koreensis, Sporosarcina luteola and
Sporosarcina ureae. According to “Antibiotic Resistance and Production in
Sporosarcina ureae,” Sporosarcina is thought to play a role in the decomposition
of urea in the soil.
Clostridium
Clostridium are rod-shaped, Gram-positive (bacteria that retain a violet or dark
blue Gram staining due to excessive amounts of peptidoglycan in their cell
walls) bacteria that are capable of producing spores. According to the Health
Protecton Agency, the Clostridium genus consists of more than a hundred known
species, including harmful pathogens such as Clostridium botulinum, Clostridium
difficile, Clostridium perfringens, Clostridium tetani and Clostridium sordellii.
Some species of the bacteria are used commercially to produce ethanol
(Clostridium thermocellum), acetone (Clostridium acetobutylicum), and to
convert fatty acids to yeasts and propanediol (Clostridium diolis).
We would study in detail about the Clostridium species. Clostridium species
forms the important
Microbiology
Clostridia are Gram-positive, spore-forming and strictly anaerobic rod shaped
bacteria. The genus consists of a number of medically important pathogens,
324 MICROBIOLOGY
including Clostridium perfringens, C. tetani, C. botulinum and C. difficile. They Microbiology
can be differentiated on the basis of their biochemical activities, including
saccharolysis and proteolysis.
Pathogenesis
The key virulence factors in pathogenic clostridia are the powerful toxins
released from vegetating (growing) bacteria.
Notes
Epidemiology
Clostridia live commensally in the human and animal gut or as saprophytes in
the soil.
Clinical infections
C. perfringens causes gas gangrene and food-poisoning. C. tetani causes tetanus,
C. botulinum causes botulism and C. difficile causes antibiotic-associated
diarrhea.
Gas gangrene is diagnosed by Gram stain and culture of body specimens. Tetanus
and botulism are often diagnosed clinically. Antibiotic-associated diarrhea is
proven by isolation of the organism and demonstration of the presence of toxins
in stool samples.
Treatment
Gas gangrene is treated with antibiotics and surgical debridement of gangrenous
wounds. Tetanus and botulism require antibiotics, antitoxins and additional life-
supporting measures. Antibiotic-associated diarrhea is treated by the withdrawal
of antibiotics from patients and the use of metronidazole if necessary. C.
perfringens food-poisoning is self limiting.
Prevention and control

Good care of wounds can prevent gas gangrene. Vaccination against tetanus is
highly successful. The risk of botulism can be minimized by avoiding expired
or inadequately sterilized canned meat. Antibiotic-associated diarrhea can be
prevented by avoiding unnecessary use of antibiotics and isolation of patients
who have acquired the disease.
Microbiology
The clostridia are a group of strictly anaerobic large (4–6μm~1μm) Gram-
positive bacilli. They can be pleomorphic. Some tolerate small amounts of
oxygen. They form spores which can be centrally or terminally positioned
MICROBIOLOGY 325
Microbiology causing bulging within the cell. They are soil saprophytes or normal commensals
of the human and animal gut. However, they are capable of causing deadly
diseases, which are invariably mediated by potent exotoxins.
In addition to their colonial and microscopic morphologies, Clostridium species
can be differentiated on the basis of their biochemical activities. Many species
break down sugar (saccharolysis) and/or protein (proteolysis) molecules. These
activities can be detected and used for species differentiation. The ability to
Notes
produce aromatic fatty acid end-products (detected by gas-liquid chromatography)
can also be used for species differentiation. Toxigenicity of isolates is often
demonstrated in the laboratory using various appropriate techniques, e. g.
observing cytopathic effect on human cells, or neutralization of enzymatic effect
on substrates using antibodies.
The clostridia, like all other medically important anaerobic organisms, are
sensitive to metronidazole and clindamycin. They are also sensitive to many
other antibiotics, including penicillin and erythromycin, but resistant to
aminoglycosides, which are therefore added to culture media to enhance the
selective isolation of these organisms.
Clostridium perfringens
C. perfringens rarely forms spores under normal laboratory conditions. On blood
agar, it produces a characteristic double zone of b-hemolysis, a narrow
transparent zone and a wide shadowy zone. It is mainly saccharolytic and
produces acid and gas from milk, producing a ‘stormy clot’ in a test tube (litmus
milk test).
C. perfringens produces a number of potent toxins, the most important of
which is the a-toxin (phospholipase C) which causes host cell lysis. This toxin
is produced by all isolates of C. perfringens.
Fig. 35.5: Clostridium perfringens
Different strains of the organism produce any of another 11 well-known toxins,

including collagenase, proteinase, hyaluronidase and deoxyribunuclease. Based
on these toxins, the C. perfringens strains are divided into five types, A-E. C.
326 MICROBIOLOGY
perfringens uses these toxins to kill human muscle cells, causing necrosis Microbiology
(myonecrosis). Death of these cells creates an even more suitable anaerobic
condition where the organism can grow rapidly and release gas, hence the
disease is known as ‘gas gangrene’. Infected and discolored blood inside the
wound and under the skin turns the infected area to black. Gas gangrene can be
caused by other, less common clostridia, including C. novyi, C. septicum, C.
histolyticum and C. sordelli. Gas gangrene is treated by surgical debridement of
infected areas and the use of high doses of penicillin given intravenously. Notes
Some strains of C. perfringens also produce enterotoxins which, when ingested
in large numbers, can cause food-poisoning (diarrhea, vomiting and abdominal
pain). This is self-limiting and does not require treatment.
Clostridium tetani
C. tetani is a straight, slender, anaerobic Gram-positive bacillus with a rounded
terminal spore which looks like a ‘drumstick’ under the microscope. It is Gram
positive, but readily loses the Gram stain. The organism is motile, lives in the
human and animal gut and contaminates soil, mainly from cattle feces. C. tetani
produces an extremely potent single toxin (tetanus toxin) which underlies the
pathogenesis of tetanus. Tetanus toxin consists of two components, including
the neurotoxic ‘tetanospasmin’ and the hemolytic tetanolysin. The toxin
prevents muscle relaxation, leading to persistent contraction of facial and body
muscles. Simultaneous contraction of opposite groups of muscles leads to a
characteristic grin on the face (risus sardonicus) and spastic posture of the limbs
and body trunk.
The organism is rarely isolated from the site of bacterial entry, which may not
even be visible. Therefore, diagnosis is made mostly on clinical grounds, the
features of which are characteristic. Tetanus is fatal in the absence of rapid and
high quality management. The organism is sensitive to penicillin which is
administered along with a specific anti-tetanus antibody (antitoxin). The
infected wound should be debrided and the patient given a booster dose of
tetanus vaccine.
Fig. 35.6: Clostridium tetani terminal spores
MICROBIOLOGY 327
Microbiology
Tetanus is now extremely rare in industrialized countries, due to the availability
of a safe and highly effective vaccine offered to all children as part of a childhood
immunization programme. Booster doses are given to toddlers and young adults.
Clostridium botulinum
C. botulinum strains are motile, anaerobic, Gram-positive bacilli which form
Notes subterminal spores. They are ubiquitous and contaminate water and meat-
containing canned food, including canned fish, liver pâté and sausages. C.
botulinum causes botulism which is a severe, usually fatal, form of food-
poisoning.
Fig. 35.7: C. botulinum sub-terminal spores
The bacterium produces the most powerful toxin known to man, which is the
key virulence factor responsible for the pathogenesis of disease. Its action is the
reverse of that of tetanus toxin, i.e. it prevents muscle contraction, leading to
flaccid paralysis of important muscles. It inhibits the release of acetylcholine at
motor nerve endings in the parasympathetic nervous system. This potent
toxin is relatively heat-resistant (although destroyed by temperatures > 60°C),
hence it is considered by the military as a suitable bioweapon. There are seven
serotypes of botulinum toxin, named A-G. These act in almost identical ways;
however, only types A, B and E cause human botulism.
C. botulinum can be isolated from left-over (suspect) food items. Isolates can
be tested in the laboratory for toxin production. Traces of toxin can be found
in food items or patient’s serum. Laboratory animals are occasionally used to
confirm the diagnoses.
Treatment is by removal of undigested food, injecting antitoxins and intensive

therapy. Patients will require assistance with breathing and eating due to
muscular paralysis. Botulinum toxin is sometimes used as a medical treatment
for spastic paralysis of facial or bladder muscles.
328 MICROBIOLOGY
Clostridium difficile Microbiology
C. difficile is found in the feces of 3–5% of humans, in the gut of several animals
and in the environment. The organism produces at least two potent toxins that
are responsible for severe and occasionally fatal diarrhea. The organism is
harmless in the normal gut where it cannot compete successfully against the
resident gut flora. This competitive environment provides a useful barrier
(colonization resistance) against C. difficile and many other pathogens. Notes
Administration of broadspectrum antibiotics in vulnerable patients, e.g. elderly
inpatients, removes the competitive barrier in the gut, allowing C. difficile to
grow and produce toxins. The latter then cause ‘antibiotic-associated’ colitis,
ranging in severity from mild diarrhea to overwhelming pseudomembranous
colitis.
Treatment is by withholding antibiotics where possible, replacing body fluids,
administration of oral metronidazole or vancomycin and isolating the patient to
prevent further spread of disease. Widespread dispersal of C. difficile spores in
hospital environment can lead to nosocomial infection.

1. Spores are seen as .................. area in simple strain
2. Lactic acid bacteria is also known as ..................
3. C. perfringens causes .................. & ..................
4. C. tetani causes ..................

z Spore forming bacteria produce a unique resting cell called cndospore,
which allows them to survive in hostile environmental conditions
z Two medically important genera are bacillus & clostridium
z Bacillus & clostridium groups produce endospores
z Endospores forms inside of the bacterial cell
z Exospores forms outside the bacterial cell
z Endospores are seen as colourless area in simple strain
z Spore is surrounded by this delicate covering caused exosporium
MICROBIOLOGY 329
Microbiology z Bacillus spores also called endosporins are resistant to harsh chemical and
physical conditions
z Sporolactobacillus are also known as Lactic acid bacteria
z Clostridia live commensally in the human gut
z C. Perfringens cause gas gangrene & food poisoning
z C. tetani causes tetanus
Notes
TERMINAL QUESTIONS
1. What are endospores
2. Describe sporulation cycle
3. Describe the structure of spore

35.1
1. Endospore
2. Endo
3. Exo
4. Inside
5. Outside
35.2
1. Colourless
2. Sporolactobacillus
3. Gas gangrene & food Poisoning
4. Tetanus
330 MICROBIOLOGY
Non-Sporing Anaerobes MODULE
Microbiology
36
Notes
NON-SPORING ANAEROBES
36.1 INTRODUCTION
Anaerobic bacteria are widespread and very important. They do not require
oxygen for growth, which is often toxic for them. They lack the enzymes
superoxide dismutase, peroxidase and/or catalase, which makes them susceptible
to oxygen derived free radicals. These organisms obtain energy from fermentation
process. These bacteria form the commensal flora of mouth and oropharynx,
gastrointestinal tract and genitourinary tract.
OBJECTIVES
z classify the non-sporing anaerobes.
z describe their pathogenic potential.
z explain the laboratory diagnosis of the important pathogenic species.
36.2 NON-SPORING ANAEROBES

The anaerobic bacteria can be sporogenous (eg. Clostridium species) or non-
sporing (eg Bacteroides species). Non-sporing anaerobes constitute an important
cause of human infections. Even in seemingly anaerobic conditions as the mouth
and the skin, anaerobic bacteria are ten to thirty times more frequent than
aerobes.
These bacteria differ widely in the degree of anaerobiosis required for their
growth.
MICROBIOLOGY 331
MODULE Non-Sporing Anaerobes
Microbiology (a) Facultative anaerobes - Can grow in the presence or absence of oxygen.
Obtain energy by both respiration and fermentation. Oxygen not toxic,
some use nitrate (NO3–) or sulphate (SO42-) as a terminal electron acceptor
under anaerobic conditions. E.g. Peptostreptococcus.
(b) Obligate (strict) anaerobes - Oxygen is toxic to these organisms, do not
use oxygen as terminal electron acceptor. E.g Bacteriodes.
Notes (c) Microaerophilic organisms - require low levels of oxygen for growth, but
cannot tolerate the levels present in the atmosphere. E.g. Spirochetes
(d) Aerotolerant anaerobes: Metabolism is anaerobic but they are unaffected
by the presence of oxygen. E.g. Propionibacterium.
36.3 CLASSIFICATION OF NON-SPORING ANAEROBES

On the basis of morphology and staining characters, non-sporing anaerobes are
classified as under:
Gram –ve bacilli Gram +ve bacilli

Bacteroides Eubacterium
Prevotella Propionibacterium
Porphyromonas Lactobacillus
Fusobacterium Mobiluncus
Leptotrichia Bifidobacterium
Actinomyces
Cocci Spirochetes
Peptococci Treponema
Peptostreptococci Borrelia
Veillonella (gram -ve)
36.4 ANAEROBIC COCCI

The anaerobic cocci are commonly found as commensals on the human skin, in
the female genital tract, in the oropharynx, and in the gastrointestinal tract. Many
of them are aero-tolerant and grow well under 10% CO2 in an aerobic or micro-
aerophillic atmosphere. The important pathogenic species of anaerobic gram
positive cocci include: Peptostreptococcus anaerobius, Pst magnus, Pst
asacchrolyticus. Most of the peptococci have now been reclassified as
peptostreptococci. Peptococcus niger is the only surviving member of the genus
peptococcus. They may cause several clinical infections such as:
332 MICROBIOLOGY
z Puerperal sepsis Microbiology
z Other genital infections

z Wound infections
z Gangrenous appendicitis
z Urinary tract infections
z Osteomyelitis
z Brain, lung, hepatic and other abscess Notes
z Intra-abdominal infections
z Empyema and aspiration pneumonias
Anaerobic gram negative cocci include Veillonella parvula. These are obligate
anaerobes and are usually non-pathogenic but may occasionally invade blood
stream.
The anaerobic cocci are generally sensitive to metronidazole and penicillin and
a wide range of other antibiotics like tetracycline, erythromycin, clindamycin
and cephalosporins. These are however, resistant to streptomycin and gentamycin.

1. Anaerobic bacteria donot require ................ for growth
2. Anaerobic bacteria form the commensal flora of ................ & ................
tract of human
3. Facultative anaerobes obtain energy by ................ & ................
4. Organisms that are unaffected by the presence of oxygen are caused by
................
36.5 ANAEROBIC GRAM POSITIVE BACILLI

The medically important genera of this group are Eubacterium,
Propionibacterium, Lactobacillus, Mobiluncus and Bifidobacterium.
Eubacterium: These are strictly anaerobic and form the normal flora of mouth
and intestine.
Propionibacterium: Pleomorphic, gram positive, non-motile rods. They are
aerotolerant.
P acnes is constantly present on skin and is a common contaminant of blood
and CSF cultures. These are not normally regarded as pathogens but are found
in acne, in some cases of infective endocarditis and in infections associated with
implanted prosthesis.
MICROBIOLOGY 333
Microbiology Lactobacillus: Straight or curved gram positive rods. They are present in the
mouth, intestines and adult vagina (Doderlein’s bacilli). They are generally non-
pathogenic, but some species have been incriminated in the pathogenesis of
dental caries and bronchopulmonary infections.
Mobiluncus: These are motile, curved, anaerobic bacilli that appear as gram
variable rods. Mobiluncus mulieris and M curtisii leads to bacterial vaginosis
Notes along with other pathogens like Gardnerella vaginalis, Mycoplasma hominis
and Bacteroides species. Bacterial vaginosis is a polymicrobial infection
characterized by a thin foul smelling vaginal discharge. Its smell is accentuated
when mixed with a drop of KOH solution. The vaginal pH is more than 4.5. Clue
cells are seen in films.
Bifidobacterium: These are pleomorphic rods that show true and false
branching. The name is derived from the frequent bifid ‘Y’ shaped cells. Most
species are obligate anaerobes and are present in large numbers in the intestines
and in the mouth.
36.6 ANAEROBIC GRAM NEGATIVE BACILLI

The anaerobic, gram negative, nonsporing and non-motile bacilli, ranging from
short gram-negative rods to filamentous and fusiform shapes, belong to the
family Bacteroidaceae. This family includes three genera: (1) Bacteroides, (2)
Fusobacterium (3) Leptotricha. (4) Porphyromonas (5) Prevotella
(1) Bacteroides: These are the most common anaerobes isolated from clinical
specimens. They grow well in an anaerobic atmosphere containing 10%
CO2. They possess capsular polysaccharides which appear to be virulence
factors.
Table 36.1
Degree of Genus Species Pigment Pathogenesis
saccharolysis
Saccharolytic Bacteroides B fragilis nil Meningitis, brain abscess,

group Pleural, peritoneal
infectionsWound,
urogenital infections
Moderately Prevotella P melanino- Brick red Lung, liver abscess

saccharolytic genica (under UV Mastoiditis, intestinal
group light) lesionsLesions of mouth
and gums
Asaccharolytic Porphyro- P gingivalis Periodontal disease

group monas P endodontalis black Root canal infections
334 MICROBIOLOGY
2. Fusobacterium: These are gram negative, strict anaerobic, long, thin or Microbiology
spindle shaped bacilli with pointed ends. They are commensals in the mouth
and also cause infections of this and related sites. F nucleatum and
F.necroforum are the most commonly isolated species of this group. It may
cause infections of head and neck regions including dental and periodontal
infections and cerebral abscess.
3. Leptotricha: This genus contains only one species, L buccalis. These are
gram negative rods with tapering ends. They are regarded as essentially Notes
commnesal in human mouth. These tend to occur in mixed infections such
as putrefactive nectrotic fusospirochaetal conditions (vincent’s angina).
Most of the anaerobic gram negative bacilli are susceptible to metronidazole,
which is the drug of choice.
36.7 LABORATORY DIAGNOSIS OF ANAEROBIC

INFECTIONS
(a) Specimen collection and transport: Minimise the contact with air.
Aspirate the specimen in an airtight syringe, plunge the needle into a rubber
cork to seal it. Pus and other fluids may be collected in small bottles with air
tight caps. Swabs should not be used when anerobic infection is suspected.
Pre-reduced anaerobically sterilized (PRAS) transport media or gassed out
vials or hungate’s tubes, Robertson’s cooked meat medium (RCM) or
Thioglycollate broth (TGB) can be used for transportation of specimens for
anaerobic cultures.
(b) Direct examination of the specimen:

z In the laboratory exposure should be limited to minimum.
z Presence of foul smell is indicative of anaerobic infections.
z Examination of the specimen under ultraviolet light may show the brick
red fluorescence of P melaninogenica.
z On gram’s staining presence of abundant pus cells along with a mixed
bacterial flora is suggestive of anaerobic infections, since most of the
times anaerobic infections are polymicrobial.
z Gas liquid chromatography of the specimen may yield the presumptive
information on the types of anaerobes present.
(c) Culture: Media that can be used include blood agar,brain heart infusion
agar, phenyl ethyl alcohol agar (PEA), kanamycin / vancomycin BA,
bacteroides bile aesculin agar, thioglycollate broth, robertson’s cooked meat
MICROBIOLOGY 335
Microbiology broth (RCM). Culture can be done in anaerobic jars, Gaspak system or in
anaerobic chambers. All cultures should be incubated for minimum 72 hours
as most of the anaerobes are slow growing.

Notes
1. P.acnes is commensal of ................
2. P.acnes are common contanminant of ................ & ................ culturally
3. ................, ................ & ................ tubes can be used for transportation of
specimen
4. Presence of ................ in the specimens is indicative of anaerobic infections

z Many anaerobic bacteria are pathogenic for human beings, and they
outnumber aerobes in many habitats. They vary widely in the degree of
anaerobiosis required for growth. They are classified on the basis of
morphology and gram staining. Bacteroides species are the most commonly
isolated pathogens amongst all the non-sporing anaerobes. Diagnosis of
anaerobic infections is difficult. Most of the times such infections are
associated with a putrid smell. Presence of pus cells along with a
polymicrobial flora on microscopy is indicative of anaerobic infections.
Culture is difficult. Exposure to oxygen should be prevented and longer
incubation periods are required. Almost all the anaerobes are susceptible
to metronidazole.
TERMINAL QUESTIONS
1. Classify anaerobic bacteria on the basis of the degree of anaerobiosis
required for growth.
2. Classify non-sporing anaerobes.
3. Write short note on anaerobic gram positive cocci.
4. Discuss anaerobic gram negative rods.
5. Write briefly on the Bacteroides species.
336 MICROBIOLOGY
Microbiology
36.1
1. Oxygen
2. Gastro-intestinal & Genito urinary Notes
3. Respiration & fermentation
4. Aerotolerant anaerobes
36.2
1. Skin
2. Blood & CSF
3. Pre-reduced transportation media, gassed out violes & hungates
4. Foul smell
MICROBIOLOGY 337
MODULE Medical parasitology
Microbiology
37
Notes
MEDICAL PARASITOLOGY
37.1 INTRODUCTION
The study of protozoan and helminthic parasites of medical importance is
included in medical parasitology. The first microscope was developed by Antony
Van Leunhoek in 1683 and observed life forms in his primitive microscope
which we now know were protozoan parasite. He termed them as animalicules.
Goldfuss in 1817 coined the term protozoa. Parasites and ova were first isolated
in 1782.
OBJECTIVES
z define terms used in parasitology
z classify parasites & helminths
37.2 DEFINITIONS AND TERMS

To understand parasitology it will be good to familiarize ourselves with the
definitions and the terms used in parasitology
Definition of some medical terms used in parasitology

(a) Parasitism: Living organisms who receive their nourishment and shelter
from another organism (host) in whom the organism lives.
(b) Symbiosis: A relationship in which both parasite and the host benefit from
their association with each other.
(c) Commensalism: A relationship where the parasite derives the benefit from
the host without harming the host. Sometimes may benefit the host.
(d) Host: Those who harbour the parasite are called hosts
338 MICROBIOLOGY
Medical parasitology MODULE
(e) Definitive host: The host in which the adult stage of the parasite lives or Microbiology
where the parasite utilizes the sexual method of reproduction.
(f) Intermediate host: The host in which the larval stage of parasite lives or
the asexual multiplication takes place is called the intermediate host
(g) Paratenic hosts: A host in which a parasite merely remains without further
development is referred to as a paratenic host. They may transmit the
infection to another host and hence are also called transport host. E.g. a Notes
fly may transfer infected amoebic cysts to food for human consumption..
(h) Reservoir host: It is either an animal or man in which the parasite usually
reside or one in which a parasite that infects man is able to be maintained
in the absence of a human host.
(j) Direct (Simple): When a parasite requires only one species of host to
complete its develpoment, it is referred to as direct life cycle. e.g.
E. histolytica
(k) Indirect life cycle (Complex): When a parasite requires two or more species
of hosts to complete its development, then the life cycle is referred to as
indirect life cycle. E.g. Filariasis, Plasmodium
37.3 CLASSIFICATION OF PARASITES

Parasites are divided into two phyla:
A. Protozoa : These are unicellular parasites
B. Metazoa : These are multi cellular parasites
A. Phylum protozoa: These parasites are unicellular eukaryotic organisms and
the single cell carries out all the functions of the parasite like reproduction,
digestion, respiration and excretion.
They usually measure from 1-150 µm.
Examples of protozoa are: Entamoeba histolytica, Giardia lamblia, Plasmodia,
Leishmania and Trypanosoma
Classification
The protozoa are classified as given in the table
Phylum Subphylum Class Order Genus
Metamonada Diplomondea Diplomonadida Giardia

Enteromonadida Enteromoas
Retortamonadida Chilomastis
Retortomonas
MICROBIOLOGY 339
Microbiology
Axostylata Parabasalea Trichomonadia Trichomonas
Pentatrichomonas
Dientamoeba
Euglenozoa Kinetoplasta Trypanosomatidea Trypanosomatida Trypanosoma

Leishmania
Amoebozoa Lobosea Amoebida Entamoeba

Iodamoeba
Endolimax
Notes
Acanthamoeba
Hartmanella
Balamuthia
Heterolobosa Schizopyrenidea Schizopyrenida Naegleria
Alveolata Apicocomplexa Coccidea Emerida Toxoplasma

Isospora
Cyclospora
Sarcocystis
Cryptosporidium
Heamatozoa Haemosporida Plasmodium

Piroplasmea Piroplamida Babesia
Ciliophora Litostomatea Vestibuliferida Balantidium
Microspora Microsporea Microsporida Brachiola

Encephalitozoon
Enterocytozoon
Microsporidium
Incerta Pleistophorida Pleistophora

Trachipleistophora
Blastocyctis
According to the site of infection the protozoan paraites are classified as

under:
Blood & Tissue Flagellates : Leishmania, Trypanosoma
Intestinal Flagellates : Giardis lamblia,Trichomonas hominis
Enteromonas hominis,Retromonas intestinalis
Oral flagellates: Trichomonas tenax
Genital flagellates: Trichomonas vaginalis
B. Phylum Metazoa: These parasites are multicellular. They consist of
helminths. Examples of metazoan are Cestodes, Nematodes, and Trematodes.
Helminths are further classified into platyhelminths and nematodes. The
platyhelminths are further classified into cestodes and trematodes
340 MICROBIOLOGY
Helminths: The common morphological features of helminths are as given Microbiology
under
z No organs of locomotion
z Have tough cuticle
z Gastro intestinal tract (GIT) absent or rudimentary or developed
z Nervous system primitive
Notes
z Very well developed reproductive system
z Hermaphrodites or separate sexes
z Enormous number of eggs produced
z Do not multiply in humans (generally)
Two Phyla
Platyhelminths Nemathelminths
z Body flat dorsoventrally Body cylindrical
z Body cavity absent Present
z GIT absent or incomplete Complete
z Suckers present Absent
z Hermaphrodites Ex: Ascaris,
z Sexes separate
Platyhelminths
Cestodes Trematodes
z Tapelike, segmented Leaf like, unsegmented
z Hermaphrodite Hermaphrodite (except
Schistosoma)
z Hooks or suckers Suckers
z GIT absent Incomplete
z No body cavity No body cavity
z Ex: Tape worms Ex: Flukes
o Taenia Fasciola
MICROBIOLOGY 341
Microbiology

1. The study of protozoan and helminthic parasites of medical importance is
included in medical ..................
2. Living organism who receive their nourishment from another organism are
..................
Notes
3. .................. is where both organisms benefit from their association
4. .................. is where the parasite only derives the benefit from the host
without harming the host.
5. Phylum protozoa are .................. parasites
6. Phylum metazoan are .................. parasites
7. Helminths are further classified into .................. & ..................

z The study of protozoan and helminthic parasities of medical importance is
included in medical parasitology
z Parasites are divided into two phyla, Protozoa are unicellular parasites and
Metazoa are multi cellular parasites
z Phylum protozoa are parasites are unicellular eukaryotic organisms like
Entamoeba histolytica, Giardia lamblia, Plasmodia, Leishmania and
Trypanosoma
z According to the site of infection the protozoan parasites are classified as
Blood & Tissue Flagellates, Intestinal Flagellates, Oral flagellates, Genital
flagellates
z Phylum Metazoa are multicellular parasites and they consist of helminths.
Examples of metazoan are Cestodes, Nematodes, and Trematodes.
z Helminths are further classified into platyhelminths and nematodes.
z The platyhelminths are further classified into cestodes and trematodes
TERMINAL QUESTIONS
1. Define the following
(a) Parasitism
(b) Symbiosis
342 MICROBIOLOGY
(c) Commensalism Microbiology
(d) Paratenic hosts

(e) Reservoir host
2. Classify protozoan according to its site of infection
Notes
ANSWERS TO INTEXT QUESTIONS 37.1
1. Parasitology
2. Parasites
3. Symbiosis
4. Commensalism
5. Unicellular
6. Multicellular
7. Platyhelminths & nematodes
MICROBIOLOGY 343
MODULE Entamoeba Histolytica and Other Rhizophodia
Microbiology
38
Notes
ENTAMOEBA HISTOLYTICA AND
OTHER RHIZOPHODIA
38.1 INTRODUCTION
Amoebae can be pathogenic called Entamoeba histolytica and non pathogenic
called Entamoeba coli (large intestines), Entamoeba gingivalis (oral cavity).
These parasites are motile with pseudopodia. The pseudopodia are cytoplasmic
processes which are thrown out.
OBJECTIVES
z describe morphology, its life cycle, pathogenecity of Entameba Histolytica,
other amoebae and free living amoeba
z differentiate between amoebic and Bacillary dysentery
z differentiate between Entamoeba Histolytica and Entamoeba Coli
z demonstrate Laboratory diagnosis of Entameba
38.2 ENTAMOEBA HISTOLYTICA

It belongs to the class Rhizopoda and family Entamoebidae. It is the causative
agent of amoebiasis. Amoebiasis can be intestinal and extra intestinal like
amoebic hepatitis, amoebic liver abscess.
38.3 MORPHOLOGY
The Entamoeba is seen in three stages
344 MICROBIOLOGY
Entamoeba Histolytica and Other Rhizophodia MODULE
(a) Trophozoite: The trophozoite is 18-40 µm in size. The trophozoite is Microbiology
actively motile. The cytoplasm is demarcated into endoplasm and ectoplasm.
Ingested food particles and red blood cells are seen in the cytoplasm No
bacteria are seen in the cytoplasm. The nucleus is 6-15 µm and has a central
rounded karyosome. Nuclear membrane has chromatin granules and spoke
like radial arrangement of chromatin fibrils.
Notes
Fig. 38.1
(b) Precyst: Smaller in size. 10-20 µm in diameter. It is round to oval in shape

with blunt pseudopodium. The nuclei is similar to the trophozoite.
(c) Cyst: These are round 10-15 µm in diameter. It is surrounded by a refractile
membrane called as the cyst wall. The cyst wall makes it resistant to gastric
juices. The nuclei are similar to the trophozoite. Mature cyst has four
nuclei. The nuclei initially divides into two and then to four by binary
fission. The uninucleate and binucleate stage also has a glycogen mass.
Cysts are seen only in the lumen of the colon and in the stools.
38.4 LIFE CYCLE OF ENTAMOEBA HISTOLYTICA

The life cycle is spent in only one host i.e. man. The mature quadrinucleate cysts
are the infective forms. The cysts are ingested in food and water and reach the
ceacum or the lower part of ileum where the excystation of the cyst occurs. The
mature cyst liberates a single amoeba with four nuclei (Tetranucleate amoeba).
The nuclei further divide to produce the eight metacystic trophozoites. These
trophozoites lodge in the submucosa of large intestine. In the large intestines they
grow and multiply by binary fission.
38.5 STRAIN DIFFERENTIATION

Strain differentiation is done phenotypically on the basis of isoenzyme patterns
called zymodemes. The isoenzymes used for strain diffentiaton are glucose
MICROBIOLOGY 345
Microbiology phosphoisomerase (GPI), phosphoglucomutase (PGM). Here are twenty two

zymodemes identified
Notes
Fig. 38.2
Table 38.1 Differentiating features between amoebic and

bacillary dysentery
Differentiating feature Amoebic dysentry Bacillary dysentry
1 Causative agent Entamoeba histolytica Shigella species

2 Number of stools 6-8 10-14
3 Volume of stools Large Small
4 Odour Foul smelling No odour (as feacal matter
is absent)
5 Blood Altered brown coloured Bright red blood is present
blood present in stools
6 Tenesmus (Pain and Absent Present
discomfort on passage
of stools)
7 pH of stools Acidic Basic
8 Microscopy Trophozoites of E No trophozoites seen
histolytica seen
9 Culture Commensals grown Shigella grown
10 Pus cells Few Many
11 Eosinophils Present Absent
12 Charcot- Layden Present Absent
crystals
346 MICROBIOLOGY
Microbiology
38.6 PATHOGENECITY
After an incubation period ranging from 4-5 days to 3-4 weeks the disease starts
presenting as loose diarrhea which may have blood and stool mixed in it. E
histolytica secretes a proteolytic enzyme which causes destruction and necrosis
of intestinal mucosal tissue leading to formation of flask shaped ulcers. A large
number of trophozoites and cysts mixed with blood and mucus are excreted in
the feaces presenting as dysentery which is referred to as amoebic dysentry. Notes
Some trophozoites gain entry into the portal vein and reach the liver. In the liver
it can cause. The pus in the liver abscess is reddish brown in colour and is like
anchovy sauce.
(a) Amoebic hepatitis
(b) Amoebic liver abscess.
The trophozoites transform to precyst and cysts and cysts are excreted in feaces.
The mature cyst is the infective form of E histolytica.

Specimen
In case of amobic dysentery : loose stools mixed with blood and mucus
In case of amoebic liver abscess: Anchovy sauce like pus aspirated with
ultrasound guided fine needle aspiration from the liver abscess.
Direct microscopy :. A small amount of the stool is mixed with a drop of normal
saline and another drop is mixed with 2% iodine solution. These slides are
examined under a light microscope. Diagnosis is established by demonstrating
trophozoite or cyst forms in the stools.or pus sample The trophozoites are
recognized by the presence of red blood cells in the cytoplasm. In the case of
saline preparation motile trophozoite froms may be seen, In cold weather the
stage needs to be kept warm at 37 0C
Serology: Blood specimen is used for serological tests for the diagnosis of
amoebiasis.. Tests available are Immuno Haem Agglutination, and ELISA.
ELISA tests detect antibodies to E histolytica antigen

1. The causative agent of Amboeiasis is ...................
2. The stages in the life cycle of Entameba are .................., .................. &
..................
MICROBIOLOGY 347
Microbiology 3. The pH of stools in Ameobic dysentery is ..................

4. The extra-intestinal ameobiasis are .................. & ..................
5. The causative agent for Bacillary dysentry is .................. species
38.8 MISCELLANEOUS AMOEBAE

1. Entamoeba coli is prevalent worldwide and are non pathogenic. Its life
Notes cycle is similar to Entamoeba histolytica. It also has three stages i.e.
Trophozoites
Precystic
Cystic
Table No 38.2. Difference between E histolytica and E coli
Differentiating E histolytica E coli
features
Trophozoite
1. Size 18-40 µm 20-40 µm
2. Motility Actively motile Sluggishly motile
3. Cytoplasm Clearly demarcated into Endoplasm and ectoplasm
endoplasm and ectoplasm are not clearly demarcated
4. Cytoplasmic Red blood cells (RBCs), Bacteria & tissue debris is
inclusions leucocyte and tissue seen but no Red blood cells
debris. No bacteria is (RBCs),or leucocytes are
seen. inclusions seen.
5. Nucleus Not visible in unstained Visible in unstained
preparation preparation
6. Karyosome Central Eccentric
7. Nuclear Delicate and is lined by Thick and is lined by coarse
membrane fine chromatin chromatin
Cyst
8. Size 6-15 µm 15-20 µm
9. Nucleus 1-4, 1-8
10. Karyosome Central karyosome Eccentric karyosome
11. Chromatid bars Rounded Filamentous
2. Entamoeba gingivalis
(a) E gingivalis is a parasite of the human mouth and may be present as
commensal especially in cases of pyorrhea alveolaris (infections of
the gums).
348 MICROBIOLOGY
(b) They are 10-20 µm in size Microbiology
(c) It is actively motile

(d) Its cytoplasm is divided into a clear ectoplasm and a granular
endoplasm.
(e) The cytoplasmic inclusions consist of bacteria, leucocytes and other
materials but it never consists of red blood cells.
(f) The nucleus is spherical with central karyosome. Notes
(g) The nuclear membrane is lined with closely packed chromatin
granules
3. Entamoeba dispar:
(a) It is a non-invasive and non pathogenic amoeba.
(b) It is identical to E histolytica. The cysts are also similar.
(c) However the red cells are not seen in the cytoplasm .
4. Entamoeba hartmanii
(a) It is morphologically similar to E histolytica but is smaller in size.
(b) The trophozoites never contain red blood cells in the cytoplasm.
(c) It is non pathogenic
5. Endolimax nana
(a) They are small amoeba which are found in large intestines of man
and animals.
(b) The trophozoites are small 6-15 µm in size.
(c) The cytoplasm is demarcated into ectoplasm and endoplasm.
(d) The cytoplasmic inclusions consist of bacteria, leucocytes and other
materials but it never consists of red blood cells.
(e) The nucleus is spherical with large irregular karyosome lying
eccentrically.
(f) Several achromatic strands extend from karyosome to nuclear
membrane
(g) The cysts are oval in shape and measure 8-10 µm in diameter.
(h) The mature cysts are quadrinucleate.
(i) Chromatid bodies and glycogen are present in the cysts.
6. Iodamoeba butschlii
(a) They are present in large intestine of humans, monkeys and pigs.
(b) Its trophozoites are 6-20 µm in diameter.
(c) The ectoplasm is not well demarcated.
MICROBIOLOGY 349
Microbiology (d) The nucleus is large 2-3.5 µm. The karyosome is central, large and
circular.
(e) The cyst is ovoid or pyriform in shape. The chromatid bars are absent.

Notes State True or False(T/F)
1. E coli are actively motile
2. E. Histolytica the nucleus is visible in unstained preparation
3. The Cytoplasm is clearly demarcated into Endoplasm and Ectoplasm in E.
Histolytica
4. Karyosomes are Eccentric in E. Coli
38.9 FREE LIVING AMOEBA

Pathogenic free living amoeba are found in water bodies and soil. They can often
become the cause of Primary amoebic meningoencephalitis (PAM) and Chronic
amoebic keratitiss.
1. Naegleria fowleri:- It was named so after Fowler and Carter who described
it first from Australia in 1965. They can cause meningo encephalitis.
Trophozoites have amoeboid form and flagellate form
(a) Amoeboid
(i) They are 10-20 µm in size and appear elongated. They have
rounded pseudopodia called lobopodia.
(ii) The nucleus has a large central karyosome and no peripheral
nuclear chromatin.
(iii) They are actively motile.
(iv) The soil amoeba convert to flagellate form.
(b) Flagellate
(i) They are pear shaped with two flagella at its anterior end.
(ii) It moves rapidly forward and spins slowly.
(iii) The flagellate forms do not multiply.
(c) Cyst
(i) They are 7-10 µm in size.
(ii) They are surrounded by a thin cyst wall
(iii) They lack glycogen or chromotoidal bars
(iv) They are not seen in the cerebro spinal fluid.
350 MICROBIOLOGY
Microbiology
Notes
Fig. 38.3
2. Pathogenecity: Human beings acquire the infection during swimming or

diving into water bodies containing thee free living amoeba. The amoeba
invade the nasal mucosa and pass through the cribriform plate and reach
the olfactory nerve.. From here they invade the meninges and cause
meningitis and encephalitis. The disease if not treated in time invariably
results in fatality.
3. Laboratory Diagnosis: The laboratory diagnosis is established by examining
the cerebro spinal fluid from a suspected case of meningoencephalitis.. The
CSF is cloudy and may be purulent with prominent neutrophilic leucocytosis.
Wet preparation examination will demonstrate the trophozoites.

z Intestinal amoeba are pathogenic called Entamoeba histolytica and non
pathogenic called Entamoeba coli and Entamoeba gingivalis
z Entamoeba histolytica belongs to the class Rhizopoda and is the causative
agent of amoebiasis.
z Amoebiasis can be intestinal and extra intestinal like amoebic hepatitis,
amoebic liver disease
z Entamoeba is seen in three stages namely Trophozoite, Precyst and Cyst
stage and the life cycle is spent in only one host i.e. man.
z Entamoeba histolytica secretes a proteolytic enzyme which causes destruction
and necrosis of intestinal mucosal tissue leading to formation of flask shaped
ulcers.
z Diagnosis is established by demonstrating trophozoite or cyst forms in the
stools.
z Entamoeba coli are non pathogenic and the life cycle is similar to
Entamoeba histolytica
z Entamoeba gingivalis is a parasite of human and mouth and may be present
as commensals especially in cases of pyorrhea alveolaris
MICROBIOLOGY 351
Microbiology z Entamoeba dispar is non-invasive and on pathogenic amoeba

z Endolimax nana are small amoeba which are foud in large intestines of man
and animals
z Pathogenic free living amoeba are found in water bodies and soil and are
cause primary amoebic meningoencephalitis and chronic amoebic keratitiss.
z Human being acquire the infection during swimming or driving into water
Notes bodies containing free living amoeba
z The laboratory diagnosis of free living amoeba is established by examining
the cerebro spinal fluid from suspected case of meningoencephalitis.
TERMINAL QUESTIONS
1. Discuss the life cycle and pathogenecity of Entamoeba histolytica.
2. Discuss the laboratory diagnosis of amoebic dysenty
3. Enumerate the difference between amoebic and bacillary dysentery.
4. Name the free living amoeba and discuss their pathogenecity and laboratory
diagnosis.
38.1
1. Entameba Histolytica
2. Trophozite, Precystic stage, Cyst
3. Acidic
4. Amoebic hepatitis, Amoebic Liver abcess
38.2
1. False
2. False
3. True
4. True
352 MICROBIOLOGY
Plasmodium MODULE
Microbiology
39
Notes
PLASMODIUM
39.1 INTRODUCTION
Malaria is characterized by intermittent fever associated with chills and rigors
in the patient. There may be enlargement of the liver and spleen in the patient.
Sporozoa of the genus plasmodium which cause malaria in man are pigment
producing amoeboid parasites of vertebrates. They live in red blood cells and
hepatocytes. Transmission of parasite occurs through the bite of infected female
anopheles mosquito.
OBJECTIVES
z describe the characteristics of Malarial Parasite
z describe the morphology of Plasmodium vivax, Falciparum, Ovale
z describe the life cycle of Malarial Parasite
z discuss the Pathogenecity of Malarial Parasite
z demonstrate the Laboratory Diagnosis of Malarial Parasite
39.2 MALARIAL PARASITE

They belong to
Phylum Apicoplexa
Order Sporozoa
Genus Plasmodium
There are four species
(a) Plasmodium vivax
(b) Plasmodium falciparum
(c) Plasmodium ovale
(d) Plasmodium malariae
MICROBIOLOGY 353
MODULE Plasmodium
Microbiology
39.3 MORPHOLOGY OF PLASMODIUM FALCIPARUM
In the peripheral smear only the ring form and the gametocytes are seen The other
developmental stages of the parasite occurs in the endothelial lining of the
venules in internal organs like the brain and kidneys.
(a) The ring form: Early trophozoite- “Ring form” containing a reddish
chromatin “dot” and blue cytoplasm “ring”. A ring may contain double
Notes chromatin. The size of the ring is small and is 2-4 µm. Maurier’s dots may
be seen in the infected red blood cells. The stain deposits may be confused
with the chromatin dot of the ring form
(b) Gametocyte form: Gametocyte, is crescent or banana shaped, cytoplasm
in female is more bluish than that of male Female gametocyte has compact
nucleus while in the male the nucleus is not compact.
Fig. 39.1: Giemsa stained smear: ring form and gametocytes of

Plasmodium falciparum seen
39.4 MORPHOLOGY OF PLASMODIUM VIVAX

In the peripheral smear all the stages of the parasite are seen. These include the
trophozoite form, shizoint form and the gametocyte form. The other developmental
stages of the parasite occur in the endothelial lining of the venules in internal
organs like the brain and kidneys.
(a) The ring form: Early trophozoite- “Ring form” containing a reddish
chromatin “dot” and blue cytoplasm “ring” Note infected red cell is larger
than uninfected red cell Schuffner’s dots may be seen as fine reddish dots
on infected red cell membrane
354 MICROBIOLOGY
Plasmodium MODULE
Microbiology
Notes
Fig. 39.2: Early ring form: P vivax
Fig. 39.3: Growing trophozoite- “Amoeboid form”
(b) Growing trophozoite: “Amoeboid form” with “pseudopodia-like”

cytoplasmic extension Note infected red cell is larger than uninfected red
cell Schuffner’s dots appear as fine reddish dots on infected red cell
membrane
(c) Schizont:
(i) Immature schizont has 2-24 nuclei. Schuffner’s dots appear as fine
reddish dots on infected red cell membrane Infected red cell become
irregular in shape and pale in color and enlarged
(ii) Mature schizont, containing 12-24 merozoites. Schuffner’s dots
appear as fine reddish dots on infected red cell membrane. Infected
red cell become irregular in shape and pale in color and enlarged
MICROBIOLOGY 355
MODULE Plasmodium
Microbiology
Notes
Fig. 39.4: Immature Schizont
Fig. 39.5: Mature Schizont
(d) Gametocyte form Gametocyte, is round to oval in shape, cytoplasm in

female is more bluish than that of male Female gametocyte has compact
nucleus while in the male the nucleus is not compact.
39.5 LIFE CYCLE OF MALARIAL PARASITE

The life cycle of the plasmodium is spent in two hosts’ man and anopheles
mosquito. The mosquito is the definitive host as the sexual development and
multiplication of the parasite occurs in it. Man is the intermediate host.
Sporozoite is the infective form of malarial parasite which is passed on to man
through the bite of infected vector, the female anopheles mosquito. The various
stages of the parasite in man are:-
(a) Pre-erythrocytic schizogony
(b) Erythrocytic schizogony
(c) Gametogonyy
(d) Exoerthrocytic schizogony
356 MICROBIOLOGY
Plasmodium MODULE
(a) Pre-erythrocytic schizogony: This phase lasts for 8 days. Sporozoites are Microbiology
elongated and spindle shaped. The sporozoites enter the liver parenchymal
cells and become rounded. They undergo multiple divisions and develop
into schizontss. One schizont contains 20,000 – 30,000 merozoites. The
liver cells ruptures and releases the merozoites into blood.
(b) Erythocytic schizogony: This has a duration of 48 hours. The merozoites
penetrate the red blood cells. The merozoites enlarge in size and develop Notes
into trophozoites. The trophozoites develop into schizont which further
develops into merozoites. There may be 6-24 merozoites in red blood cells.
In the case of Plasmodium falciparum the schizonts aggregate in the
capillaries of the brain and other internal organs. So that only ring fforms
are formed in the peripheral blood.
(c) Gametogony: Some merozoites enlarge and get transformed to microgamete
and macrogamete. The malarial parasite now becomes infective as the
presence of gametocytes is a must for sexual development in the mosquito.
The mosquito gets these gametocytes after taking a blood meal from a
patient of malaria.
(d) Exo-erythrocytic schizogony: This phase resembles the pre-erythrocytic
schizogony. Some sporozoites after entering the liver cells do not undergo
multiplication but go into resting phase. The resting stage of the parasite
is known as hypnozoite. These can reactivate up to after 2 years and
become schizoints and release merozoites. This phase is responsible for
relapse of malaria

1. The life cycle of Plasmodium is spent in two hosts namely ................ and
................
2. ................ is the intermediate host in the transmission of the parasite.
3. Relapse of Malarial infection occurs in ................ phase
4. The resting stage of parasite is ................
Development in the mosquito

The female anopheles mosquito takes a blood meal from a malaria patient and
the plasmodium gametocytes (Both microgametes and macrogametes) reach the
gut of the mosquito. Only the mature gametocytes undergo further development.
Microgametes penetrate the macrogametes and fertilize it leading to the
MICROBIOLOGY 357
MODULE Plasmodium
Microbiology formation of a zygote. In the midgut of the mosquito one microgamete develops
into 4-8 filamentous structures called microgamete.
The zygote matures into an ookinete. The ookinete further develops into oocyst.
The oocyst further matures and increases in size. A large number of sporozoites
(100-1000) develop inside the oocyst.
The oocyst ruptures and releases the sporozoites in the body cavity of the
Notes mosquito. The sporozoites go to all the organs but prefer to go to the salivary
glands. The mosquito is now infective to man.
39.6 PATHOGENECITY
Malaria
The plasmodium species in man cause malaria. Plasmodium vivax causes a mild
form of the disease where the fever comes after every 72 hours and is called
benign tertian malaria. Plasmodium falciparum causes a severe form of the
disease where the fever comes after every 72 hours and is called malignant
tertian malaria.
The clinical features in malaria are characterized by high grade fever which is
associated with chills and rigors. The fever is intermittent in nature and comes
after 48-72 hours. After the period of chills and rigors comes the hot phase when
the patient starts sweating and feels very hot. The patient may develop anaemia.
The liver and spleen are also enlarged in this disease.
Complications of P falciparum malaria

Cerebral malaria: it is a serious form of the disease where the brain is severely
affected by the malarial parasite. The patient can go into coma and may die if
not treated in time.
Algid malaria is due to the involvement of the gastrointestinal system.
Black water fever is due to severe intravascular hemolysis.

Microscopy
Demonstration of parasite in blood film: The definitive diagnosis of malaria is
established when the parasite is demonstrated in the blood smear taken from a
suspected case of malaria. The blood smear should preferably be prepared during
the febrile phase of malaria, but can be taken at any time that the patient reports
358 MICROBIOLOGY
Plasmodium MODULE
for investigation. The smears are stained by Romanowsky stains. Lieshman stain Microbiology
is usually preferred . However staining with Giemsa is also acceptable. For
screening purposes a thick blood smear is prepared. This smear must be de-
hemoglobinized before staining. The disadvantage of this smear is that the
morphology of the parasite is not visualized properly. For proper morphological
identification a thin blood smear which is properly stained is required. It must
be noted that in Plasmodium falciparum infection only the ring form and
gametocytes are seen in the peripheral blood smear. While in Plasmodium vivax Notes
infection all the different stages of the trophozoite and gametocytes are seen,
Fig. 39.6: Thick and thin smearsSerology
Rapid diagnostic test (RDT)

Indirect evidence of malaria is also established by the demonstration of malarial
antigen or antibodies to malaria antigen. Tests based on immunochromatography
are available (malaria card test).
Fig. 39.7
MICROBIOLOGY 359
MODULE Plasmodium
Microbiology The antigen used for detection of P falciparum infection is HRP II.The antigen
detection test may remain positive for up to two after the infection has been
cured.
Notes
Fig. 39.8
Detection of Malaria antigen by Parasight – F test

Some tests detect the specific isoenzyme LDH (Lactate Dehydrogenase). These
test detect only the living Plasmodium parasite. This test has the advantage that
it becomes negative when specific treatment is started.
Fig. 39.9: Parasite LDH Test (Optimal- IT)
Fluorescent Microscopy
Quantitative buffy coat (QBC) examination involves the use of microcapillaries.
The blood is drawn into the microcapillary from one end and a fluorescent
reagent is drawn in from the other end. This is then centrifuged in a special
centrifuge. The centrifugation results in the formation of a buffy coat which
contains the parasitized red blood cells. The malarial parasite takes up the
fluorescent dye and the nuclei are seen as pink dots and the body of the parasite
is greenish in colour. This observed under a special fluorescent microscope.
360 MICROBIOLOGY
Plasmodium MODULE
Polymerase chain reaction: PCR technique may be employed to dignose Microbiology
malarial infections in intractable cases. This is a specializeede technique

requiring special skills and equipment and is expensive too.
Miscellaneous tests
Patient of malaria may suffer from anaemia if the disease is prolonged in nature.
Leucopenia or leucocytosis may be seen in some cases of complicated malaria. Notes

Match the following
1. Plasmodium Malaria (a) Malignant Tertian Malaria
2. Plasmodium Vivax (b) Cerebral Malaria
3. Plasmodium Falciparum (c) Benign Tertian Malaria
4. Complication of Malaria (d) Malaria

z Malaria is characterized by intermittent fever associated with chills and
rigors in the patient. Sporozoa of genus plasmodium which cause malaria
in man are pigment producing amoeboid parasites of vertebrates.
z They belong to Phylum Apicoplexa, Order Sporozoa, Genus Plasmodium
z There are four species namely Plasmodium vivax, Plasmodium falciparum,
Plasmodium ovale, Plasmodium malariae
z The life cycle of plasmodium is spent in two hosts man and anopheles
mosquito.
z The mosquito is the definitive host as the sexual development of the parasite
occurs in it. Man is the intermediate host
z Sporozoites is the infective form of malarial parasite which is passed on
to man through the bite of female anopheles mosquito
z Plasmodium species in man cause malaria. Plasmodium vivax causes a mild
form of the disease whereas Plasmodium falciparum causes a severe form
of the disease.
z Complications of malaria are cerebral malaria, Algid malaria and Black
water fever
MICROBIOLOGY 361
MODULE Plasmodium
Microbiology z The definite diagnosis of malaria is established when the parasite is

demonstrated in blood smear and the smear should preferably be prepared
during the febrile phase of malaria.
z Smears are stained by Romanowsky stains, lieshman stain is usually
preferred.
z Thick blood smear is prepared for screening purposes and thin blood smear
Notes is prepared for morphological identification.
z Indirect evidence of malaria is also established by demonstration of antigen
or antibodies to malaria antigen.
TERMINAL QUESTIONS
1. Discuss the life cycle of Plasmodium falciparum
2. Draw a labeled diagram of the ring form trophozoite and gametocyte of
P vivax
3. Discuss the pathogenecity and complications of malaria caused by P
falciparum.
4. Enumerate the various species of Plasmodium.

39.1
1. Man and Mosquito
2. Man
3. Exo-erothrocytic Schizogony
4. Hypnozoite
39.2
1. (d)
2. (c)
3. (a)
4. (b)
362 MICROBIOLOGY
Nematodes MODULE
Microbiology
40
Notes
NEMATODES
40.1 INTRODUCTION
The nematodes are parasites which are cylindrical and have a bilateral symmetry.
They cause intestinal and tissue infestation. The largest nematode is Dracunculus
medinensis and can be 1.2 meter long and the smallest nematode is Trichinella
spiralis which can be 4-5 mm long.
OBJECTIVES
z describe the characteristics of nematodes
z describe the morphology of ascariasis lumbricoides
z explain the life cycle of ascariasis lumbricoides
z discuss the pathogenecity of ascariasis lumbricoides
z explain the laboratory diagnosis of ascariasis lumbricoides
40.2 CHARACTERISTICS OF NEMATODES

1. They have an elongated, cylindrical and unsegmented body.
2. They are bilaterally symmetrical
3. The body covering is lined from out to inside by:
(a) Epicuticle
(b) Exocuticle
(c) Mesocuticle
(d) Endocuticle
MICROBIOLOGY 363
MODULE Nematodes
Microbiology (e) Basal lamina

(f) Hypodermis
4. The body covering encloses a body cavity, which is a pseudocoele. The
internal organs are suspended in the pseudocoele.
5. Musculature comprises of spindle shaped muscles which are divided into
contractile and non- contractile part.
Notes 6. The size may vary from 5 mm to 1meter
7. They have male and female sex separately
8. The reproductory organs are as under
(a) Male : Vas deferans, seminal vesical, ejaculatory duct
(b) Female: Tubular ovary, oviduct, seminal receptacle, uterus, ovijector,
vagina and vulva
9. The nervous system has sensory papillae on the cuticle.
10. Digestive system has a mouth, muscular oesophagus, intestine rectum and
anus.
The nematodes are further divided based on their habitat in the host and are
classified as
(a) Intestinal nematodes
(i) Ascaris lumbricoides also called as round worm
(ii) Ankylostoma duodenale and Necator americanus also called as hook
worm
(iii) Trichuris trichura
(iv) Entrobius vermicularis
(b) Tissue nematodes
(i) Wucheraria bancrofti
(ii) Dracunculus medinensis
(iii) Trichinella spiralis
(iv) Oncocerca volvulus
(v) Strongyloides stercoralis.
40.3 ASCARIASIS
Ascariasis is caused by Ascaris lumbricoides. It is also commonly called as round
worm. The worm is found in the small intestines of infected individuals.
Ascariasis is seen worldwide, but is more commonly seen in Asian and African
nations.
364 MICROBIOLOGY
Nematodes MODULE
Microbiology
40.4 MORPHOLOGY
The worm is oviparous and both adult form and ova are seen.
(a) Adult worm

The size of the adult worm is:
Female 200-400 mm × 3-6 mm Notes
Male 150-300 mm × 2-4 mm
Anterior end: The anterior end has the mouth which has two ventral lips and
one dorsal lip. The sensory papillae are present in the anterior end. Body cavity
contains a fluid called ascaron.
Anterior end
Mouth 2 ventral lips
1 dorsal lip
Sensory papillae
Posterior end:
Male
Curved, conical tip
Genital pore opens into cloacae
Two curved copulatory spicules
Female
Anus is sub terminal
Vulva opens at junction of ant & middle 1/3 of body (vulval waist)
(b) Ova
The ova of ascaris lumbricoides are seen in two forms
(i) Fertilized
(ii) Unfertilized
(i) Fertilized ova are 75 µm × 50 µm in size and are ovoid in shape.
They are bile stained and hence appear brown in colour in saline mount
preparation of stool specimen.
MICROBIOLOGY 365
MODULE Nematodes
Microbiology The outermost egg shell layer is a mammilated albuminous coat.

There is a thick egg shell made up of chitin.
The shell contains a single cell stage ovum
There is a clear space at the poles next to the single cell.
The fertilized ova may loose the outer mammilated albuminous coat and
is then referred to as decorticated fertilized ova.
Notes
Fig. 40.1
Fertilized Ova Unfertilized Ova
Fig. 40.2
366 MICROBIOLOGY
Nematodes MODULE
(ii) Unfertilized ova are larger and measure 90 µm × 55 µm Microbiology
It is ovoid in shape.
The egg shell is thinner.
It contains a mass of disorganized highly refractile granules of various
sizes.
Notes

1. Ascariasis is caused by ................
2. Round worm is also called as ................
3. Body cavity of hook worm contains a fluid called ................
4. Fertilized ovum without the albuminous coat is referred as ................
40.5 LIFE CYCLE

The adult worms are present in the small intestines and after sexual reproduction
the gravid female Ascaris worm lays eggs, these are passed in faeces and mature
Fig. 40.3: Ascaris lumbriocoids (round workm)
MICROBIOLOGY 367
MODULE Nematodes
Microbiology within 1-2 weeks in the soil. Ova remains viable in the soil for months to a year.
The infective ova are ingested by the hands carrying the ova from soil
contaminated with excreta, vegetables or dust etc.
The ova reach the stomach where the larva comes out from the egg shell. The
larva penetrates the stomach wall and enters the lymphatics and venules.
Through the venules and blood circulation it reaches the heart and eventually
Notes reaches the lungs.
In the lungs the larva undergoes development from first stage to second stage
and then the third stage larva. The larva increases in length and reaches a length
of 1500 µm. The larva then moves up on the airway passage from the alveoli
to bronchioles. It passes through the bronchi and trachea to reach the pharynx
and from there it goes to the esophagus. From the esophagus it travels to the
stomach and duodenum to finally reach the large intestines. In the intestines the
larva develops into the adult stage. The life cycle continues after sexual
reproduction and formation of ova. The life span of an adult worm is about one
year.
40.6 PATHOGENECITY
The Ascaris infestation can cause nutritional deficiency especially in children.
Numerous worms can get entangled and cause intestinal obstruction. The worm
can obstruct small lumen organs like the bile duct, appendix. They may cause
appendicitis, pancreatitis, and peritonitis.
Children may vomit out a bolus worms. The presence of larva in the lungs may
cause an allergic pneumonia called Loeffler’s pneumonia. Granulomatous lesion
may form at an ectopic site once the worm dies.

The demonstration of ova in the stools or detection of adult worm establishes
the laboratory diagnosis. The ova are seen both in saline stool preparation as well
as in the iodine preparation.

z Ascariasis is caused by Ascaris Lumbricoides. It is also commonly called
as round worm
z The worm is found in the small intestines of infected individuals
z The worm is oviparous and both adult from and ova are seen
368 MICROBIOLOGY
Nematodes MODULE
Microbiology
z The adult worms are present in the small intestines and after sexual
reproduction the gravid female ascaris worm passes fertilized ova in the
stools on to the soil
z The ascaris infestation can cause nutritional deficiency especially in
children.
z The demonstration of ova in the stools or detection of adult worm
established the laboratory diagnosis. The ova are seen both in saline stool Notes
preparation as well as in the iodine preparation.
TERMINAL QUESTIONS
1. Discuss the morphology and pathogenecity of Ascaris lumbricoides.
2. Discuss the life cycle of Ascaris lumbricoides.
3. Draw a labeled diagram of the various forms of ova of Ascaris lumbricoides.
40.1
1. Ascaris lumbricoides
2. Ascaris lumbricoides
3. Ascaron
4. Decorticated fertilized ova
MICROBIOLOGY 369
MODULE Entrobius vermicularis
Microbiology
41
Notes
ENTROBIUS VERMICULARIS
41.1 INTRODUCTION
Entrobius vermicularis worm has a shape like a pin and so is also called as pin
worm or thread worm. The worm was first seen by Leuckart in 1865 A.D. The
pin worm infections are seen all over the world. The worm is present in the
appendix and ceacum. The anterior end of the worm attaches to the mucosa.
OBJECTIVES
z describe the morphology of pin worm
z explain the life cycle of pin worm
z discuss the pathogenecity of pin worm
z explain the laboratory diagnosis of pin worm
41.2 MORPHOLOGY
The worm is finely striated. The anterior end lacks buccal capsule. It has three
lips with a dorso-ventral bladder like expansion of the culticle.
Anterior end Ova

Fig. 41.1
370 MICROBIOLOGY
Entrobius vermicularis MODULE
Male Microbiology
z Measure 2-4 mm x 0.1 – 0.2 mm

z The posterior end of the esophagus is dilated.
z Posterior end is curved and is truncated
z There is a sharply curved terminal spicule
z It possesses a single tubular testis Notes
z The male dies after fertilization
Female
z Measure 8-12 mm × 0.3-0.5 mm
z Looks like a pin
z The reproductive organs are paired and T shaped
z The vulva opens mid-ventrally
z The worm is oviparous
Ova
z The ova measure 50-60 µm × 30 µm
z It has a unique shape which is plano-convex
z It is non bile stained and thus appears colourless
z The ova is surrounded by a transparent shell having two layers of chitin
z It contains a tadpole like larva
z It floats on saturated salt solution
z It is coated on outside by albumin gel which assists in adherence to surfaces.
z The ovum requires six hours of exposure to oxygen containing air for it
to become infective. This is the reason the worm lays the ova in the perianal
region.
z The ova are resistant to commonly used antiseptics.
Life cycle
The gravid female lays the ova in the perianal region so that the ova are exposed
to oxygen containing air. The ova become infective in six hours time. The
albuminous outer layer helps to adhere to the perianal mucosa. The infected
person, mostly a child develops severe itching. The ova get lodged in the nail
bed and are then ingested through contact with the hands.
MICROBIOLOGY 371
MODULE Entrobius vermicularis
Microbiology The larva hatches in the intestine. The larva reaches the ceacum and appendix
where it matures into an adult worm.
Pathogenecity
There is severe pruritis. The patient may suffer from nocturnal enuresis. There
may be abdominal pain and discomfort. Symptoms include anorexia (loss of
Notes appetite) and loss of weight. Some cases may suffer from appendicitis.
Sometimes patient may suffer from some ectopic infections like salpingitis,
omentitis, cervicitis, peritonitis and recurrent urinary tract infections.
Patient also develops eosinophilia.
Adult worm in caecum
Larva hatches in intestines and Female worm migrates to

develops into an adult worm peri-anal area and lays eggs
Embryonated eggs become

Infective eggs are infective after coming in
ingested by human contact with oxygen
Fig. 41.2: Life cycle of Entrobius vermicularis
the laboratory diagnosis. The ova may be obtained by using a cello tape or NIH
swab (National Institute of Health) in the peri - anal area
Eosinophilia may be seen the infected hosts.

1. Pinworm is also known as ...................
2. The shape of the ova is ...................
3. The ovum requires ................... for it to be infective
4. The worm lays ova in the ................... region of the body
5. The ova for laboratory diagnosis may be obtained by ...................
372 MICROBIOLOGY
Entrobius vermicularis MODULE
Microbiology

z Entrobius vermicularis worm has a shape like a pin and so is called as pin
worm or thread worm
z The worm is finely striated. The anterior end lacks buccal capsule. It has
three lips with a dorso-ventral bladder like expansion of the culticle. Notes
z Pin worm causes severe pruritis and suffer from nocturnal enuresis. There
may be abdominal pain and discomfort.
z Demonstration of ova in the stools or detection of adult worm established
the laboratory diagnosis and the ova may be obtained by using a cello tape
or NIH swab in the peri –anal area.
TERMINAL QUESTIONS
1. Discuss the morphology and pathogenecity of E vermicularis.
2. Discuss the life cycle of E vermicularis.
3. Draw a labeled diagram of ova of E vermicularis.
41.1
1. Entrobius vermicularis
2. Plano-convex
3. Oxygen
4. Perianal
5. Cello tape or NIH swab
MICROBIOLOGY 373
MODULE Leishmaniasis
Microbiology
42
Notes
LEISHMANIASIS
42.1 INTRODUCTION
Sir William Leishman and Charles Donovan demonstrated the parasite in
patients from Calcutta in year 1903. The Genus was so named by Sir Ronald
Ross-Leishmania Donovani. It was first cultured by Rogers in 1904.
Geographic distribution-It is mainly seen in South and South –East Asia, China,
Sudan tropical Africa South America. In India the regions mainly affected are
– Bengal, Bihar, and Eastern Uttar Pradesh
OBJECTIVES
z describe the morphology of Leishmaniasis
z explain the life cycle of Leishmaniasis
z discuss the pathogenecity of Leishmaniasis
z explain the laboratory diagnosis of Leishmaniasis
42.2 HABITAT
Amastigote forms are seen in human infections and are seen mainly in the cells
of reticuloendothelial system located in liver, spleen, bone marrow, and
peripheral blood
Promastigote forms are seen in the gut of sand fly Phlebotomus argentipes. It
is also seen when grown in laboratory on artificial culture media.
374 MICROBIOLOGY
Leishmaniasis MODULE
Microbiology
42.3 MORPHOLOGY
(a) Amastigote: These are non motile and round to oval 2-4 µm long.
Notes
Fig. 42.1
Nucleus is round to oval. The nucleus is red and the kinetoplast is bright
red on Leishman stain
A clear unstained space is present alongside the axoneme called the
vacuole. The parasite has the blepharoplast and axoneme. Axoneme, arises
from the blepheroplast and extends to the margin of the parasite. Amastigote
form also has a parabasal body.
(b) Promastigote: It is a spindle shaped structure measuring 15-20 um by
1-2 um. It has a flagellum arising from the axoneme and coming out of
the anterior end. There is a blepheroplast and a vacuole in the anterior end.
Nucleus is round to oval and central in location. The nucleus is red and
the kinetoplast is bright red on Leishman stain
Fig. 42.2
MICROBIOLOGY 375
Microbiology
42.4 LIFE CYCLE
The parasite is transmitted amongst humans by the bite of infected Sand Fly-
Phlebotomus and Lutzomiya Transmission can also occur through infected
blood transfusion.
After infected blood meal is taken by the vector sand fly Phlebotomus argentipes,
the aflagellates develop into flagellate promastigote form in the gut of the insect
Notes in 8-20 days. The infective sand fly transmits the disease by biting man. Due
to partial/complete blockage of mouth parts Parasites is lodged at site of bite
when it ingests blood.
The promastigotes penetrate the host macrophage and are converted to
amastigote forms. They multiply and rupture the macrophages. There is
ingestion of amastigote by other macrophages.
Incubation period: Usually 3-6 months, but can extend up to 1-2 years
Fig. 42.3
376 MICROBIOLOGY
Microbiology

1. Amastigote forms are seen in ........................
2. Promastigote forms are seen in ........................
3. Unstained space present alongside axoneme called ........................ Notes
4. Promastigote is transmitted by ........................
5. Kala azar is caused by ........................
42.5 PATHOGENECITY
Leishmania donovani causes a visceral disease called Kala azar. It is a parasite
of the reticulo endothelial (RE) system and affects the organs containing the
reticuloendothelial system like the bone marrow, liver and spleen. There is
hepatosplenomegaly. The patient gets intermittant fever. There is associated
anaemia, cachexia, loss of weight. There is dry skin, brittle hair and
pigmentation of skin. There may also be diarrhoea, dysentery. Oedema is seen
due to hypoalbuminemia.
Leishmania tropica causes Oriental sore (cutaneous leishmaniasis) On the skin

which becomes dry there may appear solitary or multiple ulcerating papules.
Healing occurs with scarification. Post kala azar dermal leishmaniasis
Leishmania brazilensis- Espundia (muco cutaneous leishmaniasis)

Demonstration of L-D bodies: The aetiological diagnosis is established by
demonstrating the parasite in the patient specimen. The amastigote form of the
parasite is seen in human infections. Parasite demonstration is done on the
following specimen after staining the smears with Romanowsky stains.like
Giemsa, Lieshman stain.
z splenic aspirate
z bone marrow smears
z thick blood film
z enriched blood leucocyte fraction
MICROBIOLOGY 377
Microbiology
Notes
Fig. 42.4
Detection of specific antibodies: Antibodies can be detected in immunocompetant

individuals in 99% of cases. ELISA, CIEP, DOT ELISA based kits are available
for the same. In immunocompromised individuals like HIV patients with AIDS
the 40-60% cases may be seronegative.
Culture on monophasic/biphasic media: The promastigote form of the
parasite is seen on artificial culture media. The patient specimen can be cultured
on the following media
z N N N media (Novy,MacNeal Nicolle)
z Brain heart infusion agar medium
z Schneider’s medium
Fig. 42.5
378 MICROBIOLOGY
Detection of antigen: Antigen detection kits based on ELISA, IFA are available Microbiology
and can be used for establishing the diagnosis.
Non-specific tests
z progressive neutropenia
z relative lymphoctosis,monocytosis
z reversal of A:G ratio
Notes
z Napiers Aldehyde test
z Chopras Antimony test
Newer tests: PCR based detection can be done in laboratories having molecular
biology facility.
Isoenzyme typing is one of the typing method which may be done in research
laboratories.

1. Kala azar (a) Leishmania Donovani
2. Oriental sore (b) Leishmania brazilensis
3. Espundia (c) Leishmania Donovani

z Amastigote forms are seen in human infections and are seen mainly in the
cells of reticuloendothelial system located in liver, spleen, bone marrow,
and peripheral blood
z Promastigote forms are seen in the gut of sand fly Phlebotomus argentipes.
It is also seen when grown in laboratory on artificial culture media.
z The parasite is transmitted amongst humans by the bite of infected Sand
Fly-Phlebotomus and Lutzomiya Transmission can also occur through
infected blood transfusion.
z Incubation period is usually 3-6 months, but can extend up to 1-2 years
z Leishmania donovani causes a visceral disease called Kala azar, Leishmania
tropica causes Oriental sore and Leishmania brazilensis causes Espundia
z The aetiological diagnosis is established by demonstrating the parasite in
the patient specimen
z Parasite demonstration is done after staining the smears with Romanowsky
stains.like Giemsa, Lieshman stain
MICROBIOLOGY 379
Microbiology z Antibodies can be detected in immunocompetant individuals, ELISA, CIEP,

DOT ELISA based kits are available for the same.
z The promastigote form of the parasite is seen on artificial culture media.
The patient specimen can be cultured on the following media N N N media
(Novy,MacNeal Nicolle), Brain heart infusion agar medium, Schneider’s
medium,
z Antigen detection kits based on ELISA, IFA are available and can be used
Notes
for establishing the diagnosis.
z PCR based detection can be done in laboratories having molecular biology
facility and Isoenzyme typing is one of the typing method which may be
done in research laboratories.
TERMINAL QUESTIONS
1. Describe the morphology of the amastigote and promastigote form of
Leishmania donovani
2. Discuss the laboratory diagnosis of a case of leishmaniasis.
42.1
1. Reticuloendothelial cells
2. Sand fly
3. Vacuole
4. Sand fly
5. Leishmania Donovani
42.2
1. (c)
2. (a)
3. (b)
380 MICROBIOLOGY
Nematodes Classification MODULE
Microbiology
43
Notes
NEMATODES CLASSIFICATION
46.1 INTRODUCTION
Nematodes belong to the phylum Nematoda. Many species of nematodes are free
living in fresh or salt water, mud or soil. They are parasites of both animals and
plants. Only medically important nematodes are discussed in the following
sections.
OBJECTIVES
z explain the general characteristics of nematodes
z describe the classification of nematodes
General characteristics
Nematodes are elongated, cylindrical, bilaterally symmetrical, unsegmented
worms with tapering ends. The name ‘ nematodes’ means thread like. The body
is covered with tough cuticle.
Their size varies from less than 5 mm (Trichinella spiralis) to 1 metre
(Dracunculus medinensis). The male is generally smaller than female and its
posterior end is curved or coiled ventrally. The sexes are separate. Female
nematodes may be divided as
Oviparous (nematodes which lay eggs)
z Laying unsegmented eggs
o Ascaris lumbricoides
o Trichuris trichura
z Laying eggs with segmented ova
MICROBIOLOGY 381
MODULE Nematodes Classification
Microbiology o Ancylostoma duodenale

o Necator americanus
Viviparous (nematodes which give birth to larvae)
z Dracunculus medinensis
z Wucheria bancrofti
Notes Ovo- viviparous (nematodes laying eggs containing larvae that immediately
hatch out)
z Strongyloides stercoralis
Modes of infection with nematode parasites

Ingestion of food and water contaminated with eggs (Ascaris lumbricoides),
ingestion of cyclops (Dracunculus medinensis) or ingestion of infected pork
(Trichinella spiralis). Penetration of skin for example larvae of A. duodenale,
N. americanus and S. stercoralis. Blood sucking insects for example Wucheria
bancrofti and Brugia malayi.
Classification of nematodes on the basis of habitat of adult worms:
Intestinal
Small intestine: A. lumbricoides, A. duedenale, N.americanus, S.stercoraris, T.
spiralis.
Large intestine: E. vermicularis, T. trichuria.
Somatic
Lymphatic system: W. bancrofti, B. malayi.
Subcutaneous tissue: Loa loa, Onchocerca volvulus, D. medinensis.
Body cavity: Mansonella perstans.
Conjunctiva: Loa loa.

1. Which of the following nematodes is ovoviviparous?
(a) Ascaris lumbricoides.
(b) Dracunculus medinensis.
382 MICROBIOLOGY
Nematodes Classification MODULE
(c) Strongyloides stercoralis. Microbiology
(d) Trichinella spiralis.

2. Which of the following nematodes lays unsegmented eggs?
(a) Necator americanus.
(b) Trichuris trichiura.
(c) Strongyloides stercoralis.. Notes
(d) Trichinella spiralis
3. Which of the following nematodes lays eggs containing larvae?
(a) Trichinella spiralis.
(b) Enterobius vermicularis.
(c) Brugia malayi.
(d) Ascaris lumbricoides.
4. Adult worm of which of the following resides in human body cavity?
(a) Wucheria bancrofti.
(b) Brugia malayi.
(c) Ascaris lumbricoides.
(d) Mansonella perstans.
5. Adult worm of which of the following resides in large intestine?
(a) Ascaris lumbricoides.
(b) Dracunculus medinensis.
(c) Strongyloides stercoralis.
(d) Enterobius vermicularis.

z Nematodes are elongated, cylindrical, bilaterally symmetrical, unsegmented
worms with tapering ends. Sexes are separate into male and female. Female
nematodes may be divided as oviparous, ovi viviparous and viviparous.
Nematodes have different modes of infection like, by ingestion of food and
water contaminated by eggs of nematodes, ingestion of and infected pork,
penetration of skin and bite of blood sucking insects. Nematodes can be
classified on the basis of habitat of adult worms.
MICROBIOLOGY 383
MODULE Nematodes Classification
Microbiology
TERMINAL QUESTION
1. Classify nematodes?
Notes ANSWERS OF INTEXT QUESTIONS
43.1
1. (c)
2. (b)
3. (b)
4. (d)
5. (d)
384 MICROBIOLOGY
Hook worm and Strongyloides MODULE
Microbiology
44
Notes
HOOK WORM AND
STRONGYLOIDES
44.1 INTRODUCTION
Hook worm is the common name given to two nematode worms which possess
hook like teeth in their mouth. The two species are Anklyostoma duodenale and
Necator americanus. The worm resides in the duodenum and the small intestines.
It attaches to the mucosa of the duodenum and intestines with the help of the
hook like teeth. It also sucks blood and also consumes the nutrition of the host
and thus causes anaemia and nutritional deficiency.
OBJECTIVES
z describe the morphology & life cycle of hookworm, strongyloides
z explain the pathogenesis of adult worm & strongyloides
z discuss the laboratory diagnosis of hookworm & strongyloides
44.2 MORPHOLOGY
(a) Adult worm: The worm measures from 8 to 13mm in length, The female
worms are longer than the male worms. The detailed morphological
features of the worms are given in the table.
Fig. 44.1
MICROBIOLOGY 385
MODULE Hook worm and Strongyloides
Microbiology (b) Ova: The hook worm are oviparous and the a gravid female after sexual
reproduction lays the ova which are passed in the stools. The ova are non
bile stained and hence appear colourless on saline preparation. The ova
measure 65 µm × 40 µm. The shell encloses a four cell staged segmented
ovum also called as the blastomere.
Notes
Ova Rhabditiform Larva
Fig. 44.2

1. Common hookworm of man is ......................
2. Hookworm live commonly in ...................... part of small intestine
3. Male worm has ...................... bursa which holds female nematodes during
mating
4. ...................... is the infective form of the parasite
Differential features of two major species of Hook worm
S No A duodenale N americanus
1 Shape Head bent in same Head bent in opposite

direction as body direction
2 Size Larger F 10-13 mm Smaller

F 10-13 mm F 9-11 mm
M 8-11mm M 5-9mm
3 Oral aperture 4 hook like ventral teeth, 2 ventral & 2 dorsal

2 knob like dorsal teeth cutting plates
4 Copulatory bursa 3 lobes (1 dorsal 2 lat) 3 lobes (1 dorsal 2 lat)

13 rays, dorsal ray tripartite 14 rays, dorsal ray bipartite
386 MICROBIOLOGY
Microbiology
5 Caudal spine in Present Absent
Female
6 Vulval opening Post to middle of body Ant to middle of body

of the body
7 Rate of Faster Slower

development
8 3rd stage larva longer Smaller Notes

9 Pathogenicity More less
44.3 LIFE CYCLE

The adult worms are present in the small intestines and after sexual reproduction
the gravid female hook worm passes fertilized ova in the stools on to the soil.
Fig. 44.3
MICROBIOLOGY 387
Microbiology Development in the soil : The larva matures in the ova within 7-8 days in the
soil. The rhabditiform larva comes out of the ova and lives in the soil where
further development takes place. The larve develops from rhabditiform larva to
filariform larva. The larva survives in the soil in shaded and moist areas. The
larva penetrates the skin of people who walk bare foot or work on soil with their
hands.
Notes Development in human host

The larva penetrates the skin and reaches the blood circulation. Through the
venules and blood circulation it reaches the heart and eventually reaches the
lungs.
In the lungs the larva undergoes development from first stage to second stage
and then the third stage filariform larva. The larva increases in length and reaches
a length of 1500 µm. The larva then moves up on the airway passage from the
alveoli to bronchioles. It passes through the bronchi and trachea to reach the
pharynx and from there it goes to the esophagus. From the esophagus it travels
to the stomach and reaches duodenum and intestines. In the duodenum and
intestines the larva develops into the adult stage. The life cycle continues after
sexual reproduction and formation of ova. The life span of an adult worm is
about one year.
44.4 PATHOGENECITY
Anaemia and nutritional deficiency: The hookworm infestation can cause
anaemia due to blood loss as the worm consumes the blood meal in the
duodenum and intestines. An adult worm is capable of consuming upto 0.4ml
of blood per day. The anaemia is mostly iron deficiency anaemia and it shows
a microcytic hypochromic blood picture in the red blood cells on peripheral
blood smear examination. Occult gastrointestinal bleeding can also occur. It also
causes nutritional deficiency in the hosts.
Itching and skin allergy may follow the worms penetration of the skin
The worm can obstruct small lumen organs like the bile duct, appendix. They
may cause appendicitis, pancreatitis, and peritonitis.

the laboratory diagnosis.
Eosinophilia may be seen the infected hosts.
388 MICROBIOLOGY
Microbiology
44.6 STRONGYLOIDES STERCORALIS
Strongyloides stercoralis, also known as the dwarf thread worm causes
strongyloidiases. It is smallest pathogen nematode to cause infection in man. The
parasite is unique in having both parasite and freeliving generations.
44.7 GEOGRAPHIC DISRTIBUTION

Notes
It is prevalent in tropical regions of Africa, Asia and South America. It is
important pathogen in immunocompromised host (HIV).
44.8 HABITAT
The female parasite inhabits the mucosa of the small intestine.
44.9 MORPHOLOGY
Adult worm
The parasitic females are approximately 2mm in length, with blunt-ended tails,
and an elongated, straight-sided (filariform) oesophagus, occupying approximately
one third of the body length . The ovary is didelphic and opens at the vulva which
is positioned approximately two thirds along the body length. The free-living
adults stages are approximately 1mm in length, with the female slightly larger
than the male. Both sexes have a rhabditiform oesophagus; the free-living female
has a didelphic ovary and a vulva at the mid-point of the body .
EGG
The egg measures 50-58µm × 30-34µm .They are thin-shelled , transparent and
oval. They contain larva ready to hatch. As soon as the eggs are laid. The
rhabditiform larve start hatching and bore their way out of the mucous membrane
into the lumen from where they are passed in the faeces.
Life Cycle
The Strongyloides’ life cycle is heterogonic - it is more complex than that of most
nematodes with its alternation between free-living and parasitic cycles, and its
potential for autoinfection and multiplication within the host. The parasitic has
a homogenic life cycle, while the free-living has a heterogonic life cycle. The
heterogonic life cycle is advantageous to the parasite because it allows
reproduction for one or more generations in the absence of a host.
MICROBIOLOGY 389
Microbiology In the free-living cycle, the rhabditiform larvae passed in the stool can either molt
twice and become infective filariform larvae (direct development) or molt four
times and become free-living adult males and females that mate and produce
eggs from which rhabditiform larvae hatch. In the direct development, L1 (1st-
stage larvae) transform into IL (infective larvae) via three molts. The indirect
route results first in the development of free-living adults that mate; the female
lays eggs, which hatch and then develop into IL. The direct route gives IL faster
Notes (3 days) versus the indirect route (7–10 days). However, the indirect route results
in an increase in the number of IL produced. Speed of development of IL is traded
off for increased numbers. The free-living males and females of S. stercoralis
die after one generation; they do not persist in the soil. The latter in turn can either
develop into a new generation of free-living adults or develop into infective
filariform larvae. The filariform larvae penetrate the human host skin to initiate
the parasitic cycle. The infectious larvae penetrate the skin when there is contact
with the soil. Some of them enter the superficial veins and ride the blood vessels
to the lungs, where they enter the alveoli. They are then coughed up and
swallowed into the gut, where they parasitise the intestinal mucosa (duodenum
and jejunum). In the small intestine, they molt twice and become adult female
worms. The females live threaded in the epithelium of the small intestine and,
by parthenogenesis, produce eggs, which yield rhabditiform larvae. Only
females will reach reproductive adulthood in the intestine. Female strongyloides
reproduce through parthenogenesis. The eggs hatch in the intestine and young
larvae are then excreted in the feces. It takes about two weeks to reach egg
development from the initial skin penetration. By this process, S. stercoralis can
cause both respiratory and gastrointestinal symptoms. The worms also participate
in autoinfection, in which the rhabditiform larvae become infective filariform
larvae, which can penetrate either the intestinal mucosa (internal autoinfection)
or the skin of the perianal area (external autoinfection); in either case, the
filariform larvae may follow the previously described route, being carried
successively to the lungs, the bronchial tree, the pharynx, and the small intestine
where they mature into adults; or they may disseminate widely in the body.
44.10 DISEASES
Many people infected are usually asymptomatic at first. Symptoms include
dermatitis: swelling, itching, larva currens, and mild hemorrhage at the site
where the skin has been penetrated. If the parasite reaches the lungs, the chest
may feel as if it is burning, and wheezing and coughing may result, along with
pneumonia-like symptoms (Löffler’s syndrome). The intestines could eventually
be invaded, leading to burning pain, tissue damage, sepsis, and ulcers. In severe
cases, edema may result in obstruction of the intestinal tract as well as loss of
peristaltic contractions. Strongyloidiasis in immunocompetent individuals is
390 MICROBIOLOGY
usually an indolent disease. However, in immunocompromised individuals, Microbiology
strongyloidiasis can cause a hyperinfective syndrome (also called disseminated
strongyloidiasis) due to the reproductive capacity of the parasite inside the host.
This hyperinfective syndrome can have a mortality rate of close to 90% if
disseminated.
The consequence of autoinfection is that the autoinfective larvae can carry gut
bacteria back into the body. About 50% of people with hyperinfection present Notes
with bacterial disease due to enteric bacteria. Also, a unique effect of
autoinfective larvae is larva currens due to the rapid migration of the larvae
through the skin. Larva currens appears as a red line that appears, moves rapidly
(>5 centimetres (2.0 in)/day), and then quickly disappears. It is pathogonomic
for autoinfective larvae and can be used as a diagnostic criterion for strongyloidiasis
due to S. stercoralis.
44.11 LABORAORY DIAGNOSIS

Parasitic diagnosis – definitive diagnosis is made by identification of the larvae
in the stool
Specimen – stool is the specimen of choice. Urine and sputum are collected in
disseminated infections.
Method of examination includes
1. Stool microscopy
2. Stool culture
3. Enterotest
Stool microscopy- only larva are demonstrated in stool. No eggs are seen.
Formalin – ether or Zinc floatation methods are employed to concentrate
strongyloides larvae in the stool
Stool culture
It is important in suscepected cases of strongyloidiasis not confirmed either by
direct smear or concentration. Stool can be cultures by a) Harada-Mori filter
paper method, b) Baermann funnel method using charcoal, and c) the agar plate
method
Enterotest
The rhabditiform larvae may also be demonstrated in the intestinal fluid
aspirated by duodenal intubation and string test (enterotest).
MICROBIOLOGY 391
Microbiology Immunodiagnosis
Serodiagnosis – ELISA, IHA AND IFA are useful in the clinical evaluation and
diagnosis of the cases.

Notes
1. ....................... technique is used for stool microscopy
2. ....................... method is used for stool culture
3. Strongyloides stercoralis is also known as .......................
4. The female parasite inhabits ....................... of human body
5. ....................... life cycle allows reproduction in the absence of host
6. Strongyloides enter human through .......................
7. Strongyloides causes ....................... syndrome in human
8. ......................., ....................... methods are commonly used in identifying
parasites in stool microscopy
9. ......................., ....................... & ....................... methods are carried out in
stool culture
44.12 TREATMENT
Ideally, prevention, by improved sanitation (proper disposal of feces), practicing
good hygiene (washing of hands), etc., is used before any drug regimen is
administered.
Thiabendazole 25mg/kg twice a day for 2 or 3 days can be used for the treatment
of strongyloides. Ivermectin has also been reported to be effective in the
treatment of chronic strongyloidiasis.

1. Which of the following stages of Ancylostoma duodenale is infective to
human beings?
(a) Rhabditiform larva (b) Filariform larva
(c) Eggs (d) Adult worm
392 MICROBIOLOGY
2. Habitat of Hookworm larva is Microbiology
(a) Small intestine (b) Large intestine

(c) Liver (d) Heart
3. Which of the following nematodes is ovoviviparous ?
(a) Ascaris lumbricoides (b) Drancunculus medinesis
(c) Strongyloides sterocoralis (d) Trichncella spiralis Notes
4. Disseminated systemic infection in AIDS patients is seen with
(a) Ancylostoma duodenale (b) Dracunculus medinensis
(c) Strongyloides stercoralis (d) Trichenella spiralis

Hookworm
z Hookworms cause intestinal infections.
z Hookworm larvae found on the soil have the ability to penetrate human skin,
thereby starting the infection process.
z Light infections are asymptomatic while heavy infections can produce a
variety of abdominal complaints.
z Treatment is relatively easy with appropriate drugs.
z There is no vaccine.
Strongyloides stercoralis
z It causes intestinal infection
z The adult parasitic stage lives in tunnels in the mucosa of the small intestine
z Many people infected are usually asymptomatic at first. Symptoms include
dermatitis: swelling, itching, larva currens, and mild hemorrhage at the site
where the skin has been penetrated
z Albendazole and mebedazole is used for treatment
TERMINAL QUESTIONS
1. Discuss the morphology and pathogenecity of Hook worm.
2. Discuss the life cycle of Hook worm.
MICROBIOLOGY 393
Microbiology 3. Draw a labeled diagram of ova of Hook worm.

4. Enumerate the differentiating feature between Ankylostoma dudenale and
Necator americanus.
5. Descibe life cycle and clinical manifestation of hook worm and strongyloides
stercoralis?
6. Write laboratory diagnosis of hook worm and strongyloides stercoralis?
Notes

44.1
1. Anklyostoma duodenale
2. Duodenum
3. Corpulatory
4. Filariform larva
44.2
1. Wet mount
2. Harada Mori
3. Dwarf thread worm
4. Mucosa of small intestine
5. Heterogonic
6. Skin
7. Loffler’s
8. Formalin-ether & Zinc floatation
9. Harada –Mori filter paper, Baermann funnel & agar plate
44.3
1. Filariform larva
2. Small intestine
3. Strongyloides sterocoralis
4. Strongyloides stercoralis
394 MICROBIOLOGY
Trichuris Trichura MODULE
Microbiology
45
Notes
TRICHURIS TRICHURA
45.1 INTRODUCTION
Trichuris trichura have a unique shape because of which they are also called as
whip worm. The worm resides in the ceacum and colon of the infected hosts.
OBJECTIVES
z describe the morphology of trichuris trichura
z explain the life cycle of trichuris trichura
z discuss the pathogenecity of trichuris trichura
z explain the laboratory diagnosis of trichuris trichura
45.2 MORPHOLOGY
The male worm measures 30-40 mm in size and the female worm measures 40-
50 mm in size. The anterior two thirds are long and slender. This end penetrates
the mucosa and the posterior one third remains out in the lumen of the colon.
In the males the terminal end has a copulatory spicule. No spicule is seen in the
female worms.
Adult worms Ova

Fig. 45.1
MICROBIOLOGY 395
MODULE Trichuris Trichura
Microbiology The ova are barrel shaped and measure 50 µm × 25 µm. There is a mucus plug
at the poles.
They are bile stained. They float on concentrated salt solution s
The ova contains an unsegmented ovum.
Notes 45.3 LIFE CYCLE

The gravid female passes ova in the stools after sexual reproduction. The ova
matures in the soil in three weeks.
The embryonated ova is ingested through contact of hands with soil or
vegetables containing the embryonated ova.
Fig. 45.2
The larva hatches in the intestine. The larva reaches the ceacum and colon and
mature into adult worms. The female worm becomes gravid after sexual
reproduction and lays eggs thus completing the life cycle. The female produces
5000-20,000 eggs/day. Children between 5-15 years have the highest prevalence
and have a higher worm load than adults.
396 MICROBIOLOGY
Trichuris Trichura MODULE
Microbiology
45.4 PATHOGENECITY
The worm infestation is mostly asymptomatic. It may cause a bloody diarrhea
also called as trichuris dysentery syndrome. It may cause iron deficiency
anaemia, growth retardation.
There may be rectal prolapsed in some cases. On endoscopic examination the

rectum gives a coconut cake appearance. Notes
Patient also develops significant eosinophilia.

Eosinophilia may be seen in the infected hosts.

1. Whip worm is also called as ...................
2. The ova of whip worm is ................... shaped
3. The larva matures into adult worm in ................... & ...................
4. Whip worm produces bloody diarrhea known as ................... syndrome

z Trichuris trichura have a unique shape because of which they are also called
as whip worm
z The worm resides in the ceacum and colon of the infected hosts.
z The gravid female passes ova in the stools after sexual reproduction and
the ova matures in the soil.
z The worm infestation is mostly asymptomatic and it may cause a bloody
diarrhea also called as trichuris dysentery syndrome
z Demonstration of ova in the stools or detection of adult worm established
MICROBIOLOGY 397
MODULE Trichuris Trichura
Microbiology
TERMINAL QUESTIONS
1. Discuss the morphology and pathogenecity of Trichuris trichura.
2. Discuss the life cycle of Trichuris trichura.
3. Draw a labeled diagram of ova of Trichuris trichura.
Notes
45.1
1. Trichuris trichura
2. Barrel
3. Ceacum & colon
4. Trichuris dysentry
398 MICROBIOLOGY
Trematodes MODULE
Microbiology
46
Notes
TREMATODES
46.1 INTRODUCTION
Trematodes are also called Platyhelminths. They are flat leaf shaped helminthes.
They are mostly hermaphrodites except for Schistosoma which have separate
male and female species. They are commonly associated with aquatic fauna like
snails, mollusks and fish.
OBJECTIVES
z classify blood trematodes
z describe morphology of trematodes
z describe morphology & life cycle of schistosoma, clonorchis, fasicola &
Paragonimus
z describe the pathogenesis of schistosoma, clonorchis, fasicola & Paragonimus
z demonstrate identification of schistosoma, clonorchis, fasicola &
Paragonimus
They are classified as under

(a) Blood trematodes
Schistosoma S hematobium
S mansoni
S japonicum
MICROBIOLOGY 399
MODULE Trematodes
Microbiology S intercaltum
S mekongi
(b) Hepatic trematodes
Fasciola F hepatica
F gigantic
Notes
Clonorchis C sinensis
Opisthorchis
(c) Intestinal trematodes
Fasciola F buski
Metagonimus yokogawi
(d) Lung trematodes
Paragonimus P westermani
2. General Morphological features of Trematodes

(a) Have conspicuous suckers, hence termed “Flukes”
(b) No body cavity
(c) Oviparous-release only eggs
(d) Eggs of all Flukes except Schistosoma are oprculated
(e) Most commonly found in tissue sections
(f) Sexes are separate and eggs non-operculated
(g) Male-female paired together
(h) Eggs differentiated on basis of spine-
i. S haematobium-terminal spine
ii. S japonicum –no spine
iii. S. mansoni-lateral spine
(i) Cercarial dermatitis, tissue reaction-thrombosis on death
(j) Expulsion of eggs-eosinophilic granuloma,fibrosis,TCC
(k) Splendore-hoeppli bodies,
400 MICROBIOLOGY
Trematodes MODULE
Microbiology
Notes
Fig. 46.1
46.2 SCHISTOSOMIASIS
1. Introduction
The Schistosoma derive their name from the fact that the males body is split
and forms a gynaecophoric canal in which the female worm rests. Schisto (Split)
soma (body). The adult worm of schistosoma live in the venous plexus of the
definitive host. The inhabit the venous plexus of the urinary bladder (S
heamatobium, intestines (S japonicum and S mansoni).
2. Morphology
a) Schistosoma japonicum egg
i. 70-100 x 50-65 microns.
ii. Oval to rounded, nonoperculate egg.
iii. Contain developed miracidium.
iv. Note: Lateral knob.
S mansonii egg
Fig. 46.2
MICROBIOLOGY 401
MODULE Trematodes
Microbiology (b) S mansonii adults

i. Adult male 0.6-1.4 × 0.11 cm. in size.
ii. Grossly tuberculate integument.
iii. Note: Ventral gynecophoral canal.
iv. Intestinal ceca unite early,united intestinal long.
Notes v. 7(3-13) small testes situated dorsal and posterior to the ventral sucker.
vi. Adult female 1.2-1.6 × 0.016 cm. in size often lie in the gynecophoral
canal.
vii. Smooth integument.
viii. Slender,pointed end.
ix. Ovary opposite in anterior half of body.
x. Short uterus fill with 1-4 ova.
Fig. 46.3
(c) S haematobium Adult

i. Adult female measure 2.0-2.5 × 0.025 cm.
ii. Cylindrical shape with pointed ends
iii. Smooth integument.
iv. The oral and ventral suckers near the anterior end are of equal size.
v. Elongate ovary situated in posterior half of body.
vi. Long,voluminous uterus contains 20-30 ova
402 MICROBIOLOGY
Trematodes MODULE
(d) S haematobium egg Microbiology
i. 112-170 × 40-70 microns.

ii. Large, spindle shaped with rounded anterior and conical posterior end.
iii. Yellowish-brown, nonoperculate egg.
iv. Contains developed miracidium.
v. Note: Terminal spine. Notes
Fig. 46.4
(e) Schistosoma cercaria

i. Elongated, pear-shaped body with round ends
ii. Long tail with a terminal furca.
iii. Tactile hair are distributed over the body and tail.

1. Trematodes are also called as .......................
2. Trematodes have suckers and hence termed as .......................
3. S. japonicum has no .......................
4. Schistosoma means .......................
MICROBIOLOGY 403
MODULE Trematodes
Microbiology
46.3 LIFE CYCLE OF SCHISTOMA SPECIES
Schistosoma pass their life in two hosts. Man is the definitive host. The mollusks
or the snails are the intermediate host. Ova from the sexually mature worms are
passed in the intestinal tract or the urinary bladder. The ova are passed in the
urine or the stools into fresh water bodies. The miracidium hatch from the ova
immediately or after a short period of incubation. The miracidium infects the
Notes first intermediate host the snail. Within the snail or mollusks the miracidium
transforms into sporocysts. The sporocysts develop into second generation
sporocysts (in schistosomes). Miracidium develops into tailed larvae called
cercaria. The cercaria mature and leave the snail and become free living in water.
In case of schistosoma the cercaria have a forked tail. They infect the man by
penetrating the skin of humans who are in water. The immature worm enters the
blood stream and eventually reach the veins near the intestines and urinary
bladder. The worm reaches sexual maturity in these venous plexus of the
intestines and urinary bladder.
46.4 PATHOLOGY
(a) Penetration of skin by metacercaria

Skin penetration may not be apparent. Human and some non human species of
Schistosoma cause cercarial dermatitis (swimmers itch). This manifests with
papules, macules, vesicles and intense itching.
b) Migration and maturation of immature worms

There are general toxic and allergic symptoms including urticaria with
eosinophilia, fever, abdominal pain and tender hepatosplenomegaly. This is
known as Katayama or snail fever.
c) Damage by eggs in tissue

Resulting damage depends on the severity of the parasite load. An inflammatory
granuloma forms with epithelial, giant plasma and eosinophil cells and
fibroblasts (Hoeppli reaction). There is subsequent fibrosis and calcalcification.
Such damage may be local and/ or ectopic.
d) Urinary schistosomiasis
It is caused by Schistosoma haematobium. The initial toxic and allergic
symptoms are not marked. The urinary bladder and the ureter are typically
involved with hyperemia and terminal hematuria, dysuria and increased
frequency of micturation, papules, papillomata and ulceration. Hypertrophy of
404 MICROBIOLOGY
Trematodes MODULE
the bladder can later lead to contraction. There may be cystitis and calculus Microbiology
formation. This may be followed by calcification and squamous cell carcinoma.
Fistula may develop. There can also be hydroureter and hydronephrosis. Ectopic
lesions are less severe than in other species of Schistosoma. Genital
Schistosomiasis may lead to lumpy semen, haematospermia or wart like lesions
on the vulva.
Notes
e) Intestinal schistosomiasis
It is caused by Schistosoma mansoni. There are marked initial toxic and allergic
symptoms. The large intestine and the rectum are typically involved with
polyposis, papules, abscesses, ulcers , papillomata, fistulae and ova in stools.
The bladder is sometimes involved with pathology similar to urinary Schistosoma
as above. There can be ectopic lesions. The liver is frequently involved
(receiving the eggs via the portal vein) with inflammatory reaction and fibrosis
leading to periportal (pipe-stem) fibrosis with portal hypertension. This may
manifest with oesophageal varices, splenomegaly and ascitis. There can also be
lesions in the brain, spinal cord and lungs.
f) Oriental schistosomiasis
This is caused by Schistosoma japonicum. There are marked initial toxic and
allergic symptoms which can lead to myocarditis and death. Intestinal lesion
are similar to those with S mansoni infection and the small intestine is involved.
The liver is involved as in S mansoni infection and hepatic lesions are similar
and may lead to portal hypertension. The brain may also be become involved.
5. Laboratory Diagnosis:
Eosinophili may be present. Ova found in terminal urine by Nucleopore filtration
or after centrifugation. Ova may also be found in semen. Ova may also be found
in faeces directly or using formol- ether concentration, rectal scrapings or
biopsies.
Serology. ELISA tests (using soluble egg antigen) are useful 6-12 weeks post
exposure. In many chronic cases, the diagnosis will be made on serology alone.

1. ........................ is the definite host of schistosoma
2. Miracidium develops into tailed larvae called ........................
MICROBIOLOGY 405
MODULE Trematodes
Microbiology 3. Schistosoma causes ........................ itch

4. Urinary schistosomiasis is caused by ........................
5. Intestinal schistosomiasis is caused by ........................
6. Oriental schistosomiasis is caused by ........................
46.5 CLONORCHIS SINENSIS

Notes
1. Introduction
These are leaf shaped and flat helminthes. They are hermaphrodites. Twenty
eight million people are infected worldwide.
2. Morphology
a) 12-20 x 3-5 mm. in size.
b) Flat, elongated, aspinous, flabby, tapering anteriorly and somewhat rounded
posteriorly.
c) Note: Lancet appearance.
d) Small, slightly lobate ovary anterior to the branched testes.
Fig. 46.5
406 MICROBIOLOGY
Trematodes MODULE
3. Life Cycle of Clonorchis species: Microbiology
Ova from the sexually mature worms are passed in the intestinal tract. The ova
are passed in the stools into fresh water bodies. The ova are ingested by the snails
and the miracidium hatch from the ova in the first intermediate host the snail.
Miracidium develops into cercaria. The cercaria leave the snail. Free swimming
cercaria encyst in the fish which is the second intermediate host. Cercaria
develop into metacercaria and reach the muscles of the fish. The fish may be
Notes
eaten by the dogs and cats which serve as the animal reservoir of the disease.
Humans get infected by consuming under cooked fish. Encysted metacercaria
release the immature worm in the duodenum. The immature worm penetrates
the intestinal mucosa and reaches the portal circulation through which it reaches
the liver. In the liver the worm lodges in the intrahepatic bile ducts where it
reaches sexual maturity.
4. Pathology and Clinical features

Adult flukes inhabit the distal bile duct with epithelial proliferation, surrounding
inflammatory reaction and ascending cholangitis. Sometimes there is secondary
bacterial infection with jaundice and septicemia. There can also be eosinophilia.
All this can lead to thick, dilated fibrous ducts with adenomata of epithelium,
bile duct stenosis and cholangiocarcinoma. Many cases are asymptomatic. Acute
infection may lead to tender hepatomegaly. Chronic infection can result in
anorexia, low-grade fever, epigastric pain and tender hepatomegaly.
The ova are found in the feaces and in the bile duct (via duodenal aspiration or
‘string test’)
FASCIOLIASIS
1. Introduction
The fluke is found in all sheep- rearing countries. About one million people are
infected worldwide. F.gigantica reside in bile ducts. Larvae might get “lost”
a) Adults are large with cephalic cone, shoulders, cuticular spines
b) Only Eggs are seen in stools
c) Liver shows coagulative necrosis, abscess, patchy haemorrhage
d) Granulomas can form
e) Cholelithiasis and cholecystitis are the usual complication in the liver.
MICROBIOLOGY 407
MODULE Trematodes
Microbiology 2. Fasciolepsis buskii

a) Adult measure 20-75 x 8-20 mm and is about 2 mm. thick.
b) They are thick, fleshy, ovate worm.
c) Two highly branched testes are situated one behind the other in the middle
of the posterior half of the body.
d) Branched ovary is anterior to the testes.
Notes
e) Unbranched ceca is present.
Fig. 46.6
3. Fasciolepsis buskii Egg

a) Eggs are 130-140 x 80-85 microns in size
b) They are Yellowish-brown, ellipsoidal egg.
c) Thick-shell with a small operculum at one end.
d) They contain an undeveloped ovum.
4. Life Cycle of flukes species

Schistosoma pass their life in two hosts. Man is the definitive host. The mollusks
or the snails are the intermediate host. Ova from the sexually mature worms are
passed in the intestinal tract or the urinary bladder. The ova are passed in the
urine or the stools into fresh water bodies. The miracidium hatch from the ova
immediately or after a short period of incubation. The miracidium infects the
408 MICROBIOLOGY
Trematodes MODULE
first intermediate host the snail. Within the snail or mollusks the miracidium Microbiology
transforms into sporocysts. The sporocysts develop into second generation
sporocysts (in schistosomes). Miracidium develops into tailed larvae called
cercaria or into rediae (in hermaphrodites like the flukes). In hermaphrodite
trematodes the cercaria have unsplit tails and they bencyst on vegetables or
within a second intermediate host (fish or crab) to form meta cercaria which
are the infective form. Humans become infected by ingestion of raw or
undercooked vegetables, fish or crabs. The encysted metacercaria excysts in the Notes
intestines and migrates to organs and tissue where they mature into worms.
5. Fluke (Fasciola) infection:
Pathology and Clinical features

Transit of immature worms through the liver can cause mechanical and toxic
irritation with toxaemia, necrosis and secondary fibrosis. Development in the
bile ducts causes cystic enlargement, endothelial hyperplasia and adenomata and
secondary inflammatory infiltration causing fibrosis and cholangitis. There can
be secondary bacterial infection causing abscesses. Eosinophilia is marked.
Worms can appear ectopically in the lungs, brain, eyes etc. with similar reactions.
If raw sheep or goats liver infected by the adult fluke, is eaten there can be local
irritation and pharyngeal infection ( Halzoun).
Acute infection may present with fever, tender hepatomegaly, epigastric pain,
anorexia and vomiting. Jaundice may occur. In chronic infection, there may be
no symptoms or epigastric / right upper quadrant pain, hepatomegaly and
vomiting
The ova are found in the feaces. Serological test (IFAT) is available.
46.7 PAROGONIMUS WESTERMANII

1. Introduction
They are also called lung flukes. It is endemic in parts of East and South East
Asia and Africa.
2. Morphology
a) Adult
i. The Paragonimus are plump and coffee bean like.
MICROBIOLOGY 409
MODULE Trematodes
Microbiology ii. 7.5-12 x 4-6 mm. in size.

iii. Note: Coffee bean appearance.
iv. Spinous cuticle.
v. Finger-like lobed ovary.
vi. Irregularly lobed testes oblique to each other, in posterior third of worm.
Notes
Fig. 46.7
b) P westermanii egg
i. They measure 80-118 x 48-60 micrometre.
ii. They are broadly ovoidal, yellowish brown, thick-shelled egg.
iii. Thickened operculum.
iv. Unembryonated at oviposition.
3. Pathology and Clinical features

Paragonimus westermanii infection:
The initial invasion has little pathological effect on the host. On localization in
the lungs, there is tissue reaction leading to formation of a fibrous tissue capsule(
of slate blue colour) containing worms( generally in pairs, ova and inflammatory
infiltrate. The capsule is connected with the respiratory passages Secondary
complication of these lung cysts include bronchiectasis, abscess formation and
hemoptysis. Localization in the other sites can cause cysts in any other part of
the body ( for example the brain, causing epepsy). Eosinophilia is a general
manifestation. Chronic infection may be asymptomatic. Cough, brown gelatinous
sputum, chest discomfort, shortness of breath and pleuritic chest pain may occur.
410 MICROBIOLOGY
Trematodes MODULE
4. Laboratory diagnosis Microbiology
Ova are found in the sputum after KOH digestion. Ova may also be sen in the
feaces after formol – ether concentration. Serological tests consist of ELISA
(Using extractr of adult fluke as antigen). Complement fixation tests and gel
diffusion tests are also available.Imaging studies like X ray chest and CT scan
also help in establishing the diagnosis.
Notes
Fig, 46.8: Various ova of Schistosoma and Fasciola

1. ...................... is the intermediate host of flukes
2. Human become infected with flukes by ingesting ...................... &
......................
3. Paragonimus westermanii has ...................... appearance
4. ......................, ......................, ......................, ...................... tests are used in
identification of ova

z Trematodes are also called Platyhelminths. They are flat leaf shaped
helminthes
MICROBIOLOGY 411
MODULE Trematodes
Microbiology z Blood trematodes are Schistosoma, Hepatic trematodes are fasciola,

clonorchis & Opisthorchis, Intestinal trematodes are Fasciola, Lung
trematodes are Paragonimus
z Schistosoma pass their life in two hosts and Man is the definitive host.
z Schistosoma cause swimmers itch and snail fever
Notes
TERMINAL QUESTIONS
1 Classify and enumerate the various species of Trematodes.
2 Discuss the life cycle and pathogenecity of Schistosoma heamtobium.
3 Discuss the morphological difference between the various ova of Schistosoma
species.

46.1
1. Platyhelminths
2. Flukes
3. Spine
4. Split body
46.2
1. Man
2. Cercaria
3. Swimmers
4. Schistosoma haematobium
5. Schistosoma mansoni
6. Schistosoma japonicum
46.3
1. Snails
2. Raw & cooked vegetables and fish /crabs
3. Coffee bean
4. KoH, ELISA, Complemet Fixation, Gel diffusion
412 MICROBIOLOGY
Cestodes MODULE
Microbiology
47
Notes
CESTODES
47.1 INTRODUCTION
Cestodes consist of flat tape like two dimensional worms which mostly reside
in the intestines. They are hermaphrodite and are oviparous. They do not have
a digestive system so the nutrients are taken up through the absorptive
integument. These are segmented tape worms that vary in size from a few
millimeters to several meters. They do not have a body cavity or alimentary
canal.
OBJECTIVES
z describe the general morphology of cestodes
z describe the pathology, life cycle & morphology of T.solium & T. saginata
2. The morphological features of the tape worms are:

a) All cestodes have a scolex, a neck and strobila (segments)
b) Body wall has three layers :
i. outer cuticle,
ii. middle muscle,
iii. radial tegumental cells
c) They do not have a body cavity, only loose parenchymal tissue is present
d) No digestive organs are present. The nutrients are absorbed by the body
segments.
MICROBIOLOGY 413
MODULE Cestodes
Microbiology 3. The Pathogenic species are:

a) Taenia solium,
b) Taenia saginata,
c) Echinococcus granulosus,
d) Hymenolepus nana
Notes e) Diphylobothrium latum
47.2 T SOLIUM / T SAGINATA
1. Introduction
About 5 million people are infected worldwide. T solium is endemic in pig
rearing areas of the world where hygiene and animal husbandry are poor.
T solium causes infection in pigs that are its definitive hosts while T saginata
infects cows. Man gets accidentally infected by eating pork meat or beef meat
which is under cooked. Man can get the larval infection called cysticercosis by
the ingestion of the eggs of T solium tape worm.
2 Morphology
a) T solium: It is 3-4m long and is smaller than T saginata. The scolex of
T solium has a rostellum armed with two rows of hooks in addition to the
four suckers. Inside the gravid segments the number of uterine lateral
branches are usually 7-13.
b) T saginata: It can grow as long as 10m. It has a scolex with four suckers
but no rostellum is present. The proglottids at the posterior end of the chain
are longer than its width. Each proglottid contains a branched treelike
uterus consisting of more than 15 branches. The uterus contains 80,000
to 100,000 ova.
c) Ova: the ova are released when a proglottid detaches from the tape worm
in the intestinal lumen or when a proglottid disintegrates outside the host.
The ova measure 30-40 µm and are round in shape. The outer shell forms
a thick brownish radially striped embryophore which encloses an oncosphere
with three pairs of hooklets. The ova are highly resistant and remain
infective in a moist environment for weeks or months.
414 MICROBIOLOGY
Cestodes MODULE
Microbiology
Notes
T solium : Rostellum T saginata: Four suckers,

with hooklets no rostellum
z Mature proglottid, shows trilobed ovary. Carmine stained.
Note : Less than 14 lateral uterine branches (one side).
MICROBIOLOGY 415
MODULE Cestodes
Microbiology
Notes
3. Life Cycle of T solium/ T saginata:

The adult worm is present in the intestines of host. The ova or the proglottid
containing the ova are passed in the stools. The ova are then ingested by the
intermediate host who could be pig (T. solium) or Cow (T. saginata). When a
cow or buffalo feeds ingests the eggs the oncosphere hatches in the intestines.
The larva hatches from the ova in the intestines of the intermediate host. The
larva then penetrates the intestinal mucosa and reaches the muscles and develops
into a cysticercus in three months time. Man gets infected when man eats the
undercooked beef (T saginata) or pork (T solium) containing the cysticercii. In
man the cysticercus develops into an adult tape worm. The cycle thus continues.
4. Pathology and clinical features

Infection by larvae (cysticercosis). Cysticerci, generally are multiple, and may
occur at any site in the body. They are however most frequently seen in the
muscles and the brain. They excite an inflammatory reaction in the surrounding
area especially when they die. In some infected individuals the certain
morphological changes (villous formation, enterocyte proliferation, cellular
mucosal infiltration) and functional disturbances are seen. The inflammation
then is followed by fibrosis and calcification. In the brain this manifests as focal
neurological syndromes especially epilepsy.
416 MICROBIOLOGY
Cestodes MODULE
Infection with adults: This may often be an inapparent infection. In some cases Microbiology
there may be mild irritation of the intestinal mucosa.
Cysticercosis is sometimes seen in people who do not consume meat. In these

people the infection is contracted by ingesting the ova along with raw or
uncooked salad or leafy vegetables which have not been washed well and may
contain the ova of tape worm.
Notes
5. Laboratory Diagnosis
Gravid segments and ova and scolex can be found in the stools of the infected
person. The uterine branches of the mature segments can be demonstrated by
injection of india ink through the uterine pore.
Larval infections are often discovered accidentally on histopathological

examination of subcutaneous nodules or muscle biopsies. Imaging studies like
CT scan or MRI are often helpful in diagnosing cysticercus as the cause of
epilepsy. X ray of the thigh muscles detects the cysticercus in the muscles.
Serological tests based on detecting specific IgG/ IgM antibodies to Taenia

solium are available. These tests are ELISA.

1. Cestodes are segmented .......................
2. Nutrients of cestodes are absorbed in the .......................
3. Taenia in man causes ....................... infection
4. The intermediate host in T. Solium is .......................
47.3 HYMENOLEPUS NANA
1. Introduction
The natural host of the dwarf tape worm is the mouse.
2. Morphology
The worm is 1-4 cm long and is I mm wide. The ova are elliptical and measure
40 X 50 µm and contain an oncosphere.
MICROBIOLOGY 417
MODULE Cestodes
Microbiology
Notes
z Hymenolepis nana Egg Hymenolepis nana

z 37-41 micrometer, faecal smear, wet mount. Adult dwarf tapeworm
Note : Bipolar thickenings with filaments. Scolex , Rostellum with hooklets
3. Lifecycle and pathogenecity

Man gets accidentally infected by accidental ingestion of the ova which are
passed in the stools of the rodents like mouse. The rodents are its definitive
418 MICROBIOLOGY
Cestodes MODULE
Microbiology
Notes
hosts. The oncosphere in the ova hatch in the intestine and penetrates into the
intestinal villi and develop into a larva (cysticercoids). The larva then returns
to the intestine and develops into an adult tape worm. Alternatively H nana may
also develop in an intermediate host like fleas, grain beetles. Infections are often
latent and in apparent. It may cause gastrointestinal disturbances.

z Cestodes are segmented tape worms. All cestodes have scolex, neck and
strobila
z Pathogenic species are Taenia solium, Taenia saginata, Echinococcus
granulosus, Hymenolepis nana
z T solium causes infection in pigs and man gets infected by eating pork meat
or beef meat which is under cooked.
z Man can get the larval infection called cysticercosis by the ingestion of the
eggs of tape worm
z Gravid segments and ova and scolex can be found in the stools of the
infected person.
z Serological tests based on detecting specific IgG/IgM antibodies are
available
z Tthe natural host of the dwarf tapeworm is mouse
z Man gets infected by accidental ingestion of ova which are passed in the
stools of the mouse
z Eggs and gravid segments can appear in faces and megaboblastic anaemia
is also seen.
MICROBIOLOGY 419
MODULE Cestodes
Microbiology
TERMINAL QUESTIONS
1. What are cestodes? Enumerate the different cestodes.
2. Describe the morphological characteristics of cestodes
3. Describe the life cycle of T saginat/ T solium.
Notes 4. Discuss the difference in the pathogenecity of T solium and T saginata.
47.1
1. Tapeworm
2. Body segments
3. Cysticercosis
4. Pig
420 MICROBIOLOGY
Echinococcus Granulosus MODULE
Microbiology
48
Notes
ECHINOCOCCUS GRANULOSUS
48.1 INTRODUCTION
E granulosus are small tape worms that parasitize the intestines of carnivores
like dogs. About one million people are infected with this tape worm worldwide.
E granulosus is widespread in the sheep-rearing areas of the world. The
definitive host is dog and man is the intermediate host.
OBJECTIVES
z describe the morphology of Echinococcus granulosus
z describe the life cycle of Echinococcus granulosus
z explain the pathology and laboratory diagnosis of Hydatid cyst
48.2 MORPHOLOGY
a) Adult worm: E granulosus 4-7 mm long. The scolex has rostellum like
hooks. It has three to six proglottids
b) Ova: The ova are 30-40 µm in diameter and are spherical in shape and
contain an oncosphere. It has a radially striped shell.
c) Hydatid cyst: The cyst is the metacystode stage of the worm. It is a fluid
filled cyst which may have a single or multiple chambers. The wall of the
cyst is made up of an inner cellular germinative layer and an outer acellular
laminated layer. The host connective tissue covers the outer layer. Brood
capsules develop inside the cyst on the germinative layer 5-6 months later.
Each brood capsule contain 20 or more protoscolices. The brood capsules
burst to release free protoscolices in the hydatid fluid.
MICROBIOLOGY 421
MODULE Echinococcus Granulosus
Microbiology
Notes
Echinococcus granulosus Hydatid sand protoscolices with double row

of hooklets and calcareous corpuscles.
422 MICROBIOLOGY
Microbiology
48.3 LIFE CYCLE OF E GRANULOSUS
The adult worm is present in the intestines of canine host usually dog. The ova
of the worm are passed in the stools of the dog. The ova are then ingested by
the intermediate host who could be sheep or man.
The larva hatches from the ova in the intestines of the intermediate host. The
oncosphere is released from the ova in the intestines of human beings after
ingestion. The larva then penetrates the intestinal mucosa and reaches the blood Notes
stream of the host. The larva can reach any organ but commonly reaches and
settles in the liver. Here the larva develops into a hydatid cyst. The definitive
host i.e. the dog gets infected when it eats the hydatid cyst (protoscolices),
mostly from sheep meat. In man the infection reaches a dead end. The
protoscolex attaches to the host intestine and develops into a tape worm. The
cycle thus continues.
48.4 PATHOLOGY AND CLINICAL FEATURES OF

HYDATID DISEASE
Unilocular cysts. There is usually surrounding inflammatory reaction and
fibrosis. After years, the cyst may die, shrink and calcify. There is general allergic
reaction with eosinophilia, bronchospasm etc. Pressure effects can cause local
tissue damage and obstruction of natural channels. Rupture or leakage of the cyst
can accentuate the allergic reaction. There can be anaphylactic shock and
secondary implantation in the surrounding tissues like the peritoneum. There can
be secondary infection with formation of abscesses.

Microscopy of the cyst fluid reveals the typical morphology of the protoscolices.
Histopathology of the removed specimen of the hydatid cyst also reveals its
typical morphology.
Serology: ELISA based tests are available to detect IgG and IgM antibodies to
E granulosus.
DIPHYLLOBOTHRIUM LATUM
These are also known as fish tape worms or broad tape worms.. It parasitizes
the intestines of humans and fish eating mammals such as dogs, cats, pigs. The
parasite has two elongated grooves called the bothria on its head. It is 2-15 m
longwith numerous proglottids (up to 4000). The ova measure 70 × 50 µm and
are oval yellow – brown coloured operculated and are similar to the trematodes.
MICROBIOLOGY 423
MODULE Echinococcus Granulosus
Microbiology The life cycle includes copepods as primary host and fresh water fish as
secondary intermediate hosts. Man gets the infection by eating raw or
undercooked fish.
Notes

1. Intermediate host of Echinococcus granulosus is ..................... & definite
host is .....................
2. E.granulosus commonly causes ..................... in human
3. The natural host of dwarf tapeworm is .....................
4. Tape worm causes ..................... anaemia

z E granulosus are small tape worms that parasitize the intestines of
carnivores like dogs.
424 MICROBIOLOGY
z The definitive host is dog and man is the intermediate host. Microbiology
z The adult worm is present in the intestines of canine host usually dog
z The definitive host dog gets infected when its eats the hydatid cyst mostly
from sheep meat.
z Microscopy of the cyst fluid reveals the typical morphology of the
protoscolices.
Notes
z Histopathology of the removed specimen of the hydatid cyst also reveals
its typical morphology
z ELISA based tests are available to detect IgG and IgM antibodies to E
granulosus
TERMINAL QUESTIONS
1. Describe the morphological characteristics of Echinococcus granulosus.
3 Describe the life cycle of Echinococcus granulosus.
ANSWERS TO INTEXT QUESTION
48.1
1. Man, dog
2. Hydatid
3. Mouse
4. Megaloblastic
MICROBIOLOGY 425
MODULE Tissue Nematodes
Microbiology
49
Notes
TISSUE NEMATODES
49.1 INTODUCTION
Some nematodes cause infection in the tissues and may be found in the blood
or lymphatics as well as in the muscle and other advetitious tissue. The
nematodes in this category are filarial nematodes, Dracunculus medineensis,
Loa
OBJECTIVES
z describe the characteristics of nematodes

z describe the morphology, pathogenecity, lab diagnosis of dracun culiasis
z describe the morphology, pathogenecity, lab diagnosis of micro filariasis
49.2 FILARIASIS
Introduction
Filariae are long slender thread like nematodes that infect human beings. They
reside in the lymphatics and produce symptoms related to obstruction of
lymphatic flow. The disease was described by Sushruta. In 1709 Clarke
described the disease in the natives off the Kerala coast as the “Malabar leg”.
Wucherer demonstrated the microfilaria in the blood film of a filarial patient.
In 1866 Manson in 1878 found the Culex fatigans mosquito to be the vector in
filariasis.
426 MICROBIOLOGY
Tissue Nematodes MODULE
The various species of filarial are : Microbiology
a) Wucheraria bancrofti
b) Brugia malayi
c) Loa loa
d) Mansonella perstans
e) Mansonella ozzardi Notes
f) Mansonella streptocerca
g) Onchocerca volvulus
The commonest species causing filariasis are Wucheraria bancrofti and Brgia
malayi.
49.3 GENERAL FEATURES

Slender thread like worms that Inhabit blood vessels, lymphatic systems,
connective tissue, serous cavities. Adult & microfilaria is seen in man. Embryo
could be sheathed or unsheathed.
49.4 WUCHERARIA BANCROFTI
Morphology
a) Adult
Female is longer than the male
Worm has lipless mouth, cylindrical oesophagus without bulb and simple
intestine.
Female is viviparous and releases the microfilaria into the blood stream.
b) Microfilaria: 290 x 6 -7 um in size and are colourless with blunt head and
pointed tail. It is covered by a hyaline sheath which is much longer 359um.
Somatic nuclei appear as granules. At the ant end Cephalic space. Stylet
can be shown with vital stains
Tail tip is free of nuclei
Nerve ring: Ant end an area devoid of granules
Excretory pore Anterior V spot
Genital cells: Number G1 – G4
Posterior V spot : Cloaca or anal pore
MICROBIOLOGY 427
Microbiology
Notes
Fig. 49.1
Table 49.1: Differential features of two major species of microfilaria
S No W bancrofti B malayi
1 Length 290 µm x 7 µm 230 µm x 6 µm
2 Appearance The body shows sweeping The body has sharp kinky
curves bends
3 Cephalic space Length and breadth equal Length twice as long as the
breadth
4 Anterior end Single stylet Double stylet
5 Nuclear column Discrete nuclei are seen Blurred nuclei are seen
6 Tail tip Free of nuclei Two distinct nuclei seen. One

is terminal and the other sub
terminal
7 Sheath Faintly stained Well stained
49.5 LIFE CYCLE (W BANCROFTI)

It is seen in two hosts
Intermediate host is the mosquito belonging to Culex, Aedes or Anopheles
The development period in the mosquito is called as the Extrinsic incubation
period
Microfilaria is taken by mosquito in blood meal.
It penetrates the stomach wall & enters thoracic muscles
428 MICROBIOLOGY
It develop to 1stStage Larva (sausage shaped 125-250um) Microbiology
Moults twice to form 2nd Stage larva

In a weeks time develops to 3rd Stage larva
Measuring 1500-2000 um
Man is the definitive host. Development in man is called as the biological
Notes
incubation phase. Infective stage larva are deposited near the site of puncture
The larva enters the wound on to the lymphatics, settle down in the inguinal,
scrotal and abdominal lymphatics and grow to adult worms. The adult parasite
survives for many years in the host.
They reach sexual maturity in 5-18 months. The microfilaria are released by
the female after sexual reproduction. The microfilaria are generally released at
night when the host is asleep.
49.6 PATHOGENECITY
a) The filarial infection is mostly asymptomatic
b) The clinical manifestation is in the form of lymphangitis and lymphadenitis.
Due to lymphangitis the adult worm may die in the lymphatic channels.
The ensuing inflammation followed by fibrosis leads to blockage of
lymphatic channels. This leads to lymphedema and swelling of the body
parts. The skin over the affected area becomes rough and thickened giving
it an elephant like appearance. Thus this disease is also called as
elephantiasis.
c) There is accompanying eosinophilia

The laboratory diagnosis is by demonstration of the microfilaria in the blood
smear of the infected individual. The microfilaria is however seen only two hours
after the person goes to sleep. Thus the blood smear is taken at night while the
person goes to sleep.
The blood smear can also be taken 30 minutes –one hour after giving one tablet
of hetrazan (diethyl carmazepine).
The adult worm is not normally seen, but may be accidentally detected in lymph
node or tissue biopsies.
The patient may also have eosinophilia
MICROBIOLOGY 429
Microbiology
Notes
49.8 DRACUNCULIASIS
The largest nematode measuring up to 1.2 m is Dracunculus medinensis also
called as “Guinea worm” or fiery serpent
Geographical distribution: It is seen in over 22 countries mostly in West Africa
& Asia. It is mostly seen in dry arid areas with limited water resources, where
man and animal are forced to use the same water source.
Habitat: The adult worm resides in the subcutaneous tissues of the infected
person
430 MICROBIOLOGY
Microbiology
49.9 MORPHOLOGY
Adults: They have rounded heads, terminating in a thick chitinous shield
containing a triangular mouth & papillae.
Males measure 1.2 - 1.9 cm x 0.4mm
Females measure 50 – 120 cm x 1.5mm
Notes
Posterior end is hook like. They are viviparous.
The body cavity contains a fluid that is toxic
Life span is 1 year
The embryos are unsheathed, flattened. They have a coiled rounded anterior end
and a long filariform tail. They measure 650-750 um It dies in 2 days if not
ingested by Cyclops after release into a water body.
49.10 LIFE CYCLE

The gravid female worm is present in the subcutaneous tissue of the infected
person. The female protrudes from an ulcer which may be present on the feet
and releases the larvae from the ulcer. The infected person feels very itchy in
the ulcer area and gets a relief only when the affected part is dipped under water.
The larva which are released in the water body are eaten by the Cyclops present
in it. Humans consume water from these water bodies which contain the infected
Cyclops.
Fig. 49.3
The cyclops is digested by the gastric juices and the larva is released in the
stomach. The larva penetrates the stomach wall and penetrates the tissue to reach
the retroperitoneal tissues. The larva develops into an adult worm and becomes
MICROBIOLOGY 431
Microbiology sexually mature. The male worm dies after mating. The female then migrates
to the lower limbs through the retroperitoneal tissue. In the limb the worm
reaches the feet or hands and releases certain secretions which make the part
itchy leading to the formation of an ulcer. The female the releases its larva
through the ulcers into the water bodies.
49.11 CLINICAL FEATURES

Notes
a) Symptoms due to migrating worm Papule, blister, ulcer
b) Allergy
c) Due to injured or broken worm
d) Due to calcified worm Arthritis, fibrosed joints, compression of spinal cord
49.12 DIAGNOSIS
Clinical
Detection of embryo: cold water on ulcer
Radiological
DLC: eosinophilia
Immunodiagnosis: ELISA, IHA, IFA, Western blot
Intradermal test: wheal in 24h
49.13 TREATMENT
Roll out the worm
Occlusive bandage
Niridazole, thiabendazole

1. Filariasis is caused by ....................
2. Microfilaria is taken by mosquito in their ....................
3. The definitive host of W.bancrofti is ....................
4. The larva settles down in ...................., .................... & .................... parts
of human body and grows to worms
5. Blood smear for diagnosis of filariasis is taken at .................... part of the
day
432 MICROBIOLOGY
Microbiology

z Some nematodes cause infection in the tissues and may be found in the
blood or lymphatics as well as in the muscle and other adventitious tissue
z Filariae are long slender like nematodes that infect human beings that reside
in lymphatics and produce symptoms related to obstruction of lymphatic
Notes
flow
z Commonest species causing filariasis are Wucheraria bancrofti and Brgia
malayi
z In Wucheraria Bancrofti the female is longer than the male. Female is
viviparous and releases the microfilaria into the blood stream
z The intermediate host is the mosquito belonging to Culex, Aedes or
Anopheles and the development period in the mosquito is called as extrinsic
incubation period and microfilaria is taken by mosquito in blood meal
z Man is the definitive host. Development in man is called as biological
incubation phase and infective stage larva are deposited near the size of
puncture
z Filarial infection is mostly asymptomatic and it manifests in the form of
lymphangitis and lymphadenitis and the disease is also called as elephantiasis
z The laboratory diagnosis is by demonstration of the microfilaria in the blood
smear of the infected individual. The microfilaria is however seen only two
hours after the person goes to sleep and the blood smear is taken at night
while the person goes to sleep
z Dracunculiasis is the largest nematode and Dracunculus medinensis also
called as Guinea worm or fiery serpent
TERMINAL QUESTIONS
1. Name the nematodes which cause filariasis.
2. Enumerate the difference between the microfilaria of W bancrofti and B
malayi.
3. Discuss the life cycle and pathogenecity of filariasis..
4. Discuss the life cycle and pathogenecity of D medinensis.
MICROBIOLOGY 433
MODULE Stool Examination
Microbiology
50
Notes
STOOL EXAMINATION
50.1 INTRODUCTION
Stool examination is carried out in laboratories for various diagnostic purposes.
It is a specimen which is easily obtained but, they may however be a reluctance
on the part of the patient to give the stool specimen due to its offensive nature
and foul smell. Mostly a clean container which does not contain any detergent
or disinfectant is sufficient for all types of stool examinations including stool
culture.
OBJECTIVES
After reading this lesson, you will be able to :
z explain physical examination of stool

z describe microscopic examination of stool
z explain the chemical examination of stool
z describe the preservation of stool
50.2 PHYSICAL EXAMINATION OF STOOL

The following aspects of stool should be examined
(a) Quantity: In intestinal amoebiasis the stools tend to be voluminous.

Whereas in bacillary dysentery due to Shigella the stools are scanty in
quantity
(b) Consistency and form: Normal stools are well formed. In diarrhea and
dysentery the stools are semi solid or watery in nature. In malabsorpton
434 MICROBIOLOGY
Stool Examination MODULE
states also the stool will be semi solid or watery depending on the severity Microbiology
of the disease. In cases of malabsorption of fats the stools are pale bulky
and semi solid.
(c) Colour: Normal stools are light to dark brown in colour due to the
presence of stercobilinogen which is a product of bilirubin metabolism.
In cases with bleeding into the intestinal tract the stools become dark tarry
in nature due to the formation of acid hematin if the bleeding is in the Notes
small intestines. In the bleeding in large intestines or rectum the blood may
be bright red. In cholera the stools have a rice water appearance as there
is no fecal matter and there is presence of flakes of epithelial cells in it.
In biliary tract obstruction the stools may be clay coloured due to absence
of stercobilinogen. Pateints diet may also lead to alteration in the colour
of the stools. For instance if the patient had spinach earlier the stools may
be green in colour. In those who had a barium examination the stools may
be white in colour.
(d) Odour: The fecal odour of stools may become offensive in conditions like
intestinal amoebiasis. In cases of bacillary dysentery and cholera the stools
are not foul smelling due to the absence of fecal matter.
(e) Blood: Blood should be noted in stools if present as it is indicative of

ulceration or presence of any other pathology like malignancy. It should
also be noted if the blood is bright red or is altered in colour as it may
be a clue to the site of pathology in the intestinal tract.
(f) Mucus: Mucus is present in certain conditions like amoebic or bacillary

dysentery.
(g) Parasite: Stools may contain adult helminthes. Nematodes like ascaris are
easily visible as their size is large. Hook worms and proglotids of cetodes
may also be present. These may be visible to the naked eye.
50.3 MICROSCOPIC EXAMINATION

The laboratory diagnosis of most parasitic infections is by the demonstration of
ova of the parasite in the stools of the infected person. The stool is collected in
a clean container. The stool can be examined by the following techniques.
(a) Wet mount examination

(b) Iodine preparation
MICROBIOLOGY 435
Microbiology (c) Buffered methylene blue stain: (Nuclear stain in E histolytica)

(d) Cellophane tape test:- NIH swab
(e) Concentration techniques
(a) Saline wet mount examination: The stool is emulsified in normal saline
and a large drop is placed on a glass slide and is then covered with a cover
slip. This is then examined under a light microscope. It is preferable to
Notes keep the condenser down and the intensity of the light low for proper
visualization of the ova and cysts. The thickness of the film should be such
that one is able to see the printed letters of the newspaper through it.
(b) Iodine Preparation: Iodine preparation leads to better visualization of
morphological details of ova and cysts as it stains the glycogen in them.
It however has the disadvantage that the live trophozoites of Entamoeba
histolytica cannot be seen as the iodine kills it. One gram of iodine and
two gram of potassium iodide is mixed in 100 ml of distilled water
Poassium iodide is mixed in water and then the iodine crystals are added
and it is shaken vigorously.. The solution is then filtered into a dark glass
bottle and kept away from light.
(c) Buffered methylene blue stain: It is used for the Nuclear stain in E
histolytica
(d) Cellophane tape test: NIH swab (National Institute of Health) is used to
pick up the ova of Entrobius vermicularis from the peri -anal area.
(e) Concentration methods:
Two types of concentration techniques are used for stool examination
(i) Sedimentation
(ii) Floatation
(i) Sedimentation technique
– Formol ether technique
– Formol ether SAF
– Formol ether PVA
(ii) Floatation technique
– Zinc sulfate
– Saturated salt solution
A small amount of stools are emulsified in saturated salt solution in a wide mouth
container. When the stool becomes homogenous a few drops of saturated saline
436 MICROBIOLOGY
are mixed and more saturated saline solution is added. A glass slide is placed Microbiology
across the receptacle in such a way that the slide is in contact with the surface
of the saturated solution. If the fluid is not in touch with the slide then more
saturated saline should be added till the fluid level touches the slide. The slide
is then left in place for fifteen minutes. After this the slide is gently lifted off
the container and turned upside down carefully ensuring no fluid from the slide
is spilled. A cover slip is placed over the fluid on the slide. And it is examined
Notes
under a microscope.
Principle of floatation technique: The specific gravity of ova and cysts is less
and thus will float to the top of the saturated salt solution where it will stick to
the under surface of the glass slide.
50.4 CHEMICAL EXAMINATION OF STOOL

(a) pH: This pH of stools is acidic in amoebic dysentery and is alkaline in
bacillary dysentery.
(b) Occult blood: Occult blood may be present in a number of diseases
including malignancy of the gastrointestinal tract. The reagent used is
benzidine powder. A pinch of benzidine powder is taken in a test tube and
acidified with 1-2 drops of glacial acetic acid and is mixed well in it. To
this is added 1ml of hydrogen peroxide which is again mixed well. Then
place a clean glass slide and place a small quantity of stool on it. Place
1-2 drops of the benzidine mixture prepared earlier on the stool specimen
taken on the glass slide and observe for a change of colour. Development
of green to blue colour is indicative of presence of occult blood in the stool
specimen.
50.5 PRESERVATION OF STOOL

The stool samples containing the ova and cysts of parasites can be preserved by
using one of the following methods
– 10% formol saline

– Buffered formol saline
– Merthiolate-iodine formalin
– Sodium acetate-acetic acid formalin(SAF)
– Polyvinyl alcohol (PVA)
– Schauddins preservative
MICROBIOLOGY 437
Microbiology
Notes
Fig. 50.1: Morphology of some common helminthic ova
Fig. 50.2: Common helminth ova
438 MICROBIOLOGY
Microbiology

1. .................. stain is used for nuclear stain in E.histolytica
2. .................. swab is used in cellophane tape test
3. Cellophance test is used to identify ..................
4. Two concentration techniques for stool examination are .................. & Notes
..................

z Stool examination is carried out in laboratories for various diagnostic
purposes
z A clean container which does not contain any detergent or disinfectant is
sufficient for all types of stool examinations including stool culture
z Physical Examination of the stool for the following aspects of stool should
be examined for its Quantity, Consistency and form, Colour, Odour, Blood,
Mucus, Parasite
z The laboratory diagnosis of most parasitic infections is by the demonstration
of ova of the parasite in the stools of the infected person.
z The stool can be examined by the following techniques namely Wet mount
examination, Iodine preparation, Buffered methylene blue stain, Cellophane
tape test:- NIH swab and Concentration techniques
TERMINAL QUESTIONS
1. Describe the techniques used for examination of stool
2. Describe the concentration method of stool examination
50.1
1. Buffered methylene blue
2. NIH
3. Entrobius vermicularis
4. Sedimentation & Floatation
MICROBIOLOGY 439
MODULE Morphology and General Properties of Fungi
Microbiology
51
Notes
MORPHOLOGY AND GENERAL
PROPERTIES OF FUNGI
51.1 INTRODUCTION
Fungus is a member of a large group of eukaryotic organisms that includes
microorganisms such as yeasts and molds (British English: moulds), as well as
the more familiar mushrooms. These organisms are classified as a kingdom,
Fungi, which is separate from plants, animals, protists and bacteria. One major
difference is that fungal cells have cell walls that contain chitin, unlike the cell
walls of plants and some protists, which contain cellulose, and unlike the cell
walls of bacteria. These and other differences show that the fungi form a single
group of related organisms, named the Eumycota (true fungi or Eumycetes), that
share a common ancestor (is a monophyletic group). This fungal group is distinct
from the structurally similar myxomycetes (slime molds) and oomycetes (water
molds). The discipline of biology devoted to the study of fungi is known as
mycology. Mycology has often been regarded as a branch of botany, even though
it is a separate kingdom in biological taxonomy. Genetic studies have shown that
fungi are more closely related to animals than to plants.
Fungi are not able to ingest their food like animals do, nor can they manufacture
their own food the way plants do. Instead, fungi feed by absorption of nutrients
from the environment around them. They accomplish this by growing through
and within the substrate on which they are feeding. Numerous hyphae network
through the wood, cheese, soil, or flesh from which they are growing. The
hyphae secrete digestive enzymes which break down the substrate, making it
easier for the fungus to absorb the nutrients which the substrate contains.
This filamentous growth means that the fungus is in intimate contact with its
surroundings; it has a very large surface area compared to its volume. While this
makes diffusion of nutrients into the hyphae easier, it also makes the fungus
susceptible to dehydration and ion imbalance. But usually this is not a problem,
since the fungus is growing within a moist substrate.
440 MICROBIOLOGY
Morphology and General Properties of Fungi MODULE
Most fungi are saprophytes, feeding on dead or decaying material. This helps Microbiology
to remove leaf litter and other debris that would otherwise accumulate on the
ground. Nutrients absorbed by the fungus then become available for other
organisms which may eat fungi. A very few fungi actively capture prey, such
as Arthrobotrys which snares nematodes on which it feeds. Many fungi
are parastitic, feeding on living organisms without killing them. Ergot, corn
smut, Dutch elm disease, and ringworm are all diseases caused by parasitic
fungi. Notes
OBJECTIVES
z describe the morphology of fungi

z explain the physiology of fungi
z classify fungi
z describe the colony morphology
z describe the pathogenecity of fungi
51.2 MORPHOLOGY OF FUNGI

(a) General. Fungi vary widely in size and shape, from unicellular, microscopic
organisms to multicellular forms easily seen with the naked eye. Individual
cells range from 1 µ to 30 µ. Microscopic fungi exist as either molds or
yeasts or both. Internally, fungal cells are fairly typical eucaryotic cells.
(b) Molds. The molds form large multicellular aggregates of long branching
filaments, called hyphae. There are vegetative hyphae and reproductive
hyphae. Spores are borne on the reproductive hyphae. (Fungal spores
should not be confused with bacterial spores that are resistant bodies
formed for bacterial survival rather than reproductive purposes.) Spore
size, shape and structure are used in the classification and identification
of fungi. The tube-like hyphae are responsible for the fluffy appearance
of the macroscopic mold colony. The hyphae and other structures combine
to form an elaborate network called a mycelium.
(c) Yeasts. These are large (5 to 8 µ), single-celled organisms that rarely form
filaments. Most yeasts reproduce by the asexual process of budding. Yeast
colonies are usually characterized by a smooth surface similar to that of
many bacteria.
MICROBIOLOGY 441
Microbiology
51.3 PHYSIOLOGY OF FUNGI
(a) Nutrition. Most fungi contain complex enzymes and other chemical
substances which, when diffused into the host, break down the complex
substances available – wood, vegetation, leather, bread, and so forth – into
simpler substances that can be used for food. The chemical products of
digestion are, therefore, completed outside of the organism, and the fungus
absorbs the end products.
Notes
(b) Reproduction. Fungi reproduce sexually or asexually, or both, depending
upon the species and the environmental conditions. As the name implies,
sexual reproduction is the result of the union of two spores. Most fungi
reproduce both sexually and asexually. Those that produce only asexual
spores are known as Deuteromycetes Fungi imperfecti. This group is
important because it contains most of the pathogenic fungi. The yeasts
reproduce both by spores and by a process known as budding, which is
similar to binary fission. The yeast cell forms a small knoblike protrusion,
or bud (Fig. 51.1), that separates from the mother cell and grows until it
reaches full size, at which time the process is repeated.
(c) Growth. Fungi grow well under the same conditions that favor the growth
of bacteria – warmth and moisture. It is for this reason that fungal infections
pose a serious problem to troops in the tropics. As the temperature
decreases, fungal activity also decreases; however, the spores are very
resistant to cold, some surviving freezing temperatures for long periods
of time. On the other hand, fungi are easily killed at high temperatures.
Fig. 51.1: Typical mycelium of a fungus.
51.4 CLASSIFICATION OF FUNGI

Fungi are usually classified according to biological taxonomy based upon the
type of hypha, spore, and reproduction. There are four classes of fungi, whose
characteristics are shown in Table 51.1 and figure 51.2.
(a) Class Phycomycetes. The algal fungi: bread molds and leaf molds. The
only known mycosis (fungal disease) caused by fungi of this class is
mucormycosis, a very rare fungal growth of the upper respiratory tract,
442 MICROBIOLOGY
bronchial mucosa, and lungs. It occurs largely as a complication of a Microbiology
chronic, debilitating disease, such as uncontrolled diabetes.
(b) Class Ascomycetes. The sac fungi: yeasts, mildews, and cheese molds.
Fungi of this class are implicated in only three fungus diseases, all of which
are rare.
(c) Class Basidiomycetes. Mushrooms, toadstools, rusts, and smuts. The only
pathogens in this class are the mushrooms of the genus Amanita, which
cause severe systemic poisoning (sometimes death) when eaten. Notes
(d) Class Deuteromyceters. Fungi imperfecti: a heterogeneous collection of
fungi without sexual reproduction. Most of the pathogens encountered in
medical mycology belong to this class.

1. Study of fungi is ................
2. Fungi are fed by ................ of nutrients from the environment around them
3. ................ of fungi secrete enzymes which helps in absorption of nutrients
4. Fungi that produce asexual spores are known as ................
5. ................ morphology is used to describe the characteristics of fungal
colony
Table 51.1 Characteristics of Fungi
Taxonomic Hypha Type of Characteristic Origin of Examples Pathogenicity
class of Fungi Reproduction spore Spore of Fungi
Phycomycetes Asptate Asexually Sporangio- Sporangio Nuisance Very rare

spore phore fungi Mucormycosis
Sexually Zygospore Fussion of including
or oospore nuclei general
Absidia,
Muclor,
and
Rhizopus
Ascomycetes Septate Asexually Blastospore Budding Allescheria Rare

Conidium Conidio- Aspergillus Maduromcosis
phore Piedraia Aspergillosis
Sexually Ascospore Ascus Saccharomyces Black Piedra

(perfect yeast)
Basidiomycetes Septate Sexually Basidio-spore Basidium Mushrooms, Rare

smuts and Mushroom
rusts poisoning
Deutero- Septate Asexually Thallospore Thallus Most Most Mycoses

mycetes (hypha) saprophytes encountered
{fungi and pathogens in medical
imperfecti) Conidium Conidio- encountered mycology
phore in medical
mycology
(Imperfect
mold and
yeast)
MICROBIOLOGY 443
Microbiology
Notes
Fig. 51.2
Colony Morphology
Colony morphology is a method that scientists use to describe the characteristics
of an individual colony of fungi growing on agar in a Petri dish. It can be used
to help to identify them.
444 MICROBIOLOGY
Microbiology
Notes
Different types of fungi will produce different-looking colonies, some colonies

may be coloured, some colonies are circular in shape, and others are irregular.
A specific terminology is used to describe common colony types. These are:
z Form - What is the basic shape of the colony? For example, circular,
filamentous, etc.
z Size – The diameter of the colony. Tiny colonies are referred to as
punctiform
z Elevation - This describes the side view of a colony. Turn the Petri dish
on end.
z Margin/border – The edge of a colony. What is the magnified shape of the
edge of the colony?
z Surface - How does the surface of the colony appear? For example, smooth,
glistening, rough, wrinkled, or dull.
z Opacity - For example, transparent (clear), opaque, translucent (like looking
through frosted glass), etc.
z Colour - (pigmentation) - For example, white, buff, red, purple, etc.
Yeast colonies are very similar to bacterial colonies.
Moulds often have fuzzy edges. They usually turn into a different colour, from
the centre outwards.
51.5 PATHOGENIC FUNGI

(a) Fungal infections are of two types: localized skin infections
(dermatomycoses), and systemic infections. Although the former are far
MICROBIOLOGY 445
Microbiology more common, the latter generally have more serious consequences. Table
51.2 lists the more common fungus diseases and the important etiological
agents in each. Note that frequently more than one species of organism
may cause identical symptoms.
Table 51.2
Disease Synonym or Brief Important Etiological
Notes Description Agents
Cutaneous and Superficial Mycoses
Tinea capitis’ Ringworm of the scalp Microsporum spp

Trichophyton spp
Tinea corporis Ringworm of the body Same as Tinea capitis
Tinia barbae Infection of bearded area of Trichophyton spp

face and neck
Tinea cruris Ringworm of the groin Trichophyton spp

(jock itch) Candida albicans
Epidermophyton floccosum
Tinea pedis Ringworm of the feet Same as Tinea cruris

(athlete’s foot)
Tinea versicolor Depigmented, scaly patches Malassezia furfur

of skin
Otomycosis Fungus infection of the Aspergillus spp

(aspergillosis) ear canal
Cutaneous Yeast infection of nails, skin Candida albicans and other

Candidiasis mouth,Vagina species
(moniliasis,
thrush)
Mycetoma Tumor-like swelling, draining Pseudallescheria boydii and

abscess other
Actinomycosis Chronic, suppurative or Actinomyces israelii

granulomatous disease of [actuallyclassified asbacteria,
jaw,thorax, or abdomen but causefungus-like infections]
Subcutaneous and Systemic Fungus Infections

Nocardiosis Infection of lungs, otherorgans, NocardIa asteroids[actually
and lowerextremities (Madura classified asbacteria, but cause
foot) fungus-like infections]
Chromoblastomycosis Warty nodules or vegetations N. brasIlIensIs
of skinand subcutaneous tissues Cladosporium carrionii
Sporotrichosis Ulcers of skin and underlying Fonsecaea pedrosoi
tissues and gumma-likeswelling
of regional lymph nodes.
Blastomycosis Inflamatory lesions of the skin, Phialophora verrucosa
lungs,or bones. Sporot schenkii
446 MICROBIOLOGY
Coccidioidomycosis Self-limited respiratory disease Blastomyces dermatltidis Microbiology
orchronic progresssive infection
of various organs
Histoplasmosis Fungus infection of the lungs, Coccidioides immitis

with fever; anemia; loss of Histoplasma capsulatum
weight,enlargement of lymph
nodes, liver,spleen
Cryptococcosis Systemic fungus infection of Cryptococcus noeformans

lungs or meninges
Notes
(b) Diseases caused by fungi are collectively called mycoses (singular,

mycosis). They are divided into four general categories on the basis of the
primary tissue affected by the pathogen:
1. Superficial mycoses are infections limited to the hair and dead layers
of the skin.
2. Cutaneous mycoses (dermatophytoses or ringworm) affect only the
skin, hair, and nails.
3. Subcutaneous mycoses affect the subcutaneous tissue below the skin
and occasionally bone.
4. Systemic (“deep”) mycoses infect the internal organs and may spread
throughout the host
(c) Those fungi infecting the outer layers of the skin are rarely severe and are
usually transmitted by contact with infected animals or humans. The agents
of subcutaneous and systemic mycoses, however, are normally saprophytic
fungi growing in the soil. Humans generally acquire these mycoses only
when the spores of these organisms are either inhaled or introduced into
the body through a break in the skin.
(d) Some fungi incapable of causing infectious diseases produce toxic substances
that poison the person who ingests them. These substances are collectively
called mycotoxins. The most commonly known mycotoxin poisoning is
from certain mushrooms; however, mycotoxins may be produced by fungi
growing on grain, nuts, and other agricultural products.
Medically Important Fungi

An accurate taxonomic scheme of the major fungal pathogens and contaminants
encountered in medicine is not presented here; instead, a simpler but perhaps
more useful organization will be applied. Morphology is particularly helpful in
speciating filamentous fungi and Pneumocystis carinii (see Other fungi, below),
but may also play a role in identifying certain yeasts.
MICROBIOLOGY 447
Microbiology Zygomycetes
Notes
Zygomycetes are a class of fungi that have characteristically broad, usually

aseptate hyphae; zygomycosis is usually characterized by opportunism,
invasiveness, and involvement of nasal cavity, paranasal sinuses, and orbit with
invasion into the brain (so-called rhinoorbitocerebral mucormycosis), involvement
of the lungs, gastrointestinal tract, or skin (the latter especially in burn patients),
and occasional dissemination. Its hyphae are generally broader than the
hyalohyphomyces and they lack the pigment found in dematiaceous fungi. Their
gross appearance is characterized by rapid, plate-covering growth.
Hyalohyphomyces
Hyalohyphomyces are a large, heterogeneous group of fungi characterized by
narrow, septate hyphae that are colorless on microscopic examination. This is
a morphologically diverse group.
Dematiaceous fungi
Dematiaceous fungi are a large, heterogeneous group of fungi characterized by
dark colonies grossly and pigmented fungal elements seen on microscopic
examination of involved biopsy material.
448 MICROBIOLOGY
Dermatophytes Microbiology
Fungi infecting stratum corneum, hair, and nails. Grossly, colonies often display
fluffy or fine texture and are pale colored or white. Grow moderately rapidly to
slowly and have narrow, septate hyphae.
Notes
Dimorphic fungi
Fungi that characteristically grow as a mold under certain environmental
conditions (usually 25-30°C) and as a yeast under other conditions (usually at
35-37°C). Medically important dimorphic fungi can be highly pathogenic;
special caution is warranted when handling fungal cultures largely because of
the risk of culturing one of these organisms.
Yeasts
Yeasts are unicellular fungi that reproduce by budding (with rare exceptions).
Unlike many of the other fungi presented here, biochemical tests and carbohydrate
or nitrate assimilation are disproportionately important for identification.
MICROBIOLOGY 449
Microbiology

1. Disease caused by fungi are collectively called ....................
2. Localised fungal infections are called as ....................
3. Unicellular fungi that reproduce by budding are ....................
Notes
4. Involvement of nasal cavity, paranasal sinuses with invasion to brain is
called ....................

z Fungus is a member of eukaryotic organisms which includes yeasts and
molds
z Scientific study of fungi is known as mycology
z Fungi feed by absorption of nutrients from environment
z Hyphae secrets digestive enzymes which breaks down the substrate and
makes it easier for fungus to absorb the nutrients
z Fungi vary widely in size & shape from unicellular microscopic organism
to multicellular organism
z Spore size, shape & structure are used in the classification & identification
of fungi
z Hypae and other structures form mycelium
z Fungi reproduces sexually and asexually
z Fungi that produce asexual spore are Deuteromycetes fungi imperfecti
z Fungi are classified based upon hypae, spore and reproduction
z Colony morphology are used to describe individual colony of fungi
z Fungal infections are localised skin infections and systemic infections
z Mycoses are diseases caused by fungi
TERMINAL QUESTIONS
1. Describe the morphology of fungi
2. Explain the physiology of fungi
3. Classify fungi
450 MICROBIOLOGY
Microbiology
51.1
1. Mycology
2. Absorption
3. Hypae Notes
4. Deuteromycetes fungi imperfecti
51.2
1. Mycoses
2. Dermatomycoses
3. Yeast
4. Rhinoorbitocerebral mucormycosis
MICROBIOLOGY 451
MODULE Laboratory Diagnosis of Fungi
Microbiology
52
Notes
LABORATORY DIAGNOSIS
OF FUNGI
52.1 INTRODUCTION
We have learned in earlier chapters about various fungal infections. This chapter
deals with the diagnosis and in particular laboratory diagnosis of fungal infection
As processed with bacterial infection, laboratory diagnosis of fungal infection
starts with appropriate specimen collection & transport. And in most fungal
infections the identifications are based primarily on the assessment of colony
morphology & microscopic features. Key biochemical tests may be required to
differentiate between the genes & species. Also molecular techniques like
Nucleic acid probe assays are being used with increased frequency to provide
early confirmation in suspected cases of deep seated mycoses. Serological
studies are required in some instances to establish differential diagnosis. Non
culture methods & automated system too are available for diagnosis of fungal
infections.
OBJECTIVES
z list the steps involved in the diagnosis of fungal infection
z describe the Specimen collection and transport
z explain the Direct examination and mount preparation
z describe the Selection & innoculation of culture media
z explain Incubation of fungal cultures
z describe the Presumptive diagnosis of fungal isolates
452 MICROBIOLOGY
Laboratory Diagnosis of Fungi MODULE
Microbiology
52.2 SPECIMEN COLLECTION AND TRANSPORT
For laboratory diagnosis of fungal infections various specimens can be received
in the laboratory; Physicians, Nurses, ward personnel & Laboratory technologists
needs to work together in developing protocols that ensure the proper collection
and prompt collection of specimen.
Notes
Fig. 52.1: Sterile container for collection of specimen for fungal culture
The selection of appropriate collection devices & transport containers, labeling

of the specimen & complete requisition forms are important considerations in
ensuring the correct diagnosis of fungal infections as followed in Table 1
Table 52.1: Transport condition for diagnosis of fungal infection
Specimen Transport condition
Sputum Sterile Screw capped container
Bronchoscopy Fluid Sterile Screw capped container
CSF If delay anticipated, specimen should be left at
room temperature
Urine If delay beyond 2hrs is anticipated, refrigerate
at 4°C
Blood Biphasic agar broth bottles designed especially
for fungal cultures
Tissue biopsy the specimen should not be frozen or allowed to
dehydrate prior to culture
In all cases the specimen should be transported as early as possible to the
laboratory. In general the specimen that are not processed immediately are held
at room temperature (for urine if delay more that 2 hrs refrigerate at 4°C).
Cryptococcus neoformans, cystoplasma capsulatum & Blastomyces dermatitidis
do not survive well in frozen or iced specimen.
MICROBIOLOGY 453
Microbiology
Criteria for specimen rejections
1. Absence of patient identification on the container or discrepancy between
the information
2. Sputum specimen with >25 squamous epithelial cells as per low power
field
3. A dried out swab or if the material collected is insufficient
Notes
4. The sample submitted in an improper container
5. The 24hr sputum or urine specimen for fungal culture is received
52.3 SPECIMEN PROCESSING

On receiving the specimen, it should be promptly processed. The direct wet
mounts or smears are prepared and for culture the specimen is inoculated on
culture media.
52.3.1 Direct Examination

Almost all the specimens are processed for direct microscopic examination. This
provides the presumptive diagnosis for the physician and also aid in the selection
of appropriate culture media.
Various methods for direct examinations are
z Direct wet mount of specimen
z India Ink
z KOH/calcoflurol mounts
z Lactophenol cotton blue (LPCB) mounts
z Frozen section of tissue biopsies
z Modified Kinyoun Acid Fast Stain for Nocardia
Direct Microscopic Observations Presumptive Identification

Hypae relatively small (3-6 µm) and Aspergillus spp
regular in size, dichotomously
branching at 45° angles with distinct
cross-septa
Hypae irregular in size (6-50 µm), Zygomycetes (Phycoycetes)
ribbonlike, and devoid of septa. rhizopus-Mucor
Hypae small (2-3 µm) and regular, Dermatophyte group
some branching with rectangular Microsporum spp
arthrospores sometimes seen, found Trichophytoon spp
only in skin, nail scrapings and hair Epidermophyton spp
454 MICROBIOLOGY
Microbiology
Hyphae regular in diameter (3-6 µm), Phaeohyphomyces spp
parallel walls, irregular branching, Hyalohyphomyces spp
septate, dark yellow, brown or hyaline.
Hyphae, distinct points of constriction Candida spp

simulating link sausages (pseudohyphae),
with budding yeast forms (blastospores)
often seen. Notes
Yeast forms, cell spherical and irregular Cryptococcus neoformans
in size (5-20 µm) classically with a Cryprococcus spp, nonencapsualted
thick polysaccharide capsule (not all
cells are encapsulated), with one or
more buds attached by a narrow
constriction
Small budding yeast, relatively uniform Histoplasma capsulatum

in size (3-5 µm) with a single bud
attached by a narrow base,
extracellular or within macrophages
Yeast forms, large (8-20 µm) with cells Blastomyces dermatitidis

appearing to have a thick, double-
contoured wall, with a single bud
attached by a broad base
Large, irregularly sized (10-50 µm) thick Coccidiodes immitis

walled spherules, many of which contain
small (2-4 µm) round endospores
52.3.2 Preparation of Mounts

The tease mount, transparency tape method and microslide techniques are
commonly used methods for microscopic examination.
The mold colony is mounted in a drop of Lactophenol cotton blue stain on a glass
slide and examined microscopically
The specimen are directly mounted in 40% & 10% KOH for skin and nail
specimens respectively. The skin and nail samples are mounted on the glass slide
to which two drops of KOH preparation is added and kept for sometime. KOH
helps in dissolving the epithelial cells and thus aid in fungal visibility.
MICROBIOLOGY 455
Microbiology
Notes
Fig. 52.2: KOH mount of infected skin scales showing typical dermatophyte
hyphae breaking up into arthroconidia.
India Ink - India ink can be added to specimens such as spinal fluids or exudates
to provide a dark background that will highlight hyaline yeast cells and capsular
material (halo effect). Hence, it should be used to examine specimens suspected
of containing Cryptococcus neoformans. White blood cells may be distinguished
from Cryptococcus neoformans because of the irregular edge of the halo and the
pale cell wash. The India ink preparation is not routinely offered by the
laboratory. If a request is received for it, the laboratory should call the physician
and offer a Cryptococcus Antigen Test instead. The procedure will be performed
only in particular instances with the approval of the director or supervisor.
Fig. 52.3: Cryptococcus neoformans using a light India ink staining preparation
Gram Stain
Gram stain is usually a poor stain to use when examining a specimen for a
fungus. Gram stain may be used when examining smears of Candida, Malassezia,
and Sporothrix but should not be relied upon to demonstrate the yeast of the other
dimorphic fungi. A gram stain will demonstrate the filaments of Nocardia and
Actinomyces which may produce clinical signs resembling mycotic infections.
456 MICROBIOLOGY
Microbiology
Notes
Fig. 52.4: Gram stain of Candida albicans
Modified Kinyoun Acid Fast Stain for Nocardia

a. Make a thin smear of the specimen to be stained; fix in methanol. A positive
control smear (Nocardia asteroides) and a negative control smear
(Streptomyces sp.) must be included.
b. Kinyoun carbolfushcin; 5 minutes, no heat.
c. Rinse with water.
d. 50% ethanol rinse; flood and pour off until excess carbolfuchsin is removed.
e. Rinse with water.
f. Decolorize with 0.5% (aqueous) H SO ; 3 minutes. 2 4
g. Rinse with water
h. 1% (aqueous) methylene blue; 1 minute.
i. Rinse with water.
Fig. 52.5: Modified AFB staining for Nocardia spp.
MICROBIOLOGY 457
Microbiology Selection and Inoculation of culture media

Generally two types of culture media are used, nonselective (such as brain heart
infusion heart) it permits growth of virtually all clinically relevant fungi. The
use of sabourauds dextrose agar as primary recovery medium is discouraged as
it is insufficiently rich to recover certain fastidious pathogenic species,
particularly dimorphic fungi. Rather, the use of Potato flake agar (PFA),
inhibitory mold agar (IMA), or combination of sabouraud’s dextrose agar with
heart infusion (SABHI) agar is recommended.
Notes
Sabouraud’s agar is sufficient for the recovery of dematophytes from cutaneous
samples or yeasts from vaginal culture. Czepak’s agar can be used for the
subculture of aspergillus species if colony morphology is an important identifying
criteria for any given unknown isolate. For more fastidious dimorphic fungi such
as Blastomyces dermatiditis & soistoplasma capsulatum an enrich agar like IMA
or SABHI is used and in particular for Histoplasma capsulatum media with the
addition of 5-10% sheep blood is recommended. Cryptococcus neoformans,
aspergillus fumigatus may be partially or totally inhibited by cycloheximide,
therefore a nonselective media must always be used in parallel.
Fig. 52.6: Uninoculated Sabouraud’s agar
Incubation of fungal culture

Each sample is cultured in two set of culture media and is incubated at two
different temperatures at 30oC (Room temperature) and at 35oC
All fungal cultures are incubated for a minimum of 30 days before discarding
as negative.
The choice between the use of culture tubes or plate is optional. For tube, the
media is poured in thick slants to prevent dehydration during prolonged
incubation period. After the medium is inoculated, do not screw down the cap
too tightly because fungi require breathing.
458 MICROBIOLOGY
Culture media in tube have advantage of ease of transport, while limitation is Microbiology
difficult to prepare stained mounts for microscopic examination and petridishes
have the advantages of providing larger surface for growth resulting in better
colony separation and making the cultures easier to examine and sub culture.
Also tease mounts or transparency tape preparations are effectively made from
plate cultures. The disadvantage being the plates may become dehydrated during
prolonged incubation to prevent drying the plates may be placed into a sealed,
moisturized polyester bad or the edges are sealed by oxygen permeable tape. Notes
52.4 LABORATORY APPROACH TO PRESUMPTIVE

IDENTIFICATION OF FUNGI
After the culture plates reveal that growth of probable fungi, the identification
of the colonies is done by the characteristic colony morphology. Also a LPCB
mount is prepared from the growth and observed under microscope for the
details.
Fig. 52.7: LPCB mount of Aspergillus Spp.
Colonies with smooth, creamy, viscous or pasty appearance, a yeast must be

considered. Dematiaccous molds produces colonies that are dark. Gray to black
mycelium growth and reverse of the colony is black. For molds that grow within
3-5 days have a distinct border, and are white or patel on the surface. For molds
that grow in 7-14 days or that have a cobweb aerial mycelium, one of the
dimorphic species should be considered. See the table for details.
52.5 AUTOMATED SYSTEMS

The only current commercially available system is the applied Biosystems,
Microseq OZ large subunit r DNA fungal sequencing kit. Micro scan also offer
candida species identification kit but not for other fungi isolates.
MICROBIOLOGY 459
Microbiology
52.6 MOLECULAR TECHNIQUES
Nucleic acid probe assays are being used with increasing frequency to provide
early culture confirmation, especially in deep mycoses infection.
Looking on to future, it must be mentioned that nucleic acid sequencing has

become the standard method for fungal identification especially in reference
laboratories
Notes

1. Urine specimen if delayed in transportation to laboratory needs to be
refrigerated at .................. °C
2. CSF should be stored at .................. temperature if delay in transportation
to laboratory is anticipated
3. Tissue biopsy should be .................. before sending it to laboratory
4. Fungal visibility is aided by adding .................. preparation
5. .................. preparation may be used to examine specimens suspected of
Cryptococcal Infections
6. .................. culture media is used for diagnosis of fungi
7. All fungal cultures are incubated fro a minimum of .................. days before
confirming as negative.

z Appropriate specimen collection play a major role in laboratory diagnosis
of fungal infection. The samples are collected in appropriate manner and
in appropriate container and are transported to laboratory promptly. If delay
is anticipated, the relevant consideration are observed. The first step in the
processing starts with the direct microscopic examination of the specimen.
In case of skin 40% KOH and for nail sample 10% KOH is used to dissolve
the epithelial cells. After the direct examination the specimens are cultured
on appropriate culture media and they are incubated at 300c and 350c.
Separately they are observed regularly for growth and negative report is
dispatched not before 30 days of complete incubation.
460 MICROBIOLOGY
z The identification of isolated fungi is basically based on the morphology Microbiology
of the colony. Also LPCB mount of the colony is prepared to observe the
microscopic features. In some case biochemical test’s may be required to
differentiate the genes and species. Also nucleic acid probe assays are being
used and also serologic studies are required in some instances to establish
definitive clinical diagnosis.
Notes
TERMINAL QUESTIONS
1. Enlist the steps involved in the laboratory diagnosis of Fungi.
2. Describe the specimen collection and transport
3. Why we do not refrigerate the specimen in case of anticipated delay
4. Describe the methods of direct examination of fungiPresumptive diagnosis
of Fungal infection
52.1
1. 4
2. Room
3. Frozen
4. KoH
5. Indian Ink
6. Brain heart infusion
7. 30
MICROBIOLOGY 461
HYALINE MOLD COLONIES DEMATIACEOUS YEAST COLONIES YEAST-LIKE
COLONIES
Growth < 3days Growth 3-5 days Growth 3-5 days Growth > 5 Growth 3-5 days Growth > 5 days Growth 2-5 days Growth 2-5 days
days
Hyphae broad and Hyphae hyaline and septate Colonies often Hyphae Dark colony, black Dark colony, black Smooth, pasty or Yeastlike colonies
aseptate granular and reverse, hyphae yellow- reverse, hyphae macoid colonies with low aerial
pigmented, hyphae pigmented and septate yellow-pigmented mycelium
septate, hyaline and septate
Suspect Zygomyces Suspect agents of Suspect Suspect Suspect Agent of Suspect Agent of Suspect Yeast Anthrocomidia
Rhizopax Hyalohyphomycocisis Dermatophyte Dimorphic Pharophyomycosis Chromomycosis or Common: Produced
Abxidia Conidia in Chains: Genus Microsporisia fungi Conidia Muriform: mycetoma Cadida albicans Suspect:
Syncephalasirum Aspergillas common: Shermaria Caldosprium-Type Candida Geatrichum candidum
Circinella Pencillium Microsparum Ulocladium Sporulation: Cryptococcus Trichosporom beigelil
Cunninghamella Paecilomyces ………….. Steaphylium Clodophtalophora neoformans coraplex
Mucor Scopulanopsis Uncommon: Epicoccum carrunil Cryptococcus Blastoschizomyces
Conidia in Clusters Microsporium Cladophiolophora Rhodotorida capitus
Acremonium ……….. Conidia divided by bantianum Uncommon:
Fusarium Microsporum adnum Transverse Septa Phialophora – type Hansenula anomala
Trichoderma Genus Trichophytom only: Sporulation: Malasseria furfur
Gliocladium common: Curvularia Phialophora Malassezia furfur
Conidia Borne Singly: Trichophytom rabum Bipolaris (Drechslera) verrucosa Saccharomyces
Scedosporium Trichophyton Exterohilum Phialophora cerevisiae
apiospermum mentogrophytes Pycnindia produced richardsiae Rare:
Scedosporium Prolifercans Trichophyton Phoma Exophiala jeanselmei Basidiobolus species
(inflatum) tonsutum Chaetomium Acrotheca-Type Conidiobolus species
Chrysosporium Trichophyton sporulation: Black yeasts:
Sepedonium Verracosum Fonsecaea pedrosoi Pullalaria pullalans
Uncommon: Fonsecaea compacta Phaeococcomyces
Trichophytom species
Violaceum Yea st forms of
Trichophyton dimorphic fungi
Schoenleinii
Genus
Epidermophyton:
Epidermophyton
Floccosum
Morphology and General properties of Viruses MODULE
Microbiology
53
Notes
MORPHOLOGY AND GENERAL
PROPERTIES OF VIRUSES
53.1 INTRODUCTION
Viruses occupy the twilight zone that separates the ‘living’ from the ‘non-living’.
They do not have a cellular organization and contain only one type of nucleic
acid, either DNA or RNA but never both.
The medical importance of viruses lies in their ability to cause a very large
number of human diseases. Viral diseases range from minor ailments like
common cold to terrifying diseases like rabies and AIDS.
In this chapter, we shall be discussing the morphology and general properties
of viruses.
OBJECTIVES
z explain the concept of viruses, in relation to other microorganisms
z describe the morphological features of viruses
z explain the multiplication of viruses (replication)
z describe the methods of cultivation of viruses
z explain the classification and naming (nomenclature) of viruses
53.2 SECTION
53.2.1 Concept of Viruses in relation to other Organisms
Viruses occupy the twilight zone that separates the ‘living’ from the ‘non-living’.
They do not have a cellular organization and contain only one type of nucleic
MICROBIOLOGY 463
MODULE Morphology and General properties of Viruses
Microbiology acid, either DNA or RNA but never both. Viruses are obligate intracellular
parasites. They lack the enzymes necessary for protein and nucleic acid
synthesis. They are dependent for replication on the synthetic machinery of host
cells. They multiply by a complex process and not by binary fission. They are
unaffected by antibacterial antibiotics. The differences between viruses and
bacteria are shown in Table 53.1.
Viruses cause a wide range of human diseases. They cause infections like
Notes common cold, chicken pox, measles, viral encephalitis, rabies and AIDS.
Table 53.1: Differences between bacteria and viruses

Properties Bacteria Viruses
Cellular organization Present Absent
Growth on inanimate media Yes No
Binary fission Yes No
DNA and RNA Both are present Either DNA or RNA
Ribosomes Present Absent
Sensitivity to antibacterial Yes No
antibiotics
53.2.2 Morphology of Viruses

Size: The extracellular infectious virus particle is called virion. Viruses are much
smaller than bacteria. They are too small to be seen under the light microscope.
Some large viruses like the poxviruses can be seen under the light microscope
when suitably stained.
The viruses range in size from 20 nm to 300 nm. Poxviruses are one of the largest
viruses and parvoviruses are one of the smallest viruses. The earliest method of
estimating the size of virus particles was by passing them through collodion
membrane filters of graded porosity. The average pore diameter of the finest
filter that permitted passage of the virion gave an estimate of its size. With the
development of the ultracentrifuge, a second method became available. From
the rate of sedimentation of the virus in the ultracentrifuge, the particle size could
be calculated using Stoke’s law. The third and the most direct method of
measuring virus size is electron microscopy. By this method, both the shape and
size of virions can be studied.
Structure, shape and symmetry: The virion consists essentially of a nucleic
acid surrounded by a protein coat, the capsid. The capsid with the enclosed
nucleic acid is called the nucleocapsid. The capsid protects the nucleic acid
from harmful agents in the environment. It is composed of a large number of
capsomers which form its morphological units. The chemical units of the capsid
are polypeptide molecules which are arranged symmetrically. They form a shell
around the nucleic acid.
464 MICROBIOLOGY
The capsid shows two kinds of symmetry – icosahedral (cubical) and helical. Microbiology
An icosahedron is a polygon with 12 vertices and 20 facets or sides. Each facet
is in the shape of an equilateral triangle. Two types of capsomers are present in
the icosahedral capsid. They are the pentagonal capsomers at the vertices
(pentons) and the hexagonal capsomers making up the facets (hexons). There
are always 12 pentons but the number of hexons varies with the virus group.
Examples of viruses with icosahedral symmetry of the capsid are Adenovirus
and Herpes Simplex Virus. In the nucleocapsids with helical symmetry, the Notes
capsomers and nucleic acid are wound together to form a helical or spiral tube,
for example tobacco mosaic virus. All viruses do not show the typical
icosahedral or helical symmetry. Some, like the poxviruses, show a complex
symmetry.
Virions may be enveloped or nonenveloped. The envelope of viruses is derived
from the host cell membrane. This occurs when the virus is released from the
host cell by budding. Protein subunits may be present as projecting spikes on
the surface of the envelope. They are called peplomers. The influenza virus
carries two kinds of peplomers: haemagglutinin and neuraminidase.
Haemagglutinin is a triangular spike and neuraminidase is mushroom-shaped.
Envelope is sensitive to the action of lipid solvents. Envelopes confer chemical,
antigenic and biological properties on viruses.
The overall shape of the virus particle varies in different groups of viruses. Most
animal viruses are roughly spherical. The rabies virus is bullet shaped.
Poxviruses are brick-shaped.
Chemical properties: Viruses contain only one type of nucleic acid, either DNA
or RNA. Viruses are unique because they carry genetic information on RNA.
This property is not seen in any other organism in nature. Viruses also contain
protein which makes up the capsid. Enveloped viruses contain lipids derived
from the host cell membrane. Most viruses do not have enzymes for the synthesis
of viral components or for energy production. Some viruses have enzymes, for
example the influenza virus has neuraminidase.
Resistance: Viruses are destroyed by heat except a few. They are stable at low
temperatures. For long term storage, they are kept at -70°C. A better method for
prolonged storage is lyophilisation or freeze-drying. Viruses are inactivated by
sunlight, UV rays and ionising radiation. They are, in general, more resistant than
bacteria to chemical disinfectants. Phenolic disinfectants have a weak action on
viruses.
53.2.3 Multiplication of Viruses

Multiplication of viruses is called viral replication. Viruses contain the genetic
information for their replication but they lack the enzymes. They depend on host
cell machinery for replication. The viral replication cycle can be divided into six
MICROBIOLOGY 465
Microbiology phases – adsorption, penetration, uncoating, biosynthesis, maturation and

release.
Adsorption: In this phase, the virus gets attached to the host cell. The host cell
should have specific receptors on its surface. These receptors recognize viral
surface components. This cell-virus interaction helps the virus to attach to the
host cell surface.
Notes Penetration: In this phase, the virus enters into the host cell. Bacteria have rigid
cell wall. So, viruses which infect bacteria cannot penetrate into the bacterial
cell. Only the nucleic acid of the virus enters the bacterial cell. Animal and
human cells do not have cell walls. Therefore, whole virus enters the cell. Virus
particle may be engulfed by a process called viropexis. In case of enveloped
viruses, the viral envelope may fuse with the cell membrane of the host cell. Then
the nucleocapsid is released into the cytoplasm.
Uncoating: This is the process in which the outer layers and capsid of the virus
are removed. This mostly occurs by the action of lysosomal enzymes of the host
cell. This can also occur by a viral uncoating enzyme. Finally, the viral nucleic
acid is released into the cell.
Biosynthesis: In this phase, the viral nucleic acid and capsid are synthesised.
The enzymes necessary in the various stages of viral synthesis, assembly and
release are also synthesised. Certain ‘regulator proteins’ are synthesised. They
shut down the normal metabolism of the host cell. They direct the production
of viral components. In general, most DNA viruses synthesise their nucleic acid
in the host cell nucleus. Exceptions are the poxviruses. They are DNA viruses,
but they synthesise all their components in the host cell cytoplasm. Most RNA
viruses synthesise all their components in the cytoplasm. Orthomyxoviruses and
some paramyxoviruses are exceptions. They synthesise some components in the
host cell nucleus. Biosynthesis consists essentially of the following steps:
1. Transcription of messenger RNA (mRNA) from the viral nucleic acid
2. Translation of mRNA into “early proteins” or “non-structural proteins”.
They are enzymes responsible for the synthesis of viral components.
3. Replication of viral nucleic acid
4. Synthesis of “late proteins” or “structural proteins”. They are the components
of daughter virion capsids.
Maturation: This is the assembly of daughter virions following the synthesis
of viral nucleic acid and proteins. It can take place in the host cell nucleus or
cytoplasm. Herpesviruses and adenoviruses are assembled in the nucleus.
Picornaviruses and poxviruses are assembled in the nucleus.
Release: Viruses which infect bacteria (bacteriophages) are released by lysis of
the infected bacterium. Animal viruses are usually released without cell lysis.
466 MICROBIOLOGY
Myxoviruses are released by budding from the cell membrane. The host cell is Microbiology
unaffected. Daughter virions are released into the surrounding medium and may
infect other cells. In some viruses (for eg. varicella), transmission occurs directly
from cell to cell. In this case, there is very little free virus in the medium. The
poliovirus causes cell damage and may be released by cell lysis.
From the stage of penetration till the appearance of mature daughter virions, the
virus cannot be demonstrated inside the host cell. During this period, the virus
seems to disappear. This is called the “eclipse phase”. The time taken for a single Notes
cycle of replication is about 15-30 minutes for bacteriophages. It is about 15-
30 hours for animal viruses. A single infected cell may release a large number
of progeny virions.
53.2.4 Methods of cultivation of viruses

Viruses are obligate intracellular parasites. They donot grow on culture media
used for bacteria. The methods used for cultivation of viruses are:
1. Animal inoculation: Monkeys were used for the isolation of the poliovirus
by Landsteiner and Popper in 1909. Infant mice are used for the isolation
of coxsackievirus and arboviruses (dengue, chikungunya). Mice may be
inoculated by several routes – intracerebral, subcutaneous, intraperitoneal
or subcutaneous. Other animals like guinea pigs, rabbits and ferrets are used
in some situations.
2. Embryonated eggs: The embryonated hen’s egg was first used for the
cultivation of viruses by Goodpasture in 1931. This method was further
developed by Burnet. Different parts of the egg are used for the cultivation
of different viruses. Herpes simplex virus, when inoculated into the
chorioallantoic membrane, produces visible lesions called pocks. Inoculation
into the amniotic sac is done for the isolation of influenza virus. Yolk sac
inoculation is done for the isolation of rabies virus.
3. Cell culture: Probably, the first application of tissue culture in virology was
by Steinhardt and colleagues in 1913. They maintained the vaccinia virus
in fragments of rabbit cornea. The turning point was the cultivation of
poliovirus which was demonstrated by Enders, Weller and Robbins in 1949.
They showed that poliovirus, till then considered a strictly neurotropic virus,
could be grown in tissue culture of non-neural origin.
The various types of tissue cultures are described as follows:

(i) Organ culture: Small bits of organs can be maintained in vitro for days
and weeks, preserving their original architecture and function. Organ culture
is useful for viruses which are highly specialised parasites of certain organs.
For example, tracheal ring organ culture is used for the isolation of
coronavirus, a respiratory pathogen.
MICROBIOLOGY 467
Microbiology (ii) Explant culture: Fragments of minced tissue can be grown as ‘explants’
embedded in plasma clots. They may also be cultivated in suspension.
Adenoid tissue explant cultures were used for the isolation of adenovirus.
(iii) Cell culture: This is routinely used for growing viruses. Tissues are
dissociated into the component cells by the action of enzymes and
mechanical shaking. The cells are washed, counted and suspended in a
growth medium. The growth medium consists of essential amino acids,
Notes glucose, vitamins, salts and a buffer. Antibiotics are added to prevent
bacterial contamination. The cell suspension is put into bottles, tubes and
petridishes. The cells adhere to the glass or plastic surface, divide and form
a confluent monolayer sheet within a week. Cell culture is further classified
on the basis of origin, chromosomal characters and the number of generations
through which they can be maintained. It is of three types – primary cell
culture, diploid cell strain and continuous cell lines. Primary cell cultures
are normal cells freshly taken from the body and cultured. They are capable
of only limited growth in culture. They cannot be maintained in serial
culture. Examples are monkey kidney, human embryonic kidney and chick
embryo cell cultures. Diploid cell strains are cells of a single type that retain
the original diploid chromosome number and karyotype during serial
subcultivation for a limited number of times. After about fifty serial
passages, they undergo ‘senescence’. Diploid strains developed from human
fibroblasts are a good example. Continuous cell lines are cells of a single
type, usually derived from cancer cells. They are capable of continuous
serial cultivation indefinitely. Hela cell lines are derived from carcinoma
of the cervix. Cell culture is used for the isolation of viruses and their
cultivation for vaccine production.
Viruses in cell cultures can be detected by various methods like cytopathic effect,
special staining techniques and detection of viral nucleic acid by molecular
techniques like polymerase chain reaction (PCR). Cytopathic effect is the
morphological change in the cultured cells which is produced by the virus
growing in those cells. These changes can be seen by microscopic examination
of the cell cultures. The cytopathic effects (CPE) produced by different groups
of viruses are characteristic and help in the presumptive identification of virus
isolates. For example, measles virus produces syncytium formation; adenovirus
produces large granular clumps resembling bunches of grapes; enteroviruses
produce crenation of cells and degeneration of the entire cell sheet.
53.2.5 Classification and naming of viruses

Till about 1950 little was known of the basic properties of viruses. They were
named haphazardly, based on the diseases they caused or on the place of their
isolation. They were grouped according to affinity to different systems or organs
468 MICROBIOLOGY
of the body (tropism). So, human viruses were classified as dermotropic, that Microbiology
is those producing skin lesions (smallpox, chickenpox, measles), neurotropic,
that is those affecting the nervous system (poliomyelitis, rabies), pneumotropic,
that is those affecting the respiratory tract (influenza, common cold) and
viscerotropic, that is those affecting visceral organs (hepatitis). Bawden (1941)
made the pioneering suggestion that viral nomenclature and classification
should be based on the properties of viruses and not upon host responses. From
the early 1950s, viruses began to be classified into groups based on their Notes
physiochemical and structural features. Nomenclature and classification are now
the official responsibility of the International Committee on Taxonomy of
Viruses (ICTV).
Viruses are classified into two main divisions based on the type of nucleic acid
they possess: riboviruses contain RNA and deoxyriboviruses contain DNA.
Further classification is based on other properties like strandedness of nucleic
acid, symmetry of nucleic acid, presence of envelope, size and shape of virion
and number of capsomeres.
DNA viruses: A few medically important families of DNA viruses are -
Herpesviridae, Adenoviridae, Hepadnaviridae, Parvoviridae and Papillomaviridae.
The Herpesviridae family consists of enveloped double-stranded DNA viruses
having an icosahedral capsid. Examples of this family are herpes simplex virus
and varicella zoster virus. Herpes simplex virus causes skin lesions like herpes
labialis. It can also cause viral encephalitis. Parvoviridae consists of non-
enveloped single-stranded DNA viruses, for example Parvovirus B19. The
Hepadnaviridae family includes Hepatitis B virus which is a partially double-
stranded DNA virus. Papillomaviridae family includes human papilloma virus
which is responsible for causing skin warts.
RNA viruses: Some medically important families of RNA viruses are –
Picornaviridae, Orthomyxoviridae and Paramyxoviridae, Flaviviridae,
Rhabdoviridae and Retroviridae. Members of the family Picornaviridae are
small (20-30 nm), non-enveloped, icosahedral viruses with single-stranded RNA
genome. Examples include poliovirus and coxsackievirus. The viruses included
in Orthomyxoviridae are enveloped viruses carrying haemagglutinin and
neuraminidase peplomers on the envelope. The genome consists of single-
stranded RNA in several (eight) pieces. Thus, they have a segmented genome.
An example of this family is influenza virus. Flaviviridae consists of enveloped
single-stranded RNA viruses. Examples include yellow fever virus, Japanese
encephalitis virus and dengue virus. The members of Retroviridae family are
enveloped RNA viruses which have a special enzyme called ‘reverse transcriptase’.
This enzyme is an RNA dependent DNA polymerase. It is required in the
synthesis of DNA from RNA. An example of the Retroviridae family is Human
Immunodeficiency Virus (HIV) which causes AIDS (acquired immunodeficiency
syndrome).
MICROBIOLOGY 469
Microbiology Based on the mechanism of replication, Baltimore (1970) categorised viruses

into seven categories. This is called the Baltimore classification.

1. The genetic material in viruses is:
Notes
A. DNA only B. RNA only
C. Either DNA or RNA D. Both DNA and RNA
2. Protein subunits presenting as projecting spikes on the surface of the
envelope are called:
A. Capsomeres B. Capsid
C. Nuceocapsid D. Peplomers
3. Which of the following is the correct sequence of viral replication?
A. Penetration, uncoating, adsorption, biosynthesis, maturation and release
B. Adsorption, penetration, uncoating, biosynthesis, maturation and release
C. Biosynthesis, penetration, uncoating, adsorption, maturation and release
D. Adsorption, biosynthesis, maturation, uncoating, penetration and release
4. Methods used for viral cultivation are:
A. Cell culture B. Animal inoculation
C. Embryonated eggs D. All of the above
5. Baltimore classified viruses on the basis of:
A. Diseases caused by them B. Structure
C. Replication mechanism D. Physiochemical properties

z Viruses do not strictly fall into the category of unicellular organisms
because they do not have a cellular organisation.
z Viruses are obligate intracellular parasites which contain only one type of
nucleic acid (DNA or RNA). They are dependent on the synthetic
machinery of the host cell for replication.
z The extracellular infectious virus particle is called the virion. The viruses
are smaller than bacteria ranging in size from 20-300 nanometers.
470 MICROBIOLOGY
z The virion consists of a central nucleic acid core surrounded by a protein Microbiology
coat called capsid. Nucleocapsid consists of the capsid enclosing the
nucleic acid core.
z The capsid protects the nucleic acid from inactivation and is made up of
a large number of capsomeres.
z The capsid may have icosahedral, complex or helical symmetry.
z The virions may be enveloped or non-enveloped. The envelope is a
Notes
lipoprotein. Protein subunits which occur as projecting spikes on the
envelope surface are called peplomers as seen in influenza virus
(haemagglutinin and neuraminidase).
z Enveloped viruses are susceptible to organic solvents. The envelope helps
the virus in attachment to the host cell surface.
z Most animal viruses are roughly spherical; some are irregular and
pleomorphic. Some have distinctive shapes like bullets (rabies) and bricks
(poxviruses).
z Most viruses are inactivated by heat, in seconds at 56ºC. Viruses are
inactivated if stored for several days at 4 ºC, but survive storage at -70
ºC. Viruses are inactivated by sunlight, ultraviolet rays and ionising
radiation. Viruses are easily destroyed by chemical disinfectants like
chlorine, hydrogen peroxide and hypochlorite.
z Viral multiplication consists of six sequential phases – adsorption,
penetration, uncoating, biosynthesis, maturation and release from the host
cell.
z Cultivation of viruses is important for the diagnosis of viral infections and
the production of vaccines. Viruses can be cultivated by animal inoculation,
inoculation into embryonated eggs and tissue culture. Tissue culture
consists of organ culture, explants culture and cell culture. Cell culture is
further classified into primary cell culture, diploid cell strains and continuous
cell lines. This classification is based on the origin of cells, their
chromosomal characters and the number of generations through which they
can be maintained.
z Viruses in cell cultures can be identified by cytopathic effects (CPE),
special staining techniques and molecular techniques like PCR.
z Viruses were previously classified on the basis of their affinity towards
different systems or organs. Recently, they have been classified on the basis
of their physiochemical properties and structure. The International
Committee on Taxonomy of Viruses (ICTV) is responsible for the
classification and naming of viruses.
z Viruses are broadly classified into DNA and RNA viruses.
z Baltimore (1970) classified viruses on the basis of their replication
mechanisms.
MICROBIOLOGY 471
Microbiology
TERMINAL QUESTIONS
1. Describe the differences between bacteria and viruses.
2. Describe the morphology of viruses under the following headings –
structure, shape and symmetry.
Notes 3. Describe the methods of viral cultivation.
4. Describe the various types of tissue cultures with suitable examples.
5. Define Cytopathic effect (CPE). Give suitable examples of viruses showing
CPE.
6. Enumerate three DNA viruses and three RNA viruses alongwith the disease
caused by each one of these
7. Give stepwise detailed description of viral replication.
53.1
1. C. Either DNA or RNA
2. D. Peplomers
3. B. Adsorption, penetration, uncoating, biosynthesis,maturation and
release
4. D. all of the above
5. C. Replication mechanism
472 MICROBIOLOGY
Laboratory Diagnosis of Viral Infections MODULE
Microbiology
54
Notes
LABORATORY DIAGNOSIS OF
VIRAL INFECTIONS
54.1 INTRODUCTION
Viruses are infective organisms which are responsible for many diseases in
humans. Since viruses cannot be grown in artificial media & the tissue culture
techniques may require longer periods, serodiagnosis of viral infections is the
mainstay of diagnostic virology. Along side other techniques like direct
detections of virus in clinical specimens & cultivation of viruses are also
practiced. The development of technologies like PCR (Polymerase Chain
Reaction) have provided an effective alternative for diagnosing viral infection
OBJECTIVES
z describe the importance of specimen collection
z explain the Methods to preserve and transport the sample
z describe the Various methods of isolation of viruses.
z explain the Molecular techniques used in diagnosis of viral infections.
Lab diagnosis of viral infection begins from the very step of collection of
specimen.
54.2 SPECIMEN COLLECTION

Attempts should be made to obtain material from the organs that are infected
eg. Skin for cutaneous lesions, direct secretions from respiratory & Gastro
intestinal tract that are required in case of respiratory or GI system involvement.
In fact since most viruses enter the body through the respiratory or GI tract, the
MICROBIOLOGY 473
MODULE Laboratory Diagnosis of Viral Infections
Microbiology most obvious & appropriate specimen is respiratory tract secretions or the GI
tract secretions. In case of internal organ involvement where direct organ
specimen is difficult to obtain, sampling from multiple sites seems useful. Eg.,
in case of measles, Skin is most dramatically involved. Yet measles virus may
be isolated from respiratory tract or from urine. Similarly Central Nervous
System & Cardio Vascular System are commonly affected in serious viral
infections, but the virus may be isolated from the upper respiratory tract or
Notes Gastro Intestinal Tract.
The optimal specimens for viral culture are aspirates of fluids, exudates or
secretion, tissues, washings of upper air ways or stool specimens, swab specimen
are acceptable in most situations. Nasopharyngeal washings are generally
sufficient for respiratory viruses. Blood may be useful for entero viral infections
in young children & infants.
To culture vesicular skin lesions the skin should be cleaned with an alcohol swab
and allowed to dry for at least one minute. The vesicle should then be unrolled
with a sterile scalpel, a sterile swab touched several times to the base.
As a general rule the frequency with which virus is recovered decreases as the
duration of illness increases and every effort should be made to obtain specimen
as early in the infection as possible.
54.3 TRANSPORTATION & STORAGE OF SPECIMEN

All the samples should be transported to laboratory in a sterile, leak proof
container. Interval between collection of the specimen & its inoculation should
be minimal. In case of some fragile viruses for eg RSV virus inoculation of cell
cultures at the bed side has been recommended.
Swabs should be placed in viral transport media swabs, any swabs are not
acceptable.
Specimens are refrigerated till they are further processed. The specimens should
be maintained at -70oC if they are to be stored for a very long time (weeks or
months). When the delay is short (less than 24 hours) specimens are stored at
4oC temperature.
Fig. 54.1: Transport medium
474 MICROBIOLOGY
Appropriate specimen collected from patients, preserved and transported to the Microbiology
laboratory in the proper manner along with relevant clinical & epidemiological
information. (Table 54.1)
Table 54.1
System Specimen Required
For Isolation For direct examination Notes
Respiratory Throat swab, Throat Nasopharygeal aspirate
washings,Aspirates
Central Nervous system Faeces, Blood, CSF Brain biopsy, CSF
Cardio Vascular System Faeces, Macular Vesicular/pustular fluid,
popular scrapings, Ulcer scraping.
ulcer scrapings, throat
swab
Eye Conjuctival scraping Conjuctival scraping/
and swabs. swads
Liver Blood Serum
Congenital infections Throat swab, product NIL
of conception

In the laboratory the following methods are commonly employed, microscopic
demonstration of the virus or its inclusion body. Demonstration of virus antigen,
or detection of the specific antibody & molecular techniques.
54.4.1 Microscopy
It includes
1. Detection of viral inclusion body by light microscopy
2. Detection of virus or viral particles with the help of electron microscope.
Light Microscopy
Light Microscopy has been traditionally used in directly demonstrating viral
infections by detecting the viral inclusion body in smear & tissue.
Inclusion bodies are dense aggregates of stainable substances, usually proteins.
They can be either intra nuclear (present inside the nucleus) of infected well or
intracytoplasmic (present inside the cytoplasm of the infected cell). Viruses that
MICROBIOLOGY 475
Microbiology are assembled in the nucleus (usually DNA viruses) like herpes simplex viruses,
Varicella zoster virus, Cytomegalo virus, adeno virus & papova viruses forms
intranucleus inclusions. While viruses that are assembled in the cytoplasm
(usually RNA viruses) like Respiratory syncytial virus, rabies virus & viruses
of the pox group forms intra cytoplasmic inclusion bodies. The intracytoplasmic
lesions of rabies virus are known as Negri bodies, intracytoplasmic inclusions
of the pox viruses known as Guarneri bodies
Notes
Fig. 54.2: Negri bodies
Electron Microscopy
Electron Microscopy is used in the study of clinical specimens & cell cultures.
Electron microscope has been used effectively in the detection of viral agent of
gastro enteritis especially those that are not recovered by conventional cell
cultures. Electron microscopes are useful in ultra structural observations
especially for research purposes.
The diagnosis of important viral infections, such smallpox, can be made quickly
& safely with help of Electron microscopy.
54.4.2 Demonstration of virus antigen

Direct detections of virus antigen can be done in cases where the antigen is
abundant in the lesions. It is demonstrated by serological methods such as
precipitate in gel or immunofluorescence.
Counter immune electrophoresis, radio immune assay & Enzyme Linked
immunosorbent assay have found wide application in diagnostic virology for the
detection of viral antigens in clinical samples.
Molecular methods like polymerase chain Reaction (PCR), Real time polymerase
reaction (RT-PCR), Nucleic acid. Sequence based Amplification (NASBA),
Transcription Mediated Amplification (TMA), etc provide rapid sensitive and
476 MICROBIOLOGY
specific techniques for detections of viral antigens. They are fast becoming Microbiology
routine diagnostic methods in developed countries.
54.4.3 Isolation of Virus

The method used for isolation of virus depend upon the virus sought. In general
they consist of innoculation into animals, eggs or tissue culture. After that
isolates are identified by neutralization or other suitable serological process. Notes
Isolation of virus from patient does not always means that it is the causative agent
of the patients illness. Many viruses like adeno viruses, entero virus etc. are
frequently found in normal individuals. Thus it is imperative to interpret the
isolation of virus in light of clinical data.
54.4.3.1 Egg inoculation

Embryonated eggs are among the most useful form of living animal tissue for
the isolation of viruses, for titrating viruses & for large quantity cultivation in
the production of viral vaccines.
Fig. 54.3
The embryonated egg has various sites namely choriallantric membrance,

Allantoic membrance, Amniotic sac. Yolk sac & the embryo proper. Different
virus grow at different above mentioned sites (see table)
Site Virus grown
Chorioallartic membrane Variola. Vaccinia
Allantoic membrane Influenza paramyxo virus
Amniotic sac Influenza virus
Yolk sac Chlamydia Rickettsiae
MICROBIOLOGY 477
Microbiology 54.4.3.2 Animal Inoculation

It is the earliest method for the cultivation of viruses causing human diseases.
Reed & colleagues F in 1900 used human volunteers for their work on yellow
fever. In 1909, Landsteiner & Popper used monkeys to isolate Polio virus.
Theiter used white mice which extended the scope of animal inoculation greatly.
Rabbit, Guinea pig, mouse, rat are now commonly used in virus cultivation.
Notes It needs to be noted that different animals are used for different viruses & also
there exists different routes to inoculation. The growth of virus in inoculated
animals may be indicated by death, disease or visible lesions. Disadvantages of
animal inoculation are that they may interfere with viral growth and that animals
often harbour latent viruses.
Fig. 54.4: mouse
Animal inoculation in particular is useful in the study of pathogenesis, immune

response, epidemiology & oncogenesis.
54.4.3.3 Tissue culture

Tissue culture is often a generic term. Three types of tissue cultures are available
a. Organ culture
Small bits of organs are maintained in vitro for days & weeks. They are
used for cultivation of viruses which can be grown only in specific organ.
b. Explants culture
Fragments of minced tissues is grown as explants embedded in plasma clot.
Eg. Adeno tissue explants for adenovirus.
c. Cell culture
It is routinely used based on their origin. Chromosomal characters & the
number of generation through which they can be maintained, cell cultures
are classified into 3 types
478 MICROBIOLOGY
1. Primary cell culture Microbiology
It consists of normal cells freshly taken from body & cultured. They are
capable of limited growth in culture & cannot be maintained in serial
culture. Eg. Monkey, kidney, human embryonic kidney, human amnion &
chick embryo cell culture. They are useful for isolation of viruses & for
vaccine production.
2. Diploid cell culture Notes
These are used to ensure a continuous supply of cell line. After 50 serial
passages, they undergo senescence. They are useful in viral vaccine
production. Eg. Human fibroblasts
3. Continuous cell culture
These are single type usually derived from cancer cells that are capable of
continous serial cultivation indefinitely. These cells lines many be maintained
by serial sub-cultivation or stored in the cold (-70o C) for use when necessary.
They are useful for vaccine production. Eg. Verocell for rabies vaccine, Hela,
HEp-2, KB
Fig. 54.5: Cytopathic effect. The cytopathic effect was apparent as refractile, rounded,
swollen, and semiattached cells, and areas of clearance were compared to those
of the uninfected control cells
The growth of virus in the cell culture can be detected by following methods
i. Cytoplasmic effect
ii. Metabolic inhibitors
iii. Hemadsorption
iv. Interference
v. Transformation
vi. Immunofluorescence
MICROBIOLOGY 479
Microbiology
Notes
Fig. 54.6: Indirect immunofluorescence microscopy performed on chicken sera with a

neutralization titre of 1/80 (diluted 1:1000 in PBS) on West Nile virus infected Vero cells
54.5 SEROLOGICAL DIAGNOSIS

It refers to diagnosis of disease based on reactions in the blood serum. The mere
presence of an antibody to a virus is not sufficient to diagnose a viral infection.
It only denotes that the person’s immune system has been exposed to the viral
antigen, besides a current infection by the virus, the antibody may have been
formed by the following
z A previous symptomatic / asymptomatic infection
z In response to cross reacting antigen and
z Vaccination
In all these cases the level of the antibody will remain near the same for the entire
duration of current illness. However, if the current illness is caused by the virus
then the antibody levels would rise during the course of the disease therefore,
the rule of “four fold rise” in the titre of antibodies is applied. It means that when
serum samples are tested from an individual taken at an interval of ten to fourteen
days there should be a fourfold increase of antibody tire, to be a indicative of
current infection with the particular virus
Variety of methods is available to diagnose viral infection by antibody detection
the choice of the method depends upon the sensitive of the test and intensity of
immune response. Various tests available are ELISA, complement fixation test,
neutralization, hemagglutination tests.
54.6 MOLECULAR TECHNIQUES

With development in molecular technology diagnosis of viral infections is
becoming rapid & specific. Various molecular technique which can be used in
480 MICROBIOLOGY
diagnosis of viral infections are Microbiology
1. Nucleic acid sequence based amplification (NASBA)

2. Transcription mediated amplification
3. Polymerase chain reaction
4. Real time polymerase chain reaction
Notes
Fig. 54.7: RT PCR System
The advantages of molecular techniques are that, they are rapid and are highly
sensitive & specific. The only disadvantage is that it requires technical expertise
and are resource intensive.

1. The specimen collected in case of measles is ...................
2. If delay in transport of specimen is anticipated, specimens to be stored at
................... temperatures
3. Microscopically ................... & ................... are detected
4. Intracytoplasmic lesion of rabies virus is known as ...................
5. Intracytoplasmic lesion of pox virus is known as ...................
MICROBIOLOGY 481
Microbiology

z Viruses are infective organisms which are responsible for many diseases in
humans.
z As viruses cannot be grown in artificial media & the tissue culture
Notes techniques may require longer periods, serodiagnosis of viral infections is
the mainstay of diagnostic virology
z Attempts should be made to obtain material from the organs that are infected
z The optimal specimens for viral culture are aspirates of fluids, exudates or
secretion, tissues, washings of upper air ways or stool specimens, swab
specimen are acceptable in most situations.
z As a general rule the frequency with which virus is recovered decreases as
the duration of illness increases and every effort should be made to obtain
specimen as early in the infection as possible.
z All the samples should be transported to laboratory in a sterile, leak proof
container. Interval between collection of the specimen & its inoculation
should be minimal.
z In the laboratory the following methods are commonly employed, microscopic
demonstration of the virus or its inclusion body, Demonstration of virus
antigen, or detection of the specific antibody & molecular techniques.
z Direct detections of virus antigen can be done in cases where the antigen
is abundant in the lesions. It is demonstrated by serological methods such
as precipitate in gel or immunofluorescence.
z The method used for isolation of virus depend upon the virus sought. In
general they consist of innoculation into animals, eggs or tissue culture
z Tissue culture is often a generic term. Three types of tissue cultures are
available namely Organ culture, Explants culture, Cell culture
z Cell culture are of three types namely Primary cell culture, Diploid cell
culture, Continuous cell culture
z Variety of methods is available to diagnose viral infection by antibody
detection.
z The choice of the method depends upon the sensitivity of the test and
intensity of immune response.
z Various tests available are ELISA, complement fixation test, neutralization,
hemagglutination tests.
482 MICROBIOLOGY
z Various molecular techniques which can be used in diagnosis of viral Microbiology
infections are Nucleic acid sequence based amplification (NASBA),
Transcription mediated amplification (TMA), Polymerase chain reaction
(PCR) and Real time polymerase chain reaction (RT-PCR)
TERMINAL QUESTIONS Notes

1. Enlist the various specimen which can be used for diagnosis of viral
infection
2. Describe in brief the precaution one needs to take while preserving and
transporting the specimen
3. Describe in brief various methods used for isolation of virus
4. Describe in brief the serological methods used in viral infection
5. Enlist the molecular techniques used for diagnosis of viral infections
54.1
1. Skin
2. 4oC
3. Viral inclusion & Viral particles
4. Negri bodies
5. Gaurneri bodies
MICROBIOLOGY 483
MODULE Immunity
Microbiology
55
Notes
IMMUNITY
55.1 INTRODUCTION
The term “immunity” is referred to the resistance of an individual towards injury
caused by microorganisms and their products.
Immunity against infections is of different types which will be dealt in detail in
following sections.
OBJECTIVES
z describe the concepts of innate immunity and acquired immunity
z list the types of innate immunity and acquired immunity
z explain the mechanism of innate immunity
z explain the differences between active and passive immunity
55.2 INNATE IMMUNITY : RESISTANCE TO

INFECTIONS THAT AN INDIVIDUAL POSSESSES
DUE TO HIS GENETIC MAKEUP
Nonspecific immunity – It indicates a degree of resistance to infections in
general.
z Species immunity – Refers to the resistance to a pathogen shown by all
members of a species for example all human beings are totally unsusceptible
to plant pathogens.
z Racial immunity – Different races within a species, may show differences
in susceptibility to infections for example in some parts of Africa resistance
to Plasmodium falciparum malaria is seen.
484 MICROBIOLOGY
Immunity MODULE
z Individual immunity – It is the difference in innate immunity shown by Microbiology
different individuals within a race. Several factors such as age, hormones
and nutrition influence the level of innate immunity in an individual.
Age: The very young and very old are more prone to infectious diseases than
the rest. The fetus in utero is normally protected by placental barrier. But some
pathogens cross this barrier. Some, such as rubella virus, herpes viruses,
cytomegaloviruses and parasite Toxoplasma gondii, can lead to congenital
Notes
infections. The increased susceptibility of the fetus to infection is due to
immaturity of its immune system.
Old persons are more susceptible to infections due to their waning immune
responses and other factors, like enlarged prostate leading to stasis of urine.
Hormonal influences: Endocrine disorders like diabetes mellitus are associated
with increased risk of infections. The increased risk may be related to increased
levels of carbohydrate in tissues.
Corticosteroids depress the individuals resistance by its anti inflammatory and
anti phagocytic effects. The effect of stress in increasing susceptibility to
infections may be due to release of steroids.
Nutrition: Both cell mediated and antibody mediated immune responses are
depressed when there in malnutrition. Cell mediated immune responses such as
Mantoux test for tuberculosis becomes negative in severe protein deficiency, as
in kwashiorkor.
Specific immunity – It refers to the resistance to a particular pathogen.
z Species
z Racial
z Individual
55.3 MECHANISM OF INNATE IMMUNITY

Epithelial surfaces: The intact skin and mucous membrane covering the body
protect it against invasion of microorganisms.
The bactericidal activity of skin secretions can be illustrated by frequent fungal
and bacterial infections in individuals who immerse their hands in soapy water
for long periods of time occupationally for example washer men.
The mucosa of respiratory tract has several innate mechanism of defence. The
structure of nose prevents entry of microorganism to a large extent, inhaled
particles being arrested at or near nasal orifices. Those that pass beyond are held
by mucous lining the epithelium, and are swept back to mouth were they tend
MICROBIOLOGY 485
MODULE Immunity
Microbiology to be swallowed or coughed out. The cough reflex is an important defence

mechanism. The cilia on the respiratory epithelial cells propel particles upwards.
Nasal and respiratory secretions contain substances that combine with influenza
and some other viruses. Particles that manage to reach alveoli are ingested by
alveolar macrophages.
The mouth is continuously bathed by saliva which has inhibitory effect on many
microorganisms. Particles deposited in the mouth are swallowed and subjected
Notes
to action of digestive juices and high acidity of stomach. The intestinal mucosa
is covered by lacelike network of mucus. Particles get trapped in the mucus and
form small masses which are propelled by peristalsis.
The conjunctiva is freed of foreign particles by flushing action of lachrymal

secretions. The eye becomes susceptible to infection when lachrymal secretions
are absent. Tears contain antibacterial substance lysozyme, which acts on cell
walls of susceptible bacteria.
The flushing action of urine eliminates many bacteria from the urethra. Zinc
present in semen has antibacterial activity. The acidity of adult vagina makes it
resistant to many pathogens.
Antibacterial substances in blood and tissues: Blood components like

complement system possesses bactericidal activity. Several substances like beta
lysine, leukins, plakins in blood and lactic acid in muscle tissue, possess
antibacterial properties.
A method of defence against viral infections is the production of interferon by

cells stimulated by live or killed viruses.
Microbial antagonisms: The skin and mucous membrane have resident

bacterial flora which prevents colonisation by pathogens. Alteration of normal
resident flora may lead to invasion by pathogenic microorganisms, causing
serious diseases such as staphylococcal intestinal infections following oral
antibiotics.
Cellular factors in innate immunity: Natural defence against the invasion of

blood and tissues by microorganisms and other foreign particles is mediated to
a large extent by phagocytic cells which ingest and destroy them. Phagocytic
cells are macrophages and neutrophils. Macrophages consists of histiocytes in
tissues, the reticuloendothelial cells and monocytes in blood. Phagocyte reach
the site of inflammation in large numbers attracted by chemotactic substances,
and ingest particulate material. Microorganisms are more readily phagocytosed
when trapped against a firm surface like alveolar wall than when they are free
in tissue fluids. Bacteria are phagocytosed into a vacuole (phagosome), which
486 MICROBIOLOGY
Immunity MODULE
with lysosomes to form phagolysosomes. The bacteria are subjected to the action Microbiology
of the lytic enzymes in the phagolysosomes and are destroyed. Some bacteria
like lepra bacilli resist intracellular digestion and may multiply inside phagocytes.
A class of lymphocytes called natural killer (NK) cells are important in defence
against viral infections and tumours.
Inflammation: Tissue injury or irritation initiated by the entry of pathogens or
other irritants, leads to inflammation. The arterioles at the site constrict initially Notes
and then dilate leading to an increase in blood flow. There is a slowing of blood
flow and margination of the leucocytes, which escape into the tissues and
accumulate in large numbers, attracted by the chemotactic substances released
at the site of injury. Microorganisms are phagocytosed and destroyed.
Fever: A rise of temperature following infection is a natural defence mechanism.
Therapeutic induction of fever was used in syphilis patients before use of
penicillin.
Acute phase proteins: Infection or injury leads to a sudden increase in the
plasma concentration of certain proteins, collectively called acute phase
proteins. These include C reactive protein (CRP). They are believed to enhance
host resistance, prevent tissue injury and promote repair of inflammatory lesions.
Section 1.02 Acquired immunity: The resistance that an individual

acquires during life is known as acquired immunity.
Acquired immunity is of two types:
Active immunity
z Natural
z Artificial
Passive immunity
z Natural
z Artificial
Active immunity is the resistance developed by an individual as a result of an
antigenic stimulus.
z This involves the active functioning of the host’s immune apparatus leading
to the synthesis of antibodies and the production of immunologically active
cells.
z Active immunity sets in after a latent period which is required for the
immunological machinery to sets in motion.
MICROBIOLOGY 487
MODULE Immunity
Microbiology z Once developed active immunity is long standing.

z If an individual who has been actively immunised against an antigen is
exposed to same antigen again, the immune responses occur quickly and
abundantly than during the first encounter. This is known as secondary
response.
z Active immunity is associated with immunological memory. This means
Notes that the immune system is able to retain for long periods the memory of
prior antigenic exposure.
z Active immunity confers better protection then passive immunity.
Active immunity can be natural or artificial. Natural active immunity results
from either a clinical or an inapparent infection by a microorganism. Such
immunity is usually long lasting but the duration varies with the type of
pathogen.
z Immunity is lifelong following viral diseases like measles and chickenpox.
z In influenza immunity is short lived due to antigenic variation, so as to
immunity following the first infection is not effective against second
infection caused by antigenically novel virus.
z In syphilis, a special type of immunity known as ‘premunition’ is seen. Here,
the immunity to reinfection lasts only as long as original infection remains
active.
Artificial active immunity is the resistance induced by the vaccines. Vaccines
are preparations of live or killed microorganisms or their products used for
immunisation.
Examples of vaccines are as follows:

Bacterial vaccines – live (BCG for tuberculosis), bacterial products (Tetanus
toxoid)
Viral vaccines – live (Oral polio vaccine Sabin), killed (Injectable polio vaccine
Salk), subunit (Hepatitis B vaccine)
Live vaccines initiate an infection without causing any injury or disease. The
immunity following live vaccine parallels that after natural infection though it
may be of lower order. Killed vaccines are generally less immunogenic than live
vaccines, and protection lasts only for a short period. They therefore, to be given
repeatedly; generally at least two doses are required. The first dose is called
primary dose and the subsequent doses as booster doses.
Passive immunity is the resistance transmitted passively to an individual in a
‘readymade’ form.
488 MICROBIOLOGY
Immunity MODULE
z There is no antigenic stimulus; instead, preformed antibodies are administered. Microbiology
z There is no latent period, protection being effective immediately.

z The immunity is transient, no secondary response in passive immunity.
z It is less effective then active immunisation.
z The main advantage of passive immunisation is that it acts immediately and,
therefore, can be used when immediate effect is desired, for example
antidiphtheritic serum given to a child presenting with diphtheria. Notes
Natural passive immunity is the resistance passively transferred from mother
to baby. In the human infants, maternal antibodies are transmitted predominantly
through the placenta. It is only by the age of three months that the infant acquires
some measure of immunological independence.
Artificial passive immunity is the resistance passively transferred to a recipient
by the administration of antibodies. The agents used for this purpose are
hyperimmune sera of animal or human origin (Anti tetanus serum, ATS, prepared
from hyperimmune horses) and pooled human gammaglobulin (tetanus
immuneglobulin, TIG).
Sometimes a combination of active and passive immunisation is used, known
as combined immunisation. For example, protection of a nonimmune individual
with a tetanus prone wound (both TIG and Tetanus toxoid is given).
A special type of immunisation is the injection of immunologically competent
lymphocytes, known as adoptive immunity.
Measurement of immunity
A simple method of testing immunity is to relate its level to some convenient
indicator, such as demonstration of the specific antibody. The antibodies may
be demonstrated by a variety of techniques such as agglutination, precipitation,
complement fixation, hemagglutination inhibition, neutralisation, enzyme linked
immunosorbent assay (ELISA). Where protection is associated with cell
mediated immunity, skin tests for delayed hypersensitivity and in vitro tests are
used as an indicator of immunity
Local immunity
In poliomyelitis systemic immunity provided by active immunisation with the
killed vaccine neutralises the virus when it enters the bloodstream, but it does
not prevent multiplication of virus at the site of entry (the gut mucosa) and its
fecal shedding. This is achieved by local intestinal immunity either acquired by
infection or immunisation with live oral vaccine. A special class of
immunoglobulins (IgA) forms the major component of local immunity.
MICROBIOLOGY 489
MODULE Immunity
Microbiology Herd immunity

This refers to the overall level of immunity in a community. When a large
proportion of individuals in a community (herd) are immune to a pathogen, the
herd immunity to the pathogen is satisfactory. Eradication of communicable
diseases depends on the development of a high level of herd immunity rather
than on the development of a high level of immunity in individuals.
Notes

1. Difference in innate immunity shown by different individuals within a
race is known as
a) Individual immunity
b) Racial immunity
c) Species immunity
d) Herd immunity
2. Immunoglobulin which forms the major component of local immunity is
a) IgM
b) IgG
c) IgA
d) IgE
3. BCG vaccine for tuberculosis is a type of
a) Killed vaccine
b) Live vaccine
c) Subunit vaccine
d) None of the above
4. The resistance passively transferred to a recipient by the administration of
antibodies is known as
a) Artificial active immunity
b) Natural active immunity
c) Artificial passive immunity
d) Natural passive immunity
490 MICROBIOLOGY
Immunity MODULE
5. Maternal antibodies transferred to fetus through placenta provides Microbiology
a) Artificial active immunity

b) Natural active immunity
c) Artificial passive immunity
d) Natural passive immunity
6. Immunity is lifelong after first episode of following viral disease Notes
a) Syphilis
b) Measles
c) Influenza
d) Tuberculosis
7. A special type of immunity ‘premunition’ is seen in
a) Polio
b) Hepatitis
c) Syphilis
d) Chickenpox
8. All of them are phagocytic cells except
a) Neutrophils
b) Macrophages
c) Histiocytes
d) Platelets

z The term “immunity” is referred to the resistance exhibited by the host
towards injury caused by microorganisms and their products.
z Immunity against infections is of different type’s innate immunity and
acquired immunity.
z Innate immunity is the resistance to infections that an individual possesses
by virtue of his genetic makeup, and can further be grouped as:
z Nonspecific immunity: Indicates a degree of resistance to infections in
general.
z Species immunity – Refers to the refractoriness to a pathogen shown
by all members of a species.
MICROBIOLOGY 491
MODULE Immunity
Microbiology z Racial immunity – Within a species, different races may show

differences in susceptibility to infections.
z Individual immunity - It is the difference in innate immunity shown
by different individuals within a race
z Specific immunity: Refers to the resistance to a particular pathogen, and
can also be divided into species, racial and individual immunity.
Notes z In mechanism of innate immunity epithelial surfaces of skin and mucosa,
antibacterial substances in blood and tissues, microbial antagonism and
cellular mediators have an important role. In addition to that physiological
and pathological responses like fever, inflammation and rise in acute phase
reactants; contribute to innate immune response.
z Acquired immunity is the resistance that an individual acquires during life.
Acquired immunity is of two types:
z Active immunity is the resistance developed by an individual as a result
of an antigenic stimulus. Passive immunity is the resistance transmitted
passively to an individual in a ‘readymade’ form. Both can be subdivided
into natural and artificial.
z Local immunity refers to the protection given to a potential site of entry
for a pathogen; such immunity prevents entry of pathogen.
z Herd immunity is the immunity developed in a large proportion of
individuals in a population. This is important in decreasing likelihood of
epidemics.
TERMINAL QUESTIONS
1. Discuss the type and mechanism of innate immunity
2. What are the differences between active and passive immunity? Describe
with examples.
3. Define local immunity and herd immunity.
ANSWERS OF INTEXT QUESTIONS
55.1
1. (a) 2. (c) 3. (b) 4. (c) 5. (d)
6. (b) 7. (c) 8. (d)
492 MICROBIOLOGY
Antigens MODULE
Microbiology
56
Notes
ANTIGENS
56.1 INTRODUCTION
Common perception about an antigen is that it is a substance which produces
antibodies and react with them. However, it is not entirely correct – particularly
in view of closely related groups of substances called immunogens and haptens.
Hence there is need to be clearly defined.
OBJECTIVES
z define antigen
z differentiate antigen from immunogens and haptens
z describe some of the characteristics of antigens
Antigen is defined as any substance which when introduced in the body,

stimulates the production of an antibody with which it reacts specifically. Its
ability to bind with antibodies or T-cell is referred to as antigenicity.
Immunogen is substance which produces an immune response as well as binds

to its products i.e., antibodies or sensitized T-cells, when injected into the host.
Hapten refers to a group of substances, usually very small in size, which do not
induce an immunresponse by themselves alone. But if combined with another
molecules called carries, the hapten-carrier complex induces an immune
response
MICROBIOLOGY 493
MODULE Antigens
Microbiology

1. Antigens stimulates the production of ......................
2. Ability of antigen to bind with antibody is termed as ......................
3. ...................... produces immune responses
Notes
4. ...................... if combined with carriers induce immune response
56.2 TYPES OF ANTIGEN

Endogeneous Antigen – These enters the body from outside i.e external
environment. Common examples includes microorganisms, drugs, pollen,
pollutants or even food items etc.
Endogenous Antigens - These antigens are produced within the host.
56.3 ANTIGEN AND HOST RELATIONSHIP

Based on genetic consideration antigens are divided into three types: Autoantigens,
alloantigens and heteroantigens
56.3.1 Autoantigens
These are the antigens belonging to host itself.
56.3.2 Alloantigens
These are the antigens derived from other members of species of the host, but
not from the host itself. Such antigens are important in tissue transplant and
blood transfusion processes e.g, antigens present on donor and the recipient
RBCs are alloantigens to each other.
56.3.3 Heteroantigens
These antigens are from two different species such as plants and animals or
microorganisms etc.
The smallest unit of antigenicity is known as the antigenic determinant or
epitope. The epitope is that small area on the antigen usually consisting of four
or small area on the antigen. Usually consisting of four or five aminoacid or
monosaccharaide residues, possessing a specific chemical structure, electrical
charge and steric configuration, capable of sensitising an immunocyte and of
reacting with its complementary site on the specific antibody or T cell receptor.
The combining area on the antibody molecules, corresponding to the epitope,
is called the paratope.
494 MICROBIOLOGY
Antigens MODULE
Microbiology

Match the following
1. Endogenous antigen (a) Belongs to host
2. Exogenous antigen (b) Derived from other members of species
of host Notes
3. Autoantigen (c) Produced within the host
4. Alloantigen (d) Enter from outside
56.4 DETRMINANTS OF ANTIGENICITY

A number of properties that make a substance antigenic have been identified but
the exact basis of antigenicity is still not clear
56.4.1 Size
Antigenicity is related to the molecular size. Very large molecule are highly
antigenic and particles with low antigenicity are nonantigenic. Low molecular
weight substances may be rendered antigenic by adsorbing them on a large inert
particles such as bentonite or kaolin.
56.4.2 Chemical nature

Proteins and polysaccharides are good immunogen as compared to lipids and
nucleic acids, Among them proteins are better than carbohydrates. Nucleic acids,
poor by themselves, can generate response in combination with other substances.
56.4.3 Susceptibility to tissue enzymes

Only substances which are metabolized and are susceptible to the action of tissue
enzymes behave as antigens. Antigens introduced into the body are degraded by
the host into fragments of appropriate size containing the antigenic determinants.
56.4.4 Foreignness
Only antigen which are ‘foreign’ to the individual (nonself) induce an immune
response. The antigenicity of a substance is related to the degree of its
foreignness. Antigen from related species are less antigenic than those from
distant species.
56.4.5 Antigenicity specificity

The basis of antigenic specificity is a stereochemical .Crossreaction can occur
between antigen that bear stereochemical similarities. In some instances,
apparent cross reactions may actually be due to the sharing of identical antigenic
determinants by different antigens.
MICROBIOLOGY 495
MODULE Antigens
Microbiology 56.4.6 Species specificity

Tissues of all individuals in a species contain species- specific antigens. There
exits some degree of cross - reaction between antigens of related species.
56.4.7 Isospecificity
Isoantigens are antigens found in some but not all members of a species. The
species may be grouped depending on the presence of different isoantigens in
Notes
its members
56.4.8 Autospecificity
Autologous or self antigens are ordinarily nonantigenic but there are exceptions.
Sequestrated antigens that are not normally found free in circulation or tissue
fluids are not recognised as a self antigens.. Similarly, antigens that are absent
during embryonic life and develop later are also not recognized as self antigens.
56.4.9 Organ specificity

Some organs, such as the brain, kidney and lens protein of different species,
share the same antigen. Such antigens, characteristic of organ or tissue and found
in different species, are organ – specific antigens.
56.4.10 Heterogenetic ( heterophile ) specificity

The same or closely related antigens may sometimes occur in different biological
species, classes and kingdoms. These are known as heterophile antigens .

1. Drugs and Pollen are examples of ........................ antigen
2. Antigens that are of importance in Tissue transplant and Blood Transfusion
are ........................
3. The smallest unit of antigenicity is known as ........................
4. Antigens found in some but not all members of a species are called as
........................

z A substance that induces an immune response is called an antigen. If the
antigen stimulates production of an antibody, it will react specifically,
generally in a observable manner, with antibody.
496 MICROBIOLOGY
Antigens MODULE
z An immunogen is a substance that can induces an immune response but Microbiology
which does not necessarily bind to its specific antibody.
z Most antigens are foreign to the host. They are large molecules, such as
proteins and polysaccharides. Small chemical groups on the antigens
molecules, called epitopes, constitute that are recognised by antibodies.
Notes
TERMINAL EXERCISE
1. Define antigen, immunogen and hapten?
2. Write characteristics of antigen?
56.1
1. Antibody
2. Antigenicity
3. Immunogen
4. Hapten
56.2
1. (c)
2. (d)
3. (a)
4. (b)
56.3
1. Endogenous
2. Alloantigen
3. Antigenic determinant or epitope
4. Isoantigen
MICROBIOLOGY 497
MODULE Immunoglobulins
Microbiology
57
Notes
IMMUNOGLOBULINS
57.1 INTRODUCTION
When foreign body (or) antigens, water, it activates or produces substances
called antibodies into serum and body fluids and these antibodies reacts with
antigens. The sera with high antibody levels following infection or immunization
are called immune sera.
The term immunoglobulin means proteins of animal origin endowed with known
antibody activity and for other proteins related to them by chemical structure.
OBJECTIVES
z describe the structure of immunoglobulin

z classify immunoglobulins
z describe the characteristics of different immunoglobulins
57.2 STRUCTURE
Immunoglobulins are glycoproteins each consisting of two pairs of polypeptide
chains of various sizes. The smaller chains are called light (L) chains and the
larger ones ‘heavy (H) chains. The chain is attracted to H chain by disulphide
bond. H chain has molecular weight of approximately 25,000 and H chain of
50,000.
H chains are structurally and antigentically distinct for each class and are
designated by Greece letter corresponding to immunoglobulin as
498 MICROBIOLOGY
Immunoglobulins MODULE
Immunoglobulin class H chain Microbiology
IgG γ (gamma)
IgA α (Alpha)
IgM μ (mu)
IgD ∂ (delta)
IgE ε (epsilon)
Notes
The H chains are similar in all classes of Immunoglobulins. They are of two types
kappa (κ ) and lambda (λ). The antigen combining site of the molecule is at its
aminoterminals which contains both L & H chains. The amino acid sequence of
carboxy terminal half occur only in a constant sequence and it is therefore called
as constant region whereas the amino acid sequence in the amino terminal half of
the chain is highly variable and it is therefore called variable region. The range of
antibody specificity of Immunoglobulins depends on the variability of the amino
acid sequences at the variable region of H & L chain which terms the antigen
combining sites.

1. Immunoglobulins are ................... proteins
2. Smaller polypeptide chain is called ...................chain & larger is called
...................chain
3. Carboxy terminal of immunoglobulin is called................... region and
amino terminal is called ................... region
4. The sera with high antibody activity following infection or immunization
is called as ................... sera
57.3 IMMUNOGLOBULIN CLASSES

Human sera contains IgG, IgA, IgM, IgD, & IgE in the descending order of
concentration.
IgG: This is the major serum Immunoglobulin constitution of about 80% of the
total and is equally distributed between intracellular and extracellular
compartments. It contains less carbohydrate than other Immunoglobulins. It has
half life of about 23 days.
IgG is the only material Immunoglobulin that is transported across placenta and
provides passive immunity in new bonds. IgG binds to micro organisms and
enhances their phagocytosis. IgG protects against infectious agents which are
active in blood and tissues. Passively administered IgG suppresses homologous
antibody synthesis by a Icedback process. This property is used in isoimmunization
MICROBIOLOGY 499
MODULE Immunoglobulins
Microbiology of women by administration of anti Rh (D) IgG during delivery. Four subclasses
have been identified namely IgG, IgG2, IgG3, IgG4,
IgA: The second most abundant class, constituting of about 10 – 13 % of serum
Immunoglobulins and its half life is 6 – 8 days. It is the major Immunoglobulin
in colostrums, saliva and tears.
The variety IgG plays an important role in lovac immunity against and intestinal
Notes pathogens and they are resistant to digestive enzymes and reducing agents. IgA
antibody inhibits the adherence of micro organisms to the surface of mucosal
cells by covering the organisms. It promotes phagocytosis and intracellular
killing of micro organisms.
IgM: IgM constitutes of 5 – 8 % of serum Immunoglobulins with a half life of
about five days. IgM is synthesized by fetus from 20 weeks of age. IgM is not
transported across the placenta, the presence of IgM in the fetus or newborn
indicates intrauterine infection and its detection is useful in diagnosis of
congenital infections such as syphilis, rubella, HIV infection and toxoplasmosis.
IgM is found in intravascular space and is responsible for protection against
blood invasion by micro organisms. IgM deficiency is often associated with
septicemias.
IgD: This resembles IgG structurally. It is intravascular and has half life of about
three days. IgD and IgM occur on the surface of unstimulated B lymphocytes
and serve as recognition receptors for antigens.
IgE: This resembles IgG structurally with half life of about two days. It is mostly
extravascular and does not pass placental barriers. Serum levels get elevated
during asthma and eczema.
IgE is produced in the linings of respiratory and intestinal tracts. IgE deficiency
is associated with IgA deficiency in individual with impaired immunity. IgE is
responsible for anaphylactic type of hypersensitivity.

Match the following
1. IgG (a) present in respiratory & Intestinal tracts
2. IgA (b) serves as recognition receptors for antigens
3. IgM (c) provides passive immunity in Newborns
4. IgD (d) present in colostrum
5. IgE (e) synthesized by fetus
500 MICROBIOLOGY
Immunoglobulins MODULE
Microbiology

z A substance that induces an immune response is called antigen.
z The sera with high antibody levels following infection or immunization are
called immune sera.
z Immunoglobulins are glycoproteins Notes
z Human sera contains IgG, IgA, IgM, IgD, & IgE in the descending order
of concentration.
z IgG is major Immunoglobulin that is transported across placenta and
provides passive immunity
z IgA, the second most abundant class, present in colostrum, saliva and tears.
z IgM is synthesized by fetus and is not transported across the placenta, and
is useful in detection of congenital infections
z IgM occur on surface of B lymphocytes and serves as antigen receptors.
z IgE are present in linings of respiratory & Intestinal tracts
TERMINAL QUESTIONS
1. Describe the structure of immunglobulins
2. Classify immunoglobulins
57.1
1. Glycol
2. Light & heavy
3. Constant & Variable
4. Immune
57.2
1. (c) 2. (d) 3. (e) 4. (b) 5. (a)
MICROBIOLOGY 501
MODULE Complement
Microbiology
58
Notes
COMPLEMENT
58.1 INTRODUCTION
The complement (C) system is part of the immune system called the innate
immune system. The term complement was used to refer to a heat-labile serum
component that was able to lyse bacteria. Today, complement is known to
contribute to host defences in other ways as well. Complement
can opsonize bacteria for enhanced phagocytosis; activate various cells including
polymorphonuclear cells (PMNs) and macrophages; it can participate in
regulation of antibody responses and it can aid in the clearance of immune
complexes and apoptotic cells. Complement can also have detrimental effects
for the host, it contributes to inflammation and tissue damage and it can
trigger anaphylaxis.
In the late 19th century, Hans Ernst August Buchner found that blood serum
contained a “factor” capable of killing bacteria. In 1894, Richard Pfeiffer, a
German scientist, had discovered that when cholera bacteria were injected into
the peritoneum of a guinea pig immunized against the infection, the pig would
rapidly die. This bacteriolysis, Bordet discovered, did not occur when the
bacteria was injected into a non-immunized guinea pig, but did so when the same
animal received the antiserum from an immunized animal. Moreover, the
bacteriolysis did not take place when the bacteria and the antiserum were mixed
in a test tube unless fresh antiserum was used. However, when Bordet heated
the antiserum to 55 degrees centigrade, it lost its power to kill bacteria. Finding
that he could restore the bacteriolytic power of the antiserum if he added a little
fresh serum from a non immunized animal, Bordet concluded that the bacteria-
killing phenomenon was due to the combined action of two distinct substances,
an antibody in the antiserum, which specifically acted against a particular kind
of bacterium, and a non-specific substance, sensitive to heat, found in all animal
serums, which Bordet called “alexine” (later named “complement”).
502 MICROBIOLOGY
Complement MODULE
The term “complement” was introduced by Paul Ehrlich in the late 1890s, Microbiology
according to him, the immune system consists of cells that have specific
receptors on their surface to recognize antigens. Upon immunisation with
an antigen, more of these receptors are formed, and they are then shed from the
cells to circulate in the blood. These receptors, which we now call “antibodies,”
were called by Ehrlich “amboceptors” to emphasise their bi-functional binding
capacity: They recognise and bind to a specific antigen, but they also recognise
and bind to the heat-labile antimicrobial component of fresh serum. Ehrlich, Notes
therefore, named this heat-labile component “complement,” because it is
something in the blood that “complements” the cells of the immune system.
OBJECTIVES
z describe the complement system.
z describe various Components of complement system
z explain the causes Complement activation
z explain Various Pathways of complement system
z describe how is C activation regulated
z describe Quantification of complement activity
z explain the effects of Deficiencies of complement system.
58.2 GENERAL PROPERTIES OF COMPLEMENT

It forms the part of normal serum protein of all humans, animals, birds,
amphibians and fishes alike. The complement is non specific meaning the
complement from one species can react with the antibodies from another species.
It is heat labile, thus serum can be deprived of its complement activity by heating
it at 56 C for 30 minutes. It ordinarily does not bind to free antigen but only with
antibodies which has combined with the antigen. Only IgM, IgG3, 1and 2 fix
complement. The complement constituents are not influenced by immunisation.
58.3 COMPONENTS OF COMPLEMENT SYSTEM

The complement system comprises a large number of plasma proteins that
mediate several functions of the inflammatory process. These circulate as
proenzymes, i.e. in an inactive form. Complement activation arises through a
cascade process, whereby activation of one proenzyme results in activation of
MICROBIOLOGY 503
MODULE Complement
Microbiology the next proenzyme in the pathway, and so on. The two most important
mechanisms for activation of the early complement components are the classical
pathway, initiated by antigen-antibody complexes, and the alternate pathway,
initiated by microbial surface molecules. These pathways result in the generation
of enzymes capable of splitting component C3. Once C3 is split into C3a and
C3b, the subsequent pathway of activation of the terminal components is
identical, regardless of the initiating stimulus. The end result is the generation
Notes of several active molecules which mediate distinct biological properties:
The serum proteins of complement system are produced by a variety of cells
including, hepatocytes, macrophages and gut epithelial cells. Some complement
proteins bind to immunoglobulins or to membrane components of cells. Others
are proenzymes that, when activated, cleave one or more other complement
proteins. Upon cleavage some of the complement proteins yield fragments that
activate cells, increase vascular permeability or opsonize bacteria.
Table 58.1: Proteins of the Complement system

Classical Pathway Lectin Pathway Alternative Pathway
Activation Proteins: C1qrs, Mannan binding C3, Factors B & D*,

C2, C3, C4 protein (MBP), Properdin (P) Factors I* & H,
Control Proteins:C1-INH, mannan-decay accelerating factor (DAF),
C4-BP asociated serine Complement receptor 1
protease (CR1),etc.
(MASP,MASP2)
Components underlined acquire enzymatic activity when activated.

Components marked with an asterisk have enzymatic activity in their native form.
58.4 PATHWAYS OF COMPLEMENT ACTIVATION

Complement activation can be divided into four pathways, the classical pathway,
the lectin pathway, and the alternative pathways. Both classical and alternative
pathways lead to the activation of C5 convertase and result in the production
of C5b which is essential for the activation of the membrane attack pathway.
58.4.1 Classical Pathway

The chain of events in which C components react in a specific sequence
following activation of C1 and typically culminate in immune cytolysis is known
as the classical pathway. The steps involved in classical pathway are as follows
504 MICROBIOLOGY
Complement MODULE
C1 activation Microbiology
C1, a multi-subunit protein containing three different proteins (C1q, C1r and
C1s), binds to the Fc region of IgG and IgM antibody molecules that have
interacted with antigen. C1 binding does not occur to antibodies that have not
complexed with antigen and binding requires calcium and magnesium ions.
(N.B. In some cases C1 can bind to aggregated immunoglobulin [e.g. aggregated
IgG] or to certain pathogen surfaces in the absence of antibody). The binding
Notes
of C1 to antibody is via C1q and C1q must cross link at least two antibody
molecules before it is firmly fixed. The binding of C1q results in the activation
of C1r which in turn activates C1s. The result is the formation of an activated
“C1qrs”, which is an enzyme that cleaves C4 into two fragments C4a and C4b.
C4 and C2 activation (generation of C3 convertase)

The C4b fragment binds to the membrane and the C4a fragment is released into
the microenvironment. Activated “C1qrs” also cleaves C2 into C2a and C2b.
C2a binds to the membrane in association with C4b, and C2b is released into
the microenvironment. The resulting C4bC2a complex is a C3 convertase,
which cleaves C3 into C3a and C3b.
C1q binding e.g. to immune complex
C1r C1r
C1s C1s
C4 C4b + C4a
C2
C4bC2 C4bC2a + C2b
The classical pathway C3 convertase
Fig. 58.1: Activation of the Classical Pathway
C3 activation (generation of C5 convertase)

C3b binds to the membrane in association with C4b and C2a, and C3a is released
into the microenvironment. The resulting C4bC2aC3b is a C5 convertase. The
generation of C5 convertase is the end of the classical pathway.
Several of the products of the classical pathway have potent biological activities
that contribute to host defenses. Some of these products may also have
detrimental effects if produced in an unregulated manner. Table summarizes the
biological activities of classical pathway components.
MICROBIOLOGY 505
MODULE Complement
Microbiology Table 58.2: Biological Activity of classical pathway products

Component Biological Activity
C2b Prokinin; cleaved by plasmin to yield kinin, which results in edema
C3a Anaphylotoxin; can activate basophils and mast cells to degranulate

resulting in increased vascular permeability and contraction of
smooth muscle cells, which may lead toanaphylaxis
Notes
C3b Opsonin; promotes phagocytosis by binding to complement
receptorsActivation of phagocytic cells
C4a Anaphylotoxin (weaker than C3a)
C4b Opsonin; promotes phagocytosis by binding to complement receptors
If the classical pathway were not regulated there would be continued production
of C2b, C3a, and C4a. Thus, there must be some way to regulate the activity
of the classical pathway. Table 58.3 summarizes the ways in which the classical
pathway is regulated.
Fig. 58.2
Table 58.2: Regulation of the Classical Pathway

Component Regulation
All C1-INH; dissociates C1r and C1s from C1q
C3a C3a inactivator (C3a-INA;Carboxypeptidase B); inactivates C3a
C3b Factors H and I; Factor H facilitates the degradation of C3b by

Factor I
506 MICROBIOLOGY
Complement MODULE
C4a C3-INA Microbiology
C4b C4 binding protein(C4-BP) and Factor I; C4-BP facilitates

degradation of C4b by Factor I; C4-BP also prevents association of
C2a with C4b thus blocking the formation of C3 convertase
The importance of C1-INH in regulating the classical pathway is demonstrated

by the result of a deficiency in this inhibitor. C1-INH deficiencies are associated
with the development of hereditary angioedema. Notes
58.4.2 Lectin Pathway

The lectinpathway is very similar to the classical pathway. It is initiated by the
binding of mannose-binding lectin (MBL) to bacterial surfaces with mannose-
containing polysaccharides (mannans). Binding of MBL to a pathogen results
in the association of two serine proteases, MASP-1 and MASP-2 (MBL-
associated serine proteases). MASP-1 and MASP-2 are similar to C1r and C1s,
respectively and MBL is similar to C1q. Formation of the MBL/MASP-1/
MASP-2 tri-molecular complex results in the activation of the MASPs and
subsequent cleavage of C4 into C4a and C4b. The C4b fragment binds to the
membrane and the C4a fragment is released into the microenvironment.
Activated MASPs also cleave C2 into C2a and C2b. C2a binds to the membrane
in association with C4b and C2b is released into the microenvironment. The
resulting C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and
C3b. C3b binds to the membrane in association with C4b and C2a and C3a is
released into the microenvironment. The resulting C4bC2aC3b is a C5
convertase. The generation of C5 convertase is the end of the lectin pathway.
The biological activities and the regulatory proteins of the lectin pathway are
the same as those of the classical pathway.
58.4.3 Alternative Pathway

The alternative pathway begins with the activation of C3 and requires Factors
B and D and Mg++ cation, all present in normal serum.
1. Amplification loop of C3b formation

In serum there is low level spontaneous hydrolysis of C3 to produce C3i. Factor
B binds to C3i and becomes susceptible to Factor D, which cleaves Factor B
into Bb. The C3iBb complex acts as a C3 convertase and cleaves C3 into C3a
and C3b. Once C3b is formed, Factor B will bind to it and becomes susceptible
to cleavage by Factor D. The resulting C3bBb complex is a C3 convertase that
will continue to generate more C3b, thus amplifying C3b production. If this
process continues unchecked, the result would be the consumption of all C3 in
the serum. Thus, the spontaneous production of C3b is tightly controlled.
MICROBIOLOGY 507
MODULE Complement
Microbiology 2. Control of the amplification loop

As spontaneously produced C3b binds to autologous host membranes, it
interacts with DAF (decay accelerating factor), which blocks the association of
Factor B with C3b thereby preventing the formation of additional C3 convertase.
In addition, DAF accelerates the dissociation of Bb from C3b in C3 convertase
that has already formed, thereby stopping the production of additional C3b.
Some cells possess complement receptor 1 (CR1). Binding of C3b to CR1
Notes facilitates the enzymatic degradation of C3b by Factor I. In addition, binding of
C3 convertase (C3bBb) to CR1 also dissociates Bb from the complex. Thus, in
cells possessing complement receptors, CR1 also plays a role in controlling the
amplification loop. Finally, Factor H can bind to C3b bound to a cell or in the
in the fluid phase and facilitate the enzymatic degradation of C3b by Factor I.
Thus, the amplification loop is controlled by either blocking the formation of
C3 convertase, dissociating C3 convertase, or by enzymatically digesting C3b.
The importance of controlling this amplification loop is illustrated in patients
with genetic deficiencies of Factor H or I. These patients have a C3 deficiency
and increased susceptibility to certain infections.
3. Stabilization of C convertase by activator (protector) surfaces

When bound to an appropriate activator of the alternative pathway, C3b will bind
Factor B, which is enzymatically cleaved by Factor D to produce C3 convertase
(C3bBb). However, C3b is resistant to degradation by Factor I and the C3
convertase is not rapidly degraded, since it is stabilized by the activator surface.
The complex is further stabilized by properdin binding to C3bBb. Activators
of the alternate pathway are components on the surface of pathogens and include:
LPS of Gram-negative bacteria and the cell walls of some bacteria and yeasts.
Thus, when C3b binds to an activator surface, the C3 convertase formed will
be stable and continue to generate additional C3a and C3b by cleavage of C3.
Fig. 58.3
508 MICROBIOLOGY
Complement MODULE
4. Generation of C5 convertase Microbiology
Some of the C3b generated by the stabilized C3 convertase on the activator surface
associates with the C3bBb complex to form a C3bBbC3b complex. This is the
C5 convertase of the alternative pathway. The generation of C5 convertase is the
end of the alternative pathway. The alternative pathway can be activated by many
Gram-negative (most significantly, Neisseria meningitidis and N. gonorrhoea),
some Gram-positive bacteria and certain viruses and parasites, and results in the
Notes
lysis of these organisms. Thus, the alternative pathway of C activation provides
another means of protection against certain pathogens before an antibody
response is mounted. A deficiency of C3 results in an increased susceptibility to
these organisms. The alternate pathway may be the more primitive pathway and
the classical and lectin pathways probably developed from it.
Remember that the alternative pathway provides a means of non-specific
resistance against infection without the participation of antibodies and hence
provides a first line of defense against a number of infectious agents.
Many gram negative and some gram positive bacteria, certain viruses, parasites,
heterologous red cells, aggregated immunoglobulins (particularly, IgA) and
some other proteins (e.g. proteases, clotting pathway products) can activate the
alternative pathway. One protein, cobra venom factor (CVF), has been extensively
studied for its ability to activate this pathway.
58.5 BIOLOGICALLY ACTIVE PRODUCTS OF

COMPLEMENT ACTIVATION
Activation of complement results in the production of several biologically active
molecules which contribute to resistance,anaphylaxis and inflammation.
Kinin production
C2b generated during the classical pathway of C activation is a prokinin which
becomes biologically active following enzymatic alteration by plasmin. Excess
C2b production is prevented by limiting C2 activation by C1 inhibitor (C1-INH)
also known as serpin which displaces C1rs from the C1qrs complex (Figure 10).
A genetic deficiency of C1-INH results in an overproduction of C2b and is the
cause of hereditary angioneuroticedema. This condition can be treated
with Danazol which promotes C1-INH production or with å-amino caproic acid
which decreases plasmin activity.
Anaphylotoxins
C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which
cause basophil/mast cell degranulation and smooth muscle contraction.
MICROBIOLOGY 509
MODULE Complement
Microbiology Undesirable effects of these peptides are controlled by carboxypeptidase B (C3a-

INA).
Chemotactic Factors
C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of
neutrophils, basophils and macrophages and causes induction of adhesion
Notes molecules on vascular endothelial cells.
Opsonins
C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on
phagocytic cells and promote phagocytosis.
Other Biologically active products of C activation

Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells by
distinct receptors and modulate their functions.
Regulation of C activation
Left unchecked the complement activity can cause not only exhaustion of
complement system but also serious damage to tissues. Several inbuilt control
mechanisms regulate the complement cascade at different levels.
Table 58.4: Activities of Complement Activation Products and
their Control Factors
Fragment Activity Effect Control Factor (s)
C2a Prokinin, accumulation of fluids Edema C1-INH
C3a Basophil and mast cells Anaphylaxis C3a-INA

degranulation; enhanced
vascular permeability, smooth
muscle contraction
C3b Opsonin, phagocyte activation Phagocytosis Factors H and I

degranulation; enhanced (least potent)
muscle contraction
C4b Opsonin Phagocytosis C4-BP and

Factor I
510 MICROBIOLOGY
Complement MODULE
Microbiology
degranulation; enhanced (most potent)
muscle contraction
Chemotaxis, stimulation of Inflammation
respiratory burst, activation of
phagocytes, stimulation of
inflammatory cytokines Notes
C5bC6C7 Chemotaxis Inflammation Protein S
(vitronectin)
Attaches to other membranes Tissue

damage
In summary, the complement system takes part in both specific and non-specific
resistance and generates a number of products of biological and pathophysiological
significance
Quantitation of Complement and its components

Complement activity of serum is measured by estimating the highest dilution
of serum lysing sheep erythrocytes sensitised by antierythrocytic antibody. C
components can also be quantified by radial immunodiffusion in agar.
Deficiencies of complement system

There are known genetic deficiencies of most individual C complement
components, but C3 deficiency is most serious and fatal. Complement deficiencies
also occur in immune complex diseases (e.g., SLE) and acute and chronic
bacterial, viral and parasitic infections.
Table 58.5: Complement deficiencies and disease
Pathway/Component Disease Mechanism
Classical Pathway
C1INH Hereditary angioedema Overproduction of C2b

(prokinin)
C1, C2, C4 Predisposition to SLE Opsonization of immune

complexes help keep them
soluble, deficiency results in
increased precipitation in
tissues and inflammation
MICROBIOLOGY 511
MODULE Complement
Microbiology Lectin Pathway
MBL Susceptibility to bacterial Inability to initiate the lectin

infections in infants or pathway
immunosuppressed
Alternative Pathway
Factors B or D Susceptibility to pyogenic Lack of sufficient opsonization

(pus-forming) bacterial of bacteria
Notes
infections
C3 Susceptibility to bacterial Lack of opsonization and

infections inability to utilize the membrane
attack pathway
C5, C6, C7 C8, and C9 Susceptibility to Gram- Inability to attack the outer
negative infections membrane of Gram-negative
bacteria
Properdin (X-linked) Susceptibility meningococcal Lack of opsonization of

meningitis bacteria
Factors H or I C3 deficiency and suscepti- Uncontrolled activation of C3

bility to bacterial infections via alternative pathway
resulting in depletion of C3

1. Heat labile serum component that lyse bacteria is ..................
2. The inactive form of complement are ..................
3. The classical complement pathway is initiated by .................. complexes
4. Alternate pathway is initiated by .................. molecules
5. Major biologically active products of complement activation are ..................,
.................., .................. and ..................
6. Overproduction of Prokinin causes ..................
7. Deficiency of Opsonin predisposes to ..................

The complement system is a set of serum proteins that play a major role in the
immune response
z Some lyse foreign cells
z Some mediated inflammation and attract and activate phagocytic cells
512 MICROBIOLOGY
Complement MODULE
z Some amplify the effects of antibodies Complement acts in a cascade Microbiology
fashion; the complement proteins are inactive, and the activation of one
leads to the sequential activation of others There are three pathways of
complement activation
z Classical pathway-results form antigen-antibody interactions that occur
during specific immune responses (discussed in chapter 32)
z Alternative complement pathway-occurs in response to intravascular invasion Notes
by bacteria and some fungi; involves interaction of complement with the
surface of the pathogen
z Lectin complement pathway-occurs when macrophages release mannose-
binding protein (a lectin), which then can activate complement via the
alternative pathway or the classical pathway
Overview of complement activation and immune responses
z Gram-negative bacteria at local tissue site interact with components of
alternative pathway
z If bacteria persist or invade a second time, antibody responses activate the
classical pathway
z Generation of C3a and C5a complement fragments leads to:
z Activation of mast cells, which release their contents, causing hyperemia
z Release of neutrophils from bone marrow into circulation, and their
chemotaxis to injury site
z Ultimately neutrophils and phagocytes ingest and destroy the bacteria
TERMINAL QUESTIONS
1. Who gave the term “complement”?
2. What do you understand by the term “cascade process” ?
3. What is the classical pathway of complement system?
4. Enlist the regulatory components of classical pathway ?
5. How is the quantitation of complement done?
6. What is the role of complement system in immunity?
7. What are various pathways by which the complement system functions?
8. Draw a flow chart explaining the classical pathway of the complement
system?
9. How can we measure the complement?
MICROBIOLOGY 513
MODULE Complement
Microbiology 10. Give differences between various pathways of the complement systems?
11. What are the effects of deficiency of complement system?
Notes 1. Complement
2. Proenzyme
3. Antigen-antibody
4. Microbial surface
5. Kinin, Anaphlotoxins, Chemotactic factor, Opsonins
6. Angioedema
7. SLE
514 MICROBIOLOGY
Immunology Structure and Function of Immune System MODULE
Microbiology
59
Notes
IMMUNOLOGY
STRUCTURE AND FUNCTION
OF IMMUNE SYSTEM
59.1 INTRODUCTION
The immune system is engaged in a constant surveillance of the body for
pathogens or tumors. Whether disease develops depends on the virulence of the
pathogen and the competence of the immune system. To prevent disease, the
immune system must recognize, attack, and remember substances that threaten
health, either foreign pathogens or mutations of the bodies own cells. To do so,
it must be able to distinguish self from non-self substances called antigens
(meaning “antibody generators”). To efficiently eliminate antigens, the immune
system must respond as quickly and as strongly as possible to kill abnormal cells
or infectious agents. At the same time, it must be tightly regulated to avoid
destroying healthy tissues. When immune regulation breaks down, excessive
inflammation can cause collateral damage resulting in autoimmune and allergic
diseases. Conversely, immunosuppression can result in increased susceptibility
to infection, and malignant tumors can arise when there is unchecked growth
of mutant cells.
OBJECTIVES
z describe the components of immune system
z explain the functions of immune system
z describe innate immunity, specific immunity
z discuss immune related diseases
MICROBIOLOGY 515
MODULE Immunology Structure and Function of Immune System
Microbiology All cells of the immune system are derived from stem cells in the bone marrow.
These cells give rise two classes of progenitor cells: (1) lymphoid progenitors
are precursors to antigen specific T and B lymphocytes, and (2) myeloid
progenitors are the precursors for the nonspecific macrophages, monocytes,
dendritic cells, mast cells, and granulocytes (neutrophils, eosinophils, basophils).
B-cells remain in the bone marrow during development, selection, and maturation,
whereas T-cells migrate to the thymus to mature. Once mature, T and B-cells
Notes emerge from these primary immune organs to reside in secondary immune
organs, e.g., lymph nodes, spleen, tonsils, and lymphoid mucosa. T and B-cells
circulate from lymphoid organs to the tissues through lymphatic and blood
vessels, monitoring sites where pathogens are likely to invade the body (airways,
gastrointestinal tract, reproductive tract, skin). Pathogens are normally taken up
by antigen-presenting cells (macrophages and dendritic cells), which process
and transport the antigen to secondary lymphoid organs where they induce T and
B-cell responses.
Macrophages are widely distributed throughout the body where they act as a first
line of defense to engulf and digest antigens, a process known as phagocytosis.
They are derived from circulating precursor cells known as monocytes, which
differentiate into macrophages once they enter tissues. Immature dendritic cells
also circulate in the blood until they migrate into the tissues and mature after
ingesting pathogen. Once mature, dendritic cells migrate to the lymph nodes to
present antigen. Mast cells also differentiate in the tissues where they are located
near small blood vessels and act to alter vascular permeability during allergic
reactions. Neutrophils, eosinophils, and basophils are collectively known as
granulocytes. They normally circulate in the blood until they are recruited to
sites of infection and inflammation. Neutrophils play an important role in
controlling bacterial infections, whereas eosinophils and basophils are involved
in parasitic infections and allergic inflammation.

1. All cells of immune system are derived from ............. cells in.............
2. Lymphoid progenitors are precursors to ............., ............. lymphocytes
3. Myeloid progenitors are precursors to ............., .............,............. & .............
4. ............. cells mature in bone marrow
5. ............. cells mature in thymus gland
516 MICROBIOLOGY
6. Pathogens are taken up by ............. & ............. Microbiology
7. ............. induces phagocytosis

8. Granulocytes are ............., ............. and .............
59.2 FUNCTIONS OF THE IMMUNE SYSTEM

The functions of the immune system can be divided into two systems: (1) innate
Notes
or nonspecific immunity, and (2) specific or adaptive immunity. These interacting
systems differ in terms of the timing and specificity of their responses. Innate
immunity provides an immediate but relatively nonspecific response to contain
pathogens at the site of entry into the body. Innate immune defenses include
inflammatory and acute phase responses, as well as the anatomical and chemical
barriers provided by the skin and mucous membranes. Specific immunity is
characterized by antigen-specificity through T and B lymphocytes. It also
exhibits immunological memory, where heightened responses occur upon
subsequent exposure to the same antigen, but this is not an immediate response.
Although specific immunity is more selective and adaptive than innate
immunity, it is a slow and complex process that occurs over several days to
weeks. Conversely, innate immunity provides an immediate front line response,
but it lacks memory and can damage healthy tissue due to its nonspecific nature.
Innate Immunity
Inflammation. Inflammation is a local response designed to limit pathogen
invasion and tissue damage. Phagocytes such as macrophages and neutrophils
play a central role in the inflammatory response. They recognize foreign invaders
through nonspecific receptors that identify common features of pathogens.
Because a large pool of phagocytic cells is readily available, inflammatory
responses can be observed within 1-2 hours after infection. During this time,
macrophages use several mechanisms to contain infection. First, they release
toxic enzymes and ingest the invading cells. Activated macrophages also
synthesize and release nitric oxide, a gas that interferes with the proliferation
of bacteria and other pathogens. In addition, activated macrophages release
substances called cytokines, which are chemical messengers secreted by one cell
that communicate with other cells. Cytokines act locally to facilitate the
inflammatory response and to attract other immune cells that promote healing
at the site of infection or injury. For example, neutrophils, which normally flow
freely in the blood stream, are recruited out of the circulation to the site of
infection by cytokines such as interleukin-1 (IL- 1) that are released by activated
macrophages. A similar mechanism is used to recruit all leukocytes (white blood
cells, including monocytes, granulocytes, and lymphocytes) to the site of
infection or inflammation.
MICROBIOLOGY 517
Microbiology

1. ............. immunity produces immediate response
2. ............. immunity produces delayed responses
3. ............. is the local response to limit pathogen invasion and tissue damage
Notes 4. Macrophages release chemical messengers called .............
Natural killer cells. Natural killer cells (NK cells) are nonspecific lymphocytes
that specialize in destroying tumor cells and virus-infected cells. Although they
lack specific antigen receptors, they are able to recognize and kill some abnormal
cells. NK cells secrete perforins, chemical bullets that blow holes in the
pathogen’s cell membrane allowing granzymes to enter the cell. Granzymes
signal the target cell to commit suicide, a process known as apoptosis.
Acute Phase Response. Whereas inflammation begins as a local response
designed to contain infection, a systemic reaction known as the acute phase
response or sickness syndrome will occur if the infection spreads to other parts
of the body. This response is triggered when high concentrations of inflammatory
cytokines (e.g., tumor necrosis factor alpha, IL-1, and IL-6) enter the circulation
to initiate a series of physiological and behavioral changes that help fight
infection and promote healing. The acute phase response involves the release
of proteins by the liver that migrate to the site of infection. Interestingly, some
of these acute phase proteins act like nonspecific antibodies that bind a broad
range of pathogens. Other physiological changes include fever, increased slow
wave sleep, and increased leukocyte production and circulation.
Behavioral changes are also observed during the acute phase response, including
decreased feeding, physical activity, exploration, social interaction, sexual
activity, and aggression. Other psychological changes include increased pain
sensitivity, depressed mood, and memory impairments. The highly conserved
nature of these sickness behaviors, which are even observed in invertebrates,
suggests that they evolved to help fight infection and enhance survival. Indeed,
recent research indicates that sickness syndrome is an adaptive motivational
state coordinated by the brain rather than a collection of reflexive responses
reflecting the pathological consequences of infection or injury. Finally, activation
of the hypothalamic-pituitary adrenal (HPA) axis is part of the acute phase
response. Cytokines released during infection activate the HPA-axis to release
glucocorticoids, hormones that help to mobilize energy and decrease
inflammation. The latter negative feedback mechanism acts to counterregulate
the inflammatory cytokines to prevent damage to normal tissues. However, when
HPA-axis activity is blunted, excessive inflammation can result in
immunopathology and contribute to the development of autoimmune diseases.
518 MICROBIOLOGY
Microbiology

1. ............. cells destroy tumor and virus infected cells
2. Natural killer cells secretes .............
3. Chemicals secreted by natural killer cells initiate a process known as
............., which induces the cells to die
Notes
4. .............a systemic reaction occurs if infection spreads to other parts of the
body
59.3 SPECIFIC IMMUNITY

T and B cells use antigen-specific receptors to recognize and destroy antigens.
To recognize antigen, part of the antigen must be presented to T-cells by an
antigen presenting cell (APC), such as macrophages and dendritic cells. After
engulfing and processing the antigen, the APC displays specific parts of the
antigen on its surface. The T-cell interacts with an antigenic site on the displayed
piece of antigen. T-cells have receptors that allow them to recognize and bind
to specific antigenic sites. Thus, a large repertoire of T-cell receptors must be
produced to adequately cover the large range of pathogens that will be
encountered over the life span. When a T-cell receptor recognizes an antigenic
site, it triggers proliferation and differentiation processes which normally occur
in the lymphoid tissues. The T-cell rapidly divides to yield an army of T-cells
with antigen-specific receptors that perform different tasks. Two classes of
Tcells, helper and cytotoxic T-cells, are distinguished by CD4+ and CD8+
molecules on their surface, respectively. Both types of T-cells act to contain
intracellular pathogens, but they also perform distinct tasks. T-helper cells
coordinate the immune response by assisting in antigen recognition and by
secreting cytokines that activate of other T and B-cells to increase their numbers.
Cytotoxic T-cells are able to kill virus-infected cells or tumor cells and thus play
a major role in antiviral and antitumor activity. Another class of T-cells, known
as suppressive Tcells, can actively inhibit the actions of other T-cells through the
secretion of suppressive cytokines. In the case of B-cells, they differentiate into
plasma cells that secrete antibody. This process is normally triggered by antigen
binding and helper T-cell activity. These plasma cells rapidly divide and secrete
antibodies, immunoglobulin molecules that act as receptors for antigen. These
are soluble molecules that circulate in the blood where they can inactive antigen
through binding or mark it to be destroyed.
Thus far we have described the primary immune response that is initiated when
the immune system does not have prior experience with the antigen. During the
primary response, a subset of lymphocytes differentiates into memory T and B-
cells and remain in circulation for many years to provide immunity from
diseases. Upon exposure to the antigen, memory T and Bcells respond quickly
to eliminate the antigen, a process known as the secondary immune response.
MICROBIOLOGY 519
Microbiology Immune-related diseases

The importance of the immune system is illustrated by immunodeficiency and
autoimmune diseases. Persistent infection of the immune system by human
immunodeficiency virus (HIV) leads to acquired immune deficiency syndrome,
AIDS. HIV evades detection by hiding in immune cells. It destroys helper T-cells
through its direct cytotoxic effects and by triggering cytotoxic T-cells. When
helper T-cell counts plummet, susceptibility to opportunistic infections increase,
Notes leading to AIDS and eventual death. In autoimmune diseases, normal immune
responses are directed against a self-antigen. For example, T-cells may lose their
ability to distinguish between self and non-self due to the development of
autoreactive receptors that bind self-antigen. Such self-reactive T-cells attack
healthy tissues of the body causing diseases such as multiple sclerosis, type-I
diabetes, Lupus, and rheumatoid arthritis. Other work suggests that suppressor
T-cells may lose their ability suppress the actions of cytotoxic T cells in
autoimmune disease. By understanding the mechanisms mediating these
diseases, research may lead to the development of innovative strategies for
disease prevention and treatment.

z The immune system is in constant surveillance of pathogens
z Virulence of pathogen and competence of immune system determines the
development of disease.
z All cells of the immune system are derived from stem cells in the bone
marrow.
z B-cells remain in the bone marrow during development, selection, and
maturation. T-cells migrate in thymus gland for maturation.
z Pathogens are normally taken up by antigen-presenting cells like macrophages
and dendritic cells.
z Macrophages are widely distributed throughout the body where and act as
a first line of defence through phagocytosis.
z Neutrophils, eosinophils, and basophils are collectively known as
granulocytes.
z Innate or nonspecific immunity, and specific or adaptive immunity are two
functional system of immune systems.
z Innate immune is through inflammation and acute phase responses
z Specific immunity is by T and B lymphocytes.
z Natural killer cells are nonspecific lymphocytes for destroying tumor cells
and virus-infected cells.
520 MICROBIOLOGY
Microbiology
TERMINAL QUESTIONS
1. What the components of immune system
2. List the functions of immune system
3. What are natural killer cells
Notes
59.1
1. Stem & bone marrow
2. T & B
3. Macrophages, monocytes, mast cells & granulocytes
4. B
5. T
6. Macrophages & Dendrite cells
7. Macrophages
8. Neutrophils, eosinophils and basophils
59.2
1. Innate
2. Specific
3. Inflammation
4. Cytokines
59.3
1. Natural killer
2. Perforins
3. Apoptosis
4. Acute phase response
MICROBIOLOGY 521
MODULE Agglutination
Microbiology
60
Notes
AGGLUTINATION
60.1 INTRODUCTION
Agglutination is one of the antigen and specific antibody reactions which takes
place when the two are mixed in-vitro in laboratory in the presence of
electrolytes at a suitable temperature and pH. Agglutination word comes from
the Latin “agglutinare”, meaning "to glue,” it is also clumping of particles.
Examples of agglutination in biology are clumping of cells such as bacteria
(Widal test) or red blood cells (Blood grouping) in the presence of specific
antibody. The antibody binds multiple antigen particles and joins them, creating
a large lattice like complex which we can see with naked eye.
Agglutination reaction used for diagnosis of diseases in lab either uses the
particulate or soluble antigens. Example of agglutination reaction using
particulate antigens is Salmonella typhi bacteria to detect specific antibody in
serum from patient suffering from typhoid fever (Widal test). Example of
agglutination reaction is latex agglutination and other particle agglutination
tests. The soluble antigen is first made particulate by coating it on inert particles
like red cells, latex particles, gelatin particles and micro beads. These particles
support or carry the soluble antigens to make the reaction visible to naked eye.
Agglutination assays have good sensitivity, do not require sophisticated
equipment, are easy to perform, require no wash procedures and are cost
effective. The lattice network formed during agglutination reaction can be
visualised macroscopically or microscopically as per the directions of the
manufacturer.
OBJECTIVES
z define agglutination
522 MICROBIOLOGY
Agglutination MODULE
z discuss the process of agglutination Microbiology
z describe the various methods of agglutination

z read the result of agglutination reaction with naked eye and under the
microscope
z describe the various applications of agglutination
60.2 DEFINITION OF AGGLUTINATION Notes

Large antigens, carrying many epitopes, easily sedimented particles such as
animal cells, erythrocytes, or bacteria when mixed with specific antibodies, at
appropriate temperature and ionic strength solution result in cross-linking the
particles, forming a lattice like structure seen as clumps with naked eye. This
reaction which is sensitive and specific is termed agglutination. Agglutination
is a serological reaction like precipitation reaction; only difference is that antigen
is large and particulate in case of agglutination. Most common example of
agglutination is the testing for blood group.
You will see during practical laboratory exercise that agglutination is more
sensitive than precipitation. However, you can make precipitation reaction also
very sensitive by attaching/coating soluble antigens to large, inert carriers, such
as erythrocytes or latex beads so that precipitation reaction now becomes an
agglutination reaction.
60.3 HISTORY
Two bacteriologists namely, Herbert Edward Durham and Max von Gruber
discovered specific agglutination in 1896. This reaction was named as Gruber-
Durham reaction to honour the discoverers. Later, Gruber named any substance
that caused agglutination reaction as “agglutinin” (from the Latin).
Same year Fernand Widal (1862–1929) used agglutination for diagnosis of
typhoid fever. Widal found that blood serum from a typhoid carrier caused a
culture of typhoid bacteria to clump, whereas serum from a typhoid-free person
did not. Widal test is the first example of a sero diagnostic test for an infectious
disease.
Karl Landsteiner found another important practical application of the agglutination
reaction in 1900 i. e. for blood group (ABO) typing. This marked the beginning
of safe blood transfusion and the science of transfusion medicine.
60.4 PROCESS OF AGGLUTINATION

Agglutination is clumping together in suspension of cells bearing the antigen
(epitopes)/ antigen bearing microorganisms, or particles in the presence of
specific antibodies called “agglutinins”. We can picture a lock and Key concept
MICROBIOLOGY 523
Microbiology to understand the specificity of agglutination reaction. An antibody is a “Y”

shaped molecule. The two arms of “Y” are the Fab portion and this has the
combining site and is made of the hypervariable regions of the heavy and light
chains. The antigenic determinant nestles in a cleft formed by the combining site
of the antibody as illustrated in the Figure 60.1. So the antigenic determinant
is the “Key” which fits onto the cleft formed by the “Fab” which is the lock.
If the fit is appropriate then agglutination will happen. This concept is true for
Notes all antigen (Ag) antibody (Ab) reactions. The process of agglutination involves
two steps. First step is sensitization and second is lattice formation.
Fig. 60.1
60.4.1 Sensitization
It is attachment of specific antibody to corresponding antigen. pH, temperature
and time of incubation influence the reaction. IgM antibodies react best at 4 to
22 degrees C and IgG antibodies react best at 37 degrees C. Time of incubation
can range from 15 to 60 minutes.
60.4.2 Lattice formation:

Lattice is just like a “Jaal”. It is formed by cross linking between sensitized
particles. It takes more time than sensitization and we may be able to see the
result with naked eyes. IgM best at this type of reaction because of large size
but IgG antibodies may need enhancement.

1. Agglutination means ...................
2. Most common example of agglutination is testing for ...................
524 MICROBIOLOGY
3. Agglutination is clumping together of antigen with their specific antibodies Microbiology
called ...................
4. Attachment of specific antibody to antigen is ...................
60.4.3 Methods of enhancing agglutination

These include centrifugation (Bridges distance); treatment with enzymes
(Reduces Zeta Potential); colloids (Albumin, reduces zeta potential) and use of Notes
anti- human globulin. Electrokinetic potential in colloidal systems is termed as
Zeta potential. It is actually the degree of repulsion between adjacent, similarly
charged particles in a solution. A high zeta potential confers so it will resist
agglutination. So, reducing Zeta potential will favour agglutination.
60.4.4 Grading agglutination reactions

Grading may be macroscopic or microscopic. Follow the criteria given in the
package insert of the commercial kit. Example of blood grouping used in blood
banks is given below.
60.4.5 Macroscopic
Example blood grouping:
4+ One solid aggregate or clump of cells.
3+ Several large aggregates, clear background.
2+ Small to medium sized aggregates, clear background.
1+ Small aggregate, turbid reddish background.
+W Tiny aggregates, turbid reddish background.
MF Mixed Field – Any degree of agglutination in a sea of un-agglutinated cells.
Hem - Hemolysis is interpreted as a positive reaction and may be graded as
complete or partial. Both hemolysis and agglutination may be recorded on the
same tube.
Ø Negative - no agglutination, smooth reddish background.
60.4.6 Microscopic
+ Positive - aggregates of at least 3-5 cells.
Ø Negative - no agglutination.
MICROBIOLOGY 525
Microbiology
60.5 METHODS OF AGGLUTINATION
Agglutination test can be performed using three different techniques. These
include: rapid agglutination tests; slow agglutination tests in tubes; slow
agglutination tests in micro titration plates.
60.5.1 The rapid agglutination tests:

Notes This method involves mixing un-diluted patient/client serum and antigen on a
glass slide or plate, rotation or agitation of the plate as per the instructions given
in the kit literature, and macroscopic examination, usually after 2 minutes for
the presence of agglutination. The antigen and serum are usually mixed in fixed
proportion. The intensity of the agglutination indicates the concentration of
antibody in the serum. Sometimes strong agglutination reactions need to be
confirmed by heating the sera (56 C. for 30 minutes) to destroy non-specific
agglutinins or by repeating the test with various dilutions of the serum.
60.5.2 Slow agglutination in tubes/tube agglutination:

This involves dilution of the serum and mixing with fixed amount of unstained
antigen. The tubes are kept at temperature and for time (usually overnight) as
per instructions in the kit literature. The positive results are visualized by the
presence of a precipitate in the bottom of the tube and a clearing of the
supernatant (as compared to antigen without any serum).
60.5.3 Micro-agglutination:
This test is carried out using small amounts of antigen and patient serum as per
kit literature in a micro-titration plate. A large number of samples/ various
dilutions can be tested at a time in one plate. The positive reaction is indicated
by formation of a ragged blanket of coloured antigen covering the bottom of the
U-shaped micro-titre well. The negative result appears as a button of un-reacted
antigen in the well.
Note: The agglutination tests can be “Qualitative agglutination test” -
agglutination test used to detect the presence of an antigen or an antibody. The
antibody is mixed with the particulate antigen and a positive test is indicated by
the agglutination of the particulate antigen. e.g. a patient’s red blood cells mixed
with antibody to a blood group antigen to determine a person’s blood type.
“Quantitative agglutination test” - agglutination tests used to quantitate the level
of antibodies to particulate antigens. Serial dilutions of a sample to be tested for
antibody are mixed with fixed number of red blood cells or bacteria or other such
particulate antigen and the last/highest dilution showing agglutination is the
amount of antibody in the sample and is expressed as the titer. The results are
reported as the reciprocal of the maximal dilution that gives visible agglutination.
526 MICROBIOLOGY
Microbiology
60.6 MISCELLANEOUS TYPES OF AGGLUTINATION
The agglutination of a particulate antigen by antibody raised against a different
but related antigen is termed Cross agglutination; agglutination of members of
a group of biologically related organisms (bacteria) or corpuscles by an
agglutinin specific for that group is called “Group agglutination” and clumping
of particulate elements within the blood vessels/red blood cell aggregation
within the blood vessels is called “Intravascular agglutination” Notes

1. Qualitative Agglutination (a) to measure the level of antibodies
2. Cross (b) members of biologically related organism
3. Group (c) to detect presence of antigen / antibody
4. Quantitative (d) clumping within blood vessels
5. Intra vascular (e) antibody against related antigen
60.7 PROZONE AND POST ZONE PHENOMENA

False negative antigen antibody reaction, either agglutination or precipitation,
can occur if antigen and antibody are not mixed in the right proportions. This
can happen if either antibody is in excess (Prozone) or when antigen is in excess
(Post zone).
60.7.1 Prozone phenomenon:

Some sera when tested un-diluted, do not show agglutination. The same sera
when tested after making dilution show a positive agglutination/precipitation
reaction. This phenomenon is called “Prozone phenomenon” in which
agglutination or precipitation occurs at higher dilution ranges of serum, but is
not visible at lower dilutions or when undiluted. Excessive levels of antibody
result in false negative reaction as antibody excess results in formation of very
small complexes which do not clump to form visible agglutination. Prozone
reaction is the probable cause of false-negative result. Prozone reaction can also
result from presence of blocking antibody or to nonspecific inhibitors in serum.
When different antigens are located close to each other, the antibodies
corresponding to each antigen may block binding by and competing with each
other
MICROBIOLOGY 527
Microbiology 60.7.2 Post-zone phenomenon:

This refers to the reaction wherein excess of antigen results in no lattice
formation and a false negative agglutination reaction. Antigen excess is also the
probable cause of false-negative antigen-antibody agglutination/precipitation
reaction.
Notes
1. Electrokinetic potential in colloidal system is termed as ...............
2. Reduction of ............... favours agglutination
3. In rapid agglutination test the intensity of agglutination indicates ...............in
the serum
4. In slow agglutination test, a positive results is by presence of ...............
5. In Micro agglutination, positive reaction is indicated by formation of
...............
6. False negative reaction because of high antibody is termed as ...............
7. False negative reaction because of high antigen is term as ...............
60.8 APPLICATIONS OF AGGLUTINATION

We hope that by now you understand what agglutination is and how it happens
and how we read the results. Let us now try to understand what the importance of
agglutination reaction in medicine is. Agglutination testing helps you to find
either antigens or antibodies in a sample. The sample can be any body fluids such
as urine, blood, saliva, and cerebrospinal fluid (CSF) in which either we want to
detect antigen or antibody, the sample can also be bacterial culture which we
want to type to know whether it is pathogenic or not. The first step is to collect the
sample. Blood is collected by venipuncture following standard work precautions;
CSF is collected by the clinician by lumbar puncture and urine is collected by
patient using the clean catch method. In blood agglutination test, a sample of
blood is collected from the patient vein by a method called venipuncture.
Applications of agglutination in clinical medicine include:
60.8.1 Typing blood cells of the recipient and donor for blood transfusion
60.8.2 To identify and type bacterial cultures
60.8.3 To detect the presence of specific antibody and quantitate the amount of
antibody in patient’s serum
528 MICROBIOLOGY
60.8.4 Latex agglutination Microbiology
60.8.5 Haemagglutination.
Let us discuss these applications one by one.
60.8.1 Typing blood cells of the recipient and donor for blood transfusion:
ABO and Rh blood grouping
Notes
Principle and application
Agglutination of red cells with known anti sera (antibody) indicates presence of
the corresponding antigen on the red cells. Serum can also be tested with known
red cells (antigen) to determine the presence or absence of specific antibodies.
ABO blood groups are classified as A, B, AB, or O depending on the presence
or absence of the A or B antigens on the red cells and presence or absence of
the corresponding anti-A or anti-B antibodies. However, individuals are
classified as Rh (D) positive or negative depending on the presence or absence
of Rh (D) antigen only. There are various methods of performing this test, such
as slide, test tube, microplate or Column Agglutination Technique. Hence it is
a process to determine presence or absence of ABO & Rh (D) antigens on donor/
patient red cells and presence or absence of corresponding antibodies in serum.
We describe here the conventional slide, test tube and Column Agglutination
Technique.
Materials required
z EDTA blood 2 ml. from donor and patient in EDTA bottle;
z Commercial blood grouping anti sera (Anti-A, -B, -D, -D blend)
z Reagent (pooled) red cells (O-cell, A-cell, B-cell), prepared in-house.
z Anti-A/Anti-B/Anti-D cards and Reverse Diluent Cards
z Test tubes 10 × 75 mm
z Normal saline (0.9 % NaCl)
z Test tube rack
z Pasteur pipette/Automated pipettes
z Table top centrifuge
z 370C dry incubator
z Incubator, Centrifuge
z Gloves
z Glass slides/applicator sticks
MICROBIOLOGY 529
Microbiology Procedure
Procedure for ABO cell grouping (slide method)

z Confirm the identity of blood sample by checking registration number and
name of the patient/donor.
z Label the slide with donor/patient name or number.
Notes z Put 1 drop of Anti-A, 1 drop of Anti-B and 1 drop of anti D anti serum
on left, middle and right portion of slide.
z Add a small drop of the approximately 50 % suspension of red cells/
capillary blood to each portion of the slide.
z Mix well with a applicator stick.
z Rock the slide in clock-wise/anti-clock-wise direction to see agglutination.
z Record in the register/form.
z Rock the slide in clock-wise/anti-clock-wise direction to see agglutination.
z Record in the register/form.
Procedure for ABO cell grouping (tube method)

z Confirm the identity of blood sample by checking registration number and
name of the patient/donor.
z Prepare 2 - 5% suspension of the EDTA red cell in normal saline
z Label three test tubes with patient name or number and tube contents (-A,
-B and –D).
z To the test tube labeled A, add 1 drop of Anti-A anti serum. To the tube
B, add 1 drop of Anti-B and to the tube labeled D add 1 drop of anti D
anti serum.
z Add 1 drop of the 2-5 % suspension of red cells to each of the test tube.
z Mix well and centrifuge the test tubes for 1 min at 1000 rpm.
z Re-suspend the cells with gentle agitation and examine macroscopically for
agglutination
Determination of and Rh (D) by column agglutination technology

z Identify the appropriate microtube of ABD and Reverse Diluent Cassette
with the donor/patient’s name/UHID number.
z Remove aluminum foil from the top of microcolumn(s).
z Prepare 2-5 % of red cell suspension of donor/patient/reagent red
cells(Ac,Bc,Oc) by adding 40µl of packed washed red cells to 1000 µl of
NS
530 MICROBIOLOGY
z Add 10 µl of reagent red cell suspension in the identified microcolumn Microbiology
along the wall
z Add 40 µl of serum in reverse diluents cassette.
z Centrifuge the cassette X 5 min in column agglutination centrifuge
z Read and grade the reaction as per manufacturer’s instructions.
Procedural notes Notes
z All the test tubes must be properly labeled.

z It is important to follow the manufacturer’s instruction for the specific
antisera in use.
z Do not perform the test at the temperature higher than room temperature
(22--24°C).
z Observe agglutination against a well lighted back ground.
z Remember that contaminated blood specimens, reagents or supplies may
interfere with the test results.
60.8.2 To identify and type bacterial cultures

When we isolate bacteria from a sample say blood of a patient suffering from
fever, it is important to know whether the bacteria isolated are Salmonella typhi
or Acinetobacter or any other bacteria. To identify the bacteria we carry out
biochemical reactions which will suggest/to some extent the bacteria we are
dealing with. However, to know the exact identity of bacteria we have to type
the bacteria using the Salmonella typhi “O antigen” specific and “H antigen
specific” sera available commercially. For this identification a drop of suspension
of bacterial isolate in normal saline is mixed with a drop of Salmonella typhi
“O antigen” specific and “H antigen specific” sera on a glass slide. Mix the two
with nichrome loop or a wooden stick and look for macroscopic agglutination
with naked eye. Positive agglutination with Salmonella typhi “O antigen”
specific and “H antigen specific” sera means the isolated organism is Salmonella
typhi and patient is suffering from typhoid fever. Same way using genus specific,
species specific, strain specific commercially available antisera against many
pathogens (e. g. Pneumococci, Meningococci and others), the bacteria isolated
from patient samples can be identified.
60.8.3 To detect the presence of specific antibody and quantitate the

amount of antibody in patient’s serum
One such example is the slide and tube Widal test. The agglutinins against ‘0’
(somatic) and ‘H’ (flagellar) antigens of Salmonella typhi, paratyphi A and
MICROBIOLOGY 531
Microbiology paratyphi B are estimated qualitatively (slide test) and quantitatively (Tube test)
employing killed suspension of appropriate organisms.
Widal-quantitative tube agglutination test

This test is done to detect antibody against Salmonella typhi, S paratyphi A and
S paratyphi B to aid in the diagnosis of enteric fever.
Notes Specimen: Blood sample 3-5 mL is collected in sterile dry screw capped
unbreakable tubes and transported to the lab in upright position or stored in
refrigerator (2-8°C) in case of delay. Sample can be centrifuged at 3000 rpm for
10 minutes at room temperature to remove particulate matter if required, before
performing the test.
Material and instruments required

z S. Typhi ‘o’ antigen suspension.
z S. Typhi ‘h’ antigen suspension.
z S. Typhi ‘ah’ antigen suspension.
z S. Typhi ‘bh’ antigen suspension.
z Blood sample 3-5 ml
z Plain vacutainer/sst
z Normal saline
z Micropipettes
z Glass test tubes
Reagents: Commercially available test kits are used. The kit must be stored at
2 – 80°C. Follow the instructions given in the kit insert to perform the Widal
tube agglutination test.
Test procedure
Step Action
1. For each serum sample under test ,arrange four rows of 4
tubes each in a rack
2. Prepare master dilutions by taking 4 tubes in another rack.
Place 7 ml of normal saline (0.85% sodium chloride) in the
first tube and 3.5.ml in each of the remaining four tubes.
3. Add 0.5.ml of serum to the first tube and mix well.
4. Transfer 3.5.ml from the first tube to the next tube and mix
well
532 MICROBIOLOGY
5. Continue successive transfer of 3.5.ml quantities till the last Microbiology
tube is reached.
6. This will give final dilutions of 1:30,60,120,240 after the
addition of equal volume of antigen
7. Transfer 0.5.ml quantities from the master dilution tubes to
each tube of the corresponding vertical row in test rack .Put
0.5 ml of normal saline in each of the tubes in the last (i.e,5th
row) to serve as controls. Notes
8. To each of the 4 tubes in the first, second, third and fourth

horizontal rows, add 0.5.ml of S.Typhi “O” S typhi “H”,S
paratyphi A (H) and S.paratyphi B(H) antigens respectively.
9. Shake the rack well to mix and incubate at 37ºC overnight
(16-20 hours).
10. Note the highest dilution in which there is evidence of
agglutination as observed by naked eye or a hand lens .With
‘H’ antigens, the pattern of agglutination is floccular –
cottonwool type whereas with ‘O’ antigen it is granular and
appears as a granular mat at the bottom of the tube
11. In addition to the pattern of sedimented organisms, the
decrease in opacity of the supernatant as compared to the
saline control tube must be observed and taken into account
while judging the degree of agglutination.
Internal quality control

With every batch of test samples the following controls are put up
Negative Control: Saline control tubes in the last row
Positive Control: Known positive pooled serum
Interpretation
Sera from normal individuals may agglutinate these antigens in dilutions up to
1: 60. Agglutination titers of 1:120 and more are significant and rise in titers
or repetition of the test after a few days will confirm the diagnosis of enteric
fever. Agglutination titers of 1:240 and above are typically found in cases of
enteric fever. However, follow the instructions in the kit insert for
interpretation.The specific organism responsible is determined by noting the ‘H’
agglutinin titre.
Persons who have suffered from enteric infections in past or who had received
TAB vaccine may show appearance of agglutinins in moderate titer when
MICROBIOLOGY 533
Microbiology suffering from other unrelated illness. Such anamnestic appearance of agglutinins
can be differentiated from true infection by demonstrating the marked rise/fall
in the titer when the test is repeated after 7-10 days. A moderate rise in titer of
all three ‘H’ agglutinins simultaneously against all ‘H’ antigens is suggestive of
recent TAB vaccination.
Safety: All specimen used in this test should be considered potentially
infectious. Standard work precautions (gloves) should be used for handling and
Notes disposal of materials during and after use. Use soap for routine hand washing.
60.8.4 Latex agglutination

As already discussed soluble antigens are coated on inert particles like latex to
develop sensitive, specific easy to perform rapid tests termed the latex
agglutination tests. Many latex agglutination tests are commercially available
and are used to detect either specific antigens or antibody against specific
bacteria to diagnose various diseases. Always follow the instructions given in
the kit insert to perform the test.
One such test is Cryptoccus antigen detection test. Cryptoccus antigen test is
a simple, qualitative or semi-quantitative, test to detect polysaccharide antigens
associated with Cryptococcus neoformans infection. Serum or cerebrospinal
fluid (CSF) may be used as specimens. Sample of operative procedure based on
a commercial kit “Remel Cryptococcus Antigen Latex Test” is given below.
Specimen:
Serum or cerebrospinal fluid (CSF).
Principle of the test:

Cryptococcus antigen test kit incorporates the use of latex particles sensitized
with murine IgM monoclonal antibodies. Cryptococcal polysaccharide (CPS)
antigens in patients serum or CSF interacts with sensitized latex particles
producing visible agglutination.
Safety precautions:
z All specimen used in this test should be considered potentially
infectious. Standard work precautions should be followed for handling and
disposal of materials during and after processing the specimen.
z Use soap for routine hand washing.
z Follow Hospital Guidelines for biohazard waste disposal.
534 MICROBIOLOGY
Reagents and materials Microbiology
Reagents
z Test latex: Latex particles sensitized with IgM anti-CPS monoclonal
antibody suspended in a buffer and preserved in 0.01% thimerosal (1 × 2.5
ml).
z Negative control: Normal human serum in a buffer, preserved in 0.1%
sodium azide (1 × 0.8 ml). Notes
z High positive control: Contains approx 50 mg/ml of C. neofomans CPS
antigen preserved in 0.1% sodium azide. (1 × 0.8 ml)
z Low positive control: Contains approx 12 mg/ml of C. neoformans CPS
antigen preserved in 0.1% sodium azide. (1 × 0.8 ml)
z Protease: 1 enzyme tablet contained in a vial. (reconstitutes to 3 ml)
z Specimen diluent: 10x NaCl/Glycine solution preserved in 1.0% sodium
azide. (1 × 10 ml-dilute to 1x).
z Reaction cards: (12 × 6 circles)
z Dispensing pipette- 50 ul
Equipments
z Centrifuge
z Micropipette for serial dilution of specimens.
z Test tubes.
z Timer
z Boiling water bath
z Graduated cylinder
z Vortex mixer.
Procedure
A) Reagent preparation
Specimen diluent: The specimen diluent is provided in 10x concentration.
Prepare a working strength solution (1x concentration) in a separate bottle by
combining the entire contents of the 10x specimen diluent bottle with 90 ml of
demineralized water. Mix the working strength specimen diluent and label with
the expiration date on the 10x specimen diluent. Use as needed or store at
2-8°C.
Protease: Prepare the protease solution by adding 3 ml of working strength
specimen diluent to the vial containing the enzyme tablet. Allow 30 minutes for
MICROBIOLOGY 535
Microbiology complete reconstitution, swirling the vial at least twice during this time. Label
the vial with the date of reconstitution. Protease solution can be stored at 2-8°C
for 1 month from the date of reconstitution. Alternatively aliquot the solution
into appropriate capped and labeled tubes and store at <=–20°C until the
expiration date of the kit.
B. Sample preparation
Notes
CSF:
z Heat CSF in a boiling water bath (100°C) for 5 min.

z Allow contents to cool to room temperature.
z Vortex contents of tube before testing.
Serum:
z If necessary dilute specimen diluent (10x) to the 1x working strength.
z If necessary reconstitute the Protease with 3 ml of 1 x specimen diluent.
z Dispense 100-200 ul of serum in a tube and add an equal of Protease.
z Cap and seal the tube. Mix by vortexing.
z Place capped tube in a boiling water bath (100°C) for 10 minutes.
z Allow contents to cool to room temp and gently mix before testing.
Note: If flocculation is observed before and/or after Protease treatment
centrifuge the specimen at 3000 rpm for 10 minutes at room temp. Decant the
supernatant (50 μl) to avoid aspirating the pellet.
Qualitative test
z Re suspend the test latex by rapidly inverting the bottle several times.
Dispence one drop of the Test latex into a sepatate test circle for each
specimen and control to be tested.
z Dispense one drop of each well mixed control into a separate, test circle
containing the test latex. Use the paddle end of a separate pipette to
thoroughly mix each control and test latex. Discard each pipette after this
step.
z Using pipettes provided in the test kit dispense one drop of each pretreated
patient specimen (approximately 50 μl) into a test circle containing Test
latex. With the paddle end of the pipette, thoroughly mix the specimen and
Test latex, spreading over the entire area of the circle. Discard the pipette
after this step.
536 MICROBIOLOGY
z Place the card on a clinical rotator set to rotate at 100 to 110 rpm for 5 Microbiology
minutes.
z Immediately following the 5-minute rotation tilt the slide to obtain a flow
pattern and carefully examine each circle for any agglutination (see results
section below) and record the results.
z Compare the specimen test reaction to a negative control reaction. Record
the results.
Notes
Semi-quantitative testing of positive specimen:
z If necessary dilute the 10x Specimen Diluent to the working strength.
z Obtain and mark eight tubes 1 to 8. Add 0.1 ml of specimen diluent to
each tube.
z Add 0.1 ml of heat-treated CSF, or protease-treated serum contents of tube
1 and transfer 0.1 ml to tube 2. Do not mix the contents of tube 2 with
the pipette.
z With a clean pipette, thoroughly mix the contents of tube 2 and deliver 0.1
ml to tube 3. Do not mix the contents of tube 3. Do not mix the contents
of tube 3 with the pipette.
z Follow this method to produce serial, doubling dilutions of the specimen
through tube 8. The serum dilutions which have been established are from
1:4 to 1:512 for tubes 1 to 8 respectively, For CSF 1:2 to 1:256 dilutions
are made. Tube 8 can be diluted further if an endpoint is not reached.
z Test each specimen dilution following the protocol as described in the
Qualitative Testing section.
Result
Qualitative test result: Any test latex clumping or cleaning, observed immediately
after the 5-minute rotation step, is considered a positive result. The absence of
agglutination of the test latex is considered a negative result. Test latex particles
should appear as a milky suspension similar to the pattern produced by the
negative control following the 5-minute rotation step.
Semi-quantitative test results: When positive specimens are examined by serial
dilution the titer is the reciprocal of the last dilution which produces a positive
result (agglutination).
Quality control
The High positive, Low positive and Negative controls should each be tested
with every test run of patient specimens as described under qualitative testing.
MICROBIOLOGY 537
Microbiology The high positive and low positive controls must agglutinate the test latex
differentially.
z The High positive control must produce strong agglutination.

z The low positive control must produce weaker but clearly visible
agglutination.
z The Negative control must not produce any agglutination, although trace
Notes
granularity is acceptable.
60.8.5 Haemagglutination:
As the name indicates this is agglutination of RBCs as such by RBC antigen
specific antibody (example of ABO blood grouping given under 8.1), or
agglutination of RBCs coated with some antigen to detect specific antibody in
patient’s serum (example is Treponema Pallidum Haemagglutination test). The
antibodies/proteins which agglutinate RBCs are called Haemagglutinins.
Materials Required
1. Adjustable multi-channel micropipette (50-300 µl)
2. Adjustable single-channel micropipette (50-200µl)
3. Adjustable single-channel micropipette (5-50µl)
4. Disposable tips
5. Reagent troughs
6. Waste discarders
Reagents and test kits

1. Micro plates (U bottom)
2. Sample diluents
3. Control cells (uncoated)
4. Test cells (sensitized with T.pallidum antigen)
5. Kit controls
Procedure
1. Approximately 30 min prior to the beginning of the test procedure; bring
kit components to room temp. (15-30°C.). Mix the liquid reagents gently.
Determine the total no. of specimens to be tested and no. of plates required
for the assay.
538 MICROBIOLOGY
2. Put a unique plate Id on the upper-middle side of plate, if required Microbiology
3. Label the plate with last three digits of donation Id on left lower side of
well, identifying the positions. Include one negative control & one positive
control per batch of specimens.
4. Arrange the samples according to plate map in a sampling rack.
5. Use first extra well according to number of specimens and add 190µl sample
diluent. Notes
6. Then using a fresh pipette tip for each addition, take 10µl of specimen and
mix it with 190µl sample diluent in the same position in the extra well/
plate.
7. Then transfer 25ml of the diluted sample in both control & test wells.
8. After sampling put the sample in the same position in the same/different
sampling rack.
9. Add 25µl negative & positive controls to their respective positions.
10. Mix the bottles of control & test cells gently to make homogenous
suspension & add 1 drop (75µl) of test cells & control cells to their
respective wells including positive & negative control wells.
11. Mix the contents of the well by rotating the plate slowly. Keep the plate
on a smooth steady surface. Read the results after 1 hr. of incubation at room
temp.
Interpretation of the test result

1. All control wells should have compact button formation. If any control well
doesn’t show button formation repeat the test to exclude any technical error.
(Agglutination of the control cells as well as the test cells indicates the
presence of anti-cell antibody and in this event the test is not valid & should
be repeated after first performing absorption of the test serum. To achieve
this dilute the test serum 1/4 with control cells & allow standing at room
temp. After centrifuging the sample (1000 rpm/5 min.). Dilute the supernatant
1/5 in diluent. Test this dilution directly, without any further dilution, using
test & control cells suspensions).
2. If there is a compact button in a test well, the result is considered non
reactive.
3. If there is a characteristics ring pattern or net of the cells in the and there
is a compact button formation in the control well, specimen is considered
as reactive for T. pallidum.
MICROBIOLOGY 539
Microbiology 4. All positive tests should be repeated by TPHA.

5. If any sample is positive by repeat test also, identify the sample and separate
out the sample and retest with RPR.
Agglutination has been commonly used to determine whether a patient had or
has a bacterial infection. For example, if a patient is suspected of having typhoid
fever, the patient’s serum is mixed with a culture of Salmonella typhi. If an
agglutination reaction occurs, shown as clumping of the bacteria, the patient
Notes
either had or has an S. typhi infection. Since certain antibodies can persist in
a patient’s blood for years after the patent has recovered from the infection, a
positive reaction does not mean that the patient currently has the infection. To
determine whether a patient is currently suffering from typhoid fever, the amount
or titer of the antibody will be determined at the onset of illness and two weeks
later.

z Agglutination word means to glue
z Agglutination is one of the antigen and specific antibody reactions in-vitro
in laboratory in the presence of electrolytes at a suitable temperature and
pH.
z Agglutination is used for diagnosis of diseases in laboratory either uses the
particulate or soluble antigens.
z Agglutination involves two steps. First step is sensitization and second is
lattice formation.
z Sensitization is the atachment of specific antibody to corresponding antigen.
z The degree of repulsion between adjacent similary charged particles in a
soluiton is termed as zeta potential.
z Reducing Zeta potential will favour agglutination.
z Rapid agglutination, slow agglutination in tubes; slow agglutination in
micro titration plates are the different techniques Agglutination
z False negative antigen antibody reaction, either agglutination or precipitation,
can hppen if either antibody is in excess (prozone) or when antigen is in
eecess (post zone).
z Clinically agglutination is used
Typing blood cells of the recipient and donor for blood transfusion
To identify and type bacterial cultures
540 MICROBIOLOGY
Microbiology
TERMINAL QUESTIONS
1. Define agglutination
2. Describe the process of agglutination.
3. Explain the methods of agglutination.
4. Explain prozone and postzone phenomena. Notes
5. List the clinical applicaiton of agglutination.
6. Describe ABO and RH blood grouping in brief

60.1
1. To glue
2. Blood group
3. Agglutinins
4. Sensitization
60.2
1. (c)
2. (e)
3. (b)
4. (a)
5. (d)
60.3
1. Zeta potential
2. Zeta potential
3. Concentration of antibody
4. Precipitate in the bottom & clearing of supernatant
5. Ragged blanket of coloured antigen
6. Pro-zone phenomenon
7. Post-zone phenomenon
MICROBIOLOGY 541
MODULE Complement Fixation Test
Microbiology
61
Notes
COMPLEMENT FIXATION TEST
61.1 INTRODUCTION
Jules Bordet’s pioneering research made clear the exact manner by which serums
and antiserums act to destroy bacteria and foreign blood cells in the body, thus
explaining how human and animal bodies defend themselves against the
invasion of foreign elements. Bordet was also responsible for developing
complement fixation tests, which made possible the early detection of many
disease-causing bacteria in human and animal blood.
OBJECTIVES
After reading this chapter, the student will be able to:
z describe the term Complement
z explain the principle of Complement Fixation Test
z describe steps involved in the Complement Fixation Test
z enlist the uses of Complement Fixation Test
z describe the modifications of Complement Fixation Test
61.2 COMPLEMENT FIXATION TEST

In 1894, Richard Pfeiffer, a German scientist, had discovered that when cholera
bacteria were injected into the peritoneum of a guinea pig immunized against
the infection, the pig would rapidly die. This bacteriolysis, Bordet discovered,
did not occur when the bacteria was injected into a non-immunized guinea pig,
but did so when the same animal received the antiserum from an immunized
animal. Moreover, the bacteriolysis did not take place when the bacteria and the
antiserum were mixed in a test tube unless fresh antiserum was used. However,
542 MICROBIOLOGY
Complement Fixation Test MODULE
when Bordet heated the antiserum to 55 degrees centigrade, it lost its power to Microbiology
kill bacteria. Finding that he could restore the bacteriolytic power of the
antiserum if he added a little fresh serum from a non-immunized animal, Bordet
concluded that the bacteria-killing phenomenon was due to the combined action
of two distinct substances: an antibody in the antiserum, which specifically acted
against a particular kind of bacterium; and a non-specific substance, sensitive
to heat, found in all animal serums, which Bordet called “alexine” (later named
“complement”). Notes
In a series of experiments conducted later, Bordet also learned that injecting red
blood cells from one animal species (rabbit cells in the initial experiments) into
another species (guinea pigs) caused the serum of the second species to quickly
destroy the red cells of the first. And although the serum lost its power to kill
the red cells when heated to 55degrees centigrade, its potency was restored when
alexine (or complement) was added. It became apparent to Bordet that
haemolytic (red cell destroying) serums acted exactly as bacteriolytic serums;
thus, he had uncovered the basic mechanism by which animal bodies defend or
immunize themselves against the invasion of foreign elements. Eventually,
Bordet and his colleagues found a way to implement their discoveries. They
determined that alexine was bound or fixed to red blood cells or to bacteria
during the immunizing process. When red cells were added to a normal serum
mixed with a specific form of bacteria in a test tube, the bacteria remained active
while the red cells were destroyed through the fixation of alexine. However,
when serum containing the antibody specific to the bacteria was destroyed, the
alexine and the solution separated into a layer of clear serum overlaying the
intact red cells. Hence, it was possible to visually determine the presence of
bacteria in a patient’s blood serum. This process became known as a complement
fixation test. Bordet and his associates applied these findings to various other
infections, like typhoid fever, carbuncle, and hog cholera. August VonWasserman
eventually used a form of the test (later known as the Wasserman test) to
determine the presence of syphilis bacteria in the human blood.
The complement fixation test (CFT) was extensively used in syphilis serology
after being introduced by Wasserman in 1909. It took a number of decades before
the CFT was adapted for routine use in virology.
CFT meet the following criteria
z it is convenient and rapid to perform
z the demand on equipment and reagents is small
z a large variety of test antigens are readily available.
However, there is now a trend to replace the CFT with more direct, sensitive
and rapid techniques, such as RIAs and EIAs. Although CFT is considered to
MICROBIOLOGY 543
Microbiology be a relatively simple test, it is a very exacting procedure because variables are
involved
Guinea pig is the commonest source of fresh complement. The serum should be
collected from guinea pig just before the test because complement is easily
destroyed by heat. However, complement can be preserved either by lipophilizing,
freezing or by adding preservatives. Preserved complement is also obtained from
Notes commercial sources. Complement should be titrated for its haemolytic activity.
One unit or minimum haemolytic dose (MHD) is the highest dilution of the
guinea pig serum that lyses one unit volume of washed sheep red blood cells
in the presence of excess of haemolysin (amboceptor) in either 30 or 60 minutes,
at 37C. Physiological saline with added magnesium and calcium ions is used
as the diluent for titration and CFT.
Fig. 61.1: Complement fixation
Complement fixation test consists of a test system and an indicator system, both
of which can activate complement. When used to detect the presence of an
antibody the test system is formed by the patient’s serum and a known antigen.
The indicator system is formed by sheep red blood cells coated with rabbit
antibody to sheep red cells (amboceptors). The sheep red blood cells will lyse
in the presence of complement.
z Sheep red cells: 5% suspension of washed sheep red blood cells should be
used.
z Haemolysin (amboceptors): it is an antibody to sheep red cells which raised
in rabbit. It should also be titrated for haemolytic activity. The MHD of the
amboceptor is the highest dilution of the inactivated an amboceptor, which
lyses one unit volume of sheep red blood cells in the presence of excess
complement in 30 or 60 minutes at 37°C.
CFT consists of two steps:

Step 1: a known antigen and inactivated patient’s serum are incubated with a
standardized, limited amount of complement. If the serum contains specific,
complement activating antibody the complement will be activated or fixed by
the antigen-antibody complex. However, if there is no antibody in the patient’s
serum, there will be no formation of antigen-antibody complex, and therefore
complement will not be fixed. But will remain free.
544 MICROBIOLOGY
Microbiology
Notes
Fig. 61.2: Complement Fixation Test
Step 2: The second step detects whether complement has been utilized in the
first step or not. This is done by adding the indicator system. If the complement
is fixed in the first step owing to the presence of antibody there will be no
complement left to fix to the indicator system. However, if there is antibody in
the patient’s serum, there will be no antigen-antibody complex, and therefore,
complement will be present free or unfixed in the mixture. This unfixed
complement will now react with the antibody- coated sheep red blood cells to
bring about their lysis. Thus, no lysis of sheep red blood cells (positive CFT)
indicates the presence of antibody in the presence of antibody in the test serum,
while lysis of sheep red blood cells (Negative CFT) indicates the absence of
antibody in the serum.
Controls should be used along with the test to ensure that
(a) Antigen and serum are not anti complimentary
MICROBIOLOGY 545
Microbiology (b) The appropriate amount of complement is used and

(c) The sheep red blood cells do not undergo autolysis.
Notes
Complement Fixation Test in Microtiter Plate, rows 1 and 2 exhibit complement

fixation obtained with acute and convalescent phase serum specimens,
respectively. (2-fold serum dilutions were used) The observed 4-fold increase
is significant and indicates infection.
Advantages of CFT
1. Ability to screen against a large number of viral and bacterial infections
at the same time.
2. Economical.
Disadvantages of CFT
1. Not sensitive - cannot be used for immunity screening
2. Time consuming and labor intensive
3. Often non-specific e.g. cross-reactivity between HSV and VZV
Modifications of complement fixation test

(a) Indirect complement fixation test: This modification is used when
serums which don’t fix guinea pig complement is to be tested. Here, the
test is set up in duplicate. After step 1, standard antiserum to antigen which
is known to fix complement is added to one set. If antibodies were not
present in the test serum then the antigen would react with the standard
antiserum fixing the complement. On the other hand if antibodies are
present in the test serum the antigen would be utilized in the first step.
So, no reaction would occur between the standard antiserum and the
antigen and therefore no fixation of complement would cause lysis of sheep
red blood cells. Thus in this case haemolysis indicates a positive result.
(b) Congulatinating complement absorption test: Here horse complement
which is non-haemolytic is used. The indicator system used is sensitized
sheep red blood cells mixed with bovine serum. Bovine serum contains
546 MICROBIOLOGY
a beta globulin called conglutinin would also combine with this complement Microbiology
causing agglutination (conglutination) of the sheep red blood cells,
indicating a negative result.
(c) Immune adherence: When some bacteria (such as vibrio cholera or
treponemapallidum) combine with their specific antibody in the presence
of complement and some particles such as erythrocytes or platelets, they
adhere to the erythrocytes or platelets. This is called immune adherence.
Notes
(d) Immobilisation test: Here antigen is incubated with patient’s serum in
presence of complement. If specific antibody is present it would immobilize
the antigen. Eg.Treponema palladium immobilization test, considered gold
standard for the serodiagnosis of syphilis.
(e) Cytolytic tests: The incubation of a live bacterium with its specific
antibody in the presence of complement leads to the lysis of the bacteria
cells. This is the basis of vibriocidal antibody test used to measure anti-
cholera antibodies.

1. The complement fixation test was initially used in ................ serology
2. The commonest source of fresh complement is ................
3. The highest dilution of guinea pig serum that lyses red blood cells is called
................
4. ................ is the antibody to sheep red cells used in complement fixation
test
5. ................ is used in coagulatinating complement absorption test
6. When bacilli combine with specific antibody in the presence of complement
is called ................
7. Gold standard serodiagnosis of syphilis is by ................ test
8. ................ test is commonly used in diagnosis of cholera

z CFT was developed by Jules Bordet. Complement fixation test consists of
a test system and an indicator system, both of which can activate complement.
When used to detect the presence of an antibody the test system is formed
by the patient’s serum and a known antigen. The indicator system is formed
MICROBIOLOGY 547
Microbiology by sheep red blood cells coated with rabbit antibody to sheep red cells
(amboceptors). The sheep red blood cells will lyse in the presence of
complement. There exits modification of complement fixation test- Indirect
complement fixation test, Congulatinating complement absorption test,
Immune adherence, Immobilisation test, Cytolytic tests.
Notes
TERMINAL QUESTIONS
1. What do you understand by the term complement?
2. Describe in brief CFT.
3. Give the advantages anf the limitations of CFT
4. Explain in brief various modifications of complement fixation test with
proper examples.
5. Explain the term amboceptor.
6. Mention the advantages and disadvantges of CFT.
7. Enlist the modifications of complement fixation test
61.1
1. Syphillis
2. Guinea Pig
3. Minimum Haemolytic Dose
4. Amboceptors
5. Horse Complement
6. Immune Adherences
7. Immobilization
8. Cytolytic
548 MICROBIOLOGY
Immunofluorescence MODULE
Microbiology
62
Notes
IMMUNOFLUORESCENCE
62.1 INTRODUCTION
Immunofluorescence (IF) is one of the very common laboratory techniques used
in almost all disciplines of Biology including Medicine for diagnostics and
research. Way back in 1942 Coons and Kaplan did some experiments and
reported that the fluorescence dyes can be conjugated with antibodies and these
labeled antibody can be used as probes to detect and locate the antigen specific
to this antibody. Later this technique was named the Direct Fluorescent Antibody
test. This technique has many applications and has been used in diagnostics and
research. Immunofluorescence (IF) is used to detect specific proteins in cells that
may be in suspension (specimen), in culture, in tissues, on microbeads and
microarrays, etc. Fluorescein isothiocyanate (FITC) or tetramethyl rhodamine
isothiocyanate (TRITC) are the common fluorescent dyes which are chemically
conjugated with the antibody. The FITC labeled antibody can be made to bind
directly with the specific antigen (Direct fluorescence antibody test) or can be
made to bind indirectly with an antigen (Indirect fluorescence antibody test).
Direct immunofluorescence is used less frequently as the antibody against the
molecule of interest is chemically conjugated to a fluorescent dye, so for every
antigen to be detected, the specific antibody will have to be conjugated with
FITC. In indirect fluorescence the antibody specific for the molecule of interest
(called the primary antibody) is unlabeled, and a second anti-immunoglobulin
antibody directed toward the constant portion of the first antibody (called the
secondary antibody) is tagged with the fluorescent dye. Indirect fluorescence is
used more commonly as the tagged secondary antibody and can be used to detect
many different antigens. However, the primary antibody will have to be specific
for the antigen to be detected.
The fluorescence can be read as a qualitative result or quantitative result using
fluorescence microscopy. The fluorescence can also be quantified using a flow
cytometer, array scanner or automated imaging instrument.
MICROBIOLOGY 549
MODULE Immunofluorescence
Microbiology Recently a couple of advances have been made in fluorescence technology. One
is an improvement in optical filters, and the second is the use of fiber optics for
measuring fluorescence emission from small organisms. The optimum wavelength
for exciting fluorescence in FITC is 495 nanometers. FITC absorbs most of light
at 495 nanometers and then emits light at 525 nanometers. Till recently and even
now mostly ultraviolet or near ultraviolet light source is used to excite
fluorescence as the wavelength usually selected (365 nanometers) can be
Notes
separated easily from the emitted light at 525 nanometers.
OBJECTIVES
z describe the history of immunofluorescence
z discuss the principle of immunofluorescence
z describe the various types of immunofluorescence
z describe the methods of various types of immunofluorescence with examples
z practice “Quality control” for immunofluorescence
z interpret the result of immunofluorescence
z describe the various applications of immunofluorescence.
62.2 DEFINITION
Immuno Fluorence is defined as various techniques used for detecting an antigen
or antibody in a sample by coupling its specifically interactive antibody or
antigen to a fluorescent dye/compound, mixing with the sample, and then
observing the reaction under an ultraviolet-light fluorescence microscope.
The antibodies and or antigens are chemically linked to a fluorescence dye to

identify or quantify interactive antigens and or antibodies in a cell/tissue sample/
culture, etc.
A fluorescence microscope (Fig. 62.1 and Fig. 62.2) is required to read the result
of the test. There are a number of fluorochromes (fluorescence dyes) which can
be used in fluorescent microscopy. Different colours are emitted by different
dyes. FITC emits bright, apple green fluorescence as shown in Fig. 62.3.
550 MICROBIOLOGY
Microbiology
Notes
Fig. 62.1: Fluorescence microscope
Fig. 62.2: Basic structure of a fluorescence Microscope
Fig. 62.3: Immunofluorescence- Positive result is bright, apple green

fluorescence in case of FITC.
MICROBIOLOGY 551
Microbiology
62.3 PRINCIPLE OF IMMUNOFLUORESCENCE
We will first understand the basics of fluorescence and then go on to understand
immunofluorescence.
62.3.1 Fluorescence
Fluorescence is a type of luminescence. The fluorescent dyes/fluorochromes
Notes (having luminescent properties) absorb light of one wavelength and emit light
of a different wavelength extremely rapidly. Absorbed light has a higher energy
than the emitted fluorescence light, so the wavelength of the emitted light is
longer than that of the excitation light. Blue light (between 450 nm and 520 nm,
495 nm is optimal) is the excitation spectrum and green light is the emission
spectrum (490 nm to 630 nm, and the emission peak is approximately 515 nm).
An atom has electrons each of which has predetermined level of energy. An
electron can absorb energy from a photon of light and become excited. The
energy level is higher in the excited stage, but this stage is unstable. The excited
electron emits fluorescence; the energy of electron at this stage is lower than
when in excited stage. This produces the magnified fluorescent image of the
object which can be visualized with fluorescence microscope.
Fig. 62.4

1. ................ are techniques used for detecting antigen/antibody by coupling
them with fluorescent dye
2. ............... absorb light of one wave length & emit light of a different
wavelength
552 MICROBIOLOGY
3. ................ is the excitation spectrum Microbiology
4. ................ is the emission spectrum
62.3.2 Immunofluorescence (IF)

In immunofluorescence usually the fluorochrome like FITC is tagged to
antibody which in the test (the fluorescence antibody technique) will bind to
corresponding specific antigen/DNA/chemical present in the sample (bacteria, Notes
virus, parasite, tissue, cells, etc.). The reaction is then read on fluorescence
microscope using blue light and appropriate filters to see the bright green
fluorescence in case of a positive result.
Some of the fluorochromes can enter the living cells and others cannot. The
former type are used to differentiate the living and dead cells.
IF is of two types direct and indirect. Indirect IF is used most commonly. In
indirect fluorescence the antibody specific for the molecule of interest (called
the primary antibody) is unlabeled, and a second anti-immunoglobulin antibody
directed toward the constant portion of the first antibody (called the secondary
antibody) is tagged with the fluorescent dye. Immunofluorescence uses antibodies
as fluorescent probes to visualize antigens within cells. The technique is highly
sensitive as well as specific.
62.4 FLUORESCENCE MICROSCOPY

For undertaking Fluorescence microscopy take into consideration the light
sources, the filters and the objectives of the Fluorescence Microscope to be used.
The light microscope used routinely in Microbiology uses visible light (400-700
nanometers) to illuminate and produce a magnified image of a sample. However,
as mentioned earlier also fluorescence microscope, uses a high intensity light
source for excitation of the fluorochrome tagged sample of interest and emits
a lower energy light of a longer wavelength that produces the magnified
fluorescent image.
Fluorescence microscopy is used in almost all fields of biology and medicine
for diagnostics and research.
62.4.1 Auto fluorescence

Certain biological structures such as mitochondria, riboflavin, melanin, elastin
and collagen, etc. fluoresce after absorbing light, emit light with fluorescence
without the addition of fluorophores like FITC. This is due to auto fluorescence
i.e. the structures are fluorescing by themselves.
MICROBIOLOGY 553
Microbiology
Auto fluorescence interferes with the reading of test result in cases where the
specimen contains structures that auto fluoresce by causing unwanted background
weak signals.
62.4.2 Photobleaching
Exposure of fluorescence stained sample to light causes photochemical destruction
Notes of the fluorescence dye (FITC or some other fluorophore) and this phenomenon
is called photobleaching. In diagnostics photobleaching can result in erroneous/
wrong result. We should be careful that after fluorescence staining the result is
read immediately or the slide is kept protected from light/in dark in refrigerator.
It is important for you to understand that the fluorescence microscopy test result
must be read on the same day.
62.5 MATERIALS REQUIRED FOR CARRYING OUT

IMMUNOFLUORESCENCE TESTS
Now you know that immunofluorescence has many applications in diagnostics
and research. It is not possible to discuss all the various applications of
immunofluorescence. We will take two examples one of detection of auto
antibodies against ds DNA and another of detecting Pneumocystis jirovecii in
broncheo alveolar Lavage.
Whenever you undertake direct or indirect IF staining follow the instructions
given in the kit insert and perform read and interpret the result accordingly.
62.5.1 Materials required for detection of auto antibody against ds DNA

in serum
Sample type: Blood Serum
Equipment and plastic wares:

z Fluorescence microscope with the light source
z Magnetic stirrer and magnetic beads
z Centrifuge
z Distilled or de-ionized water
z Pasteur pipettes = 0.5-10ul, 10-100ul, 100-1000ul
z 500 ml measuring cylinder 10. 500 ml conical flask
Reagents:
z Substrate slides: Crithidia luciliae
554 MICROBIOLOGY
z Positive control: Pooled human serum with a specific autoantibody activity Microbiology
with 1% bovine serum albumin with 0.1% sodium azide
z Negative Control: Pooled normal human serum with 1% bovine serum
albumin and 0.1% sodium azide
z FITC Conjugate: Fluorescein conjugated antiserum to human
immunoglobulins with 1% bovine serum albumin, 0.1% sodium azide;
z Mounting media
Notes
z Phosphate buffered saline (PBS): pH 7.3± 0.10
z Blotting strips.
62.5.2 Materials required for detection of Pneumocystis jirovecii in

broncheoalveolar lavage:
Sample type Broncheo Alveolar Lavage (BAL)

The sensitivity of BAL specimen for detection of P. jiroveci is 90%-99%. BAL
should be performed in two areas of lungs including the upper lobes. After the
bronchoscope is placed and wedged, normal saline is instilled in 50 ml aliquots
and immediately withdrawn with suction or syringe. The recovered specimen is
centrifuged and stained for P. jiroveci.
Equipment and other materials

z Fluorescence microscope with filter system for FITC and Evans blue
z Glass coverslips (24X60mm)
z Automatic pipettes
z Petridish with moistened filter paper for moist incubation
z Staining cuvettes
z Vortex mixer
z Screw capped centrifuge tubes
Reagents
z Commercially available kit with following reagents:
(a) FITC conjugated mouse anti-Pneumocystis jirovecii monoclonal
antibodies
(b) Buffered glycerine mounting medium
(c) Phosphate buffered saline (PBS)
The basic requirement is the fluorescence microscope for carrying out IF tests,
the other equipment and materials required will be as per the kit insert for the
test being undertaken.
MICROBIOLOGY 555
Microbiology
62.6 METHOD OF IF STAINING
The method of the IF test will be as per the details given in the kit insert of the
test being performed. We are giving below as examples, the methods for the
same two tests (detection of autoantibody against ds DNA and detection of P
jirovecii) for which the requirements have been detailed above.
Notes 62.6.1 Procedure for ds DNA auto antibody detection

z Remove appropriate number of slides from freezer. Equilibrate to room
temperature in foil bags (20 minutes). Bring all reagents to room temperature
z Reconstitute PBS with distilled water in clean and contamination free
container
z Prepare screening dilutions (1:10) of the test sera in PBS. Mix by inversion
z Remove the reagent slide from foil bag and place in Petri dish. Immediately
add 25-35 µl controls or diluted test sera to wells. Repeat this step for all
the slides
z Incubate substrate slides at room temperature for 20 minutes
z Rinse each slide briefly with a stream of PBS
z Wash slides for a total of 10 minutes in Petri dish filled with PBS
z Remove each slide from Petri dish and blot excess PBS from around wells
z Place slide in new Petri dish and immediately dispense approximately 25µl
FITC Conjugate to each well. Repeat this step for all the slides
z Incubate 20 minutes at room temperature in dark
z Rinse each slide briefly with a stream of PBS
z Wash slides for a total of 10 minutes in Petri dish filled with PBS. Remove
each slide from PBS and drain briefly on a paper towel. Apply drops of
mounting media per slide, making sure to cover all wells and mount the
slides, cover with coverslip
z Analyze the slides under a fluorescent microscope in a dark room. Evaluate
each substrate well for the presence or absence of auto antibodies
z Note the result
62.6.2 Detection of P jirovecii in BAL:

z Centrifuge 5 ml of the Lavage or bronchial washings in screw capped tubes
at 3000 xg for 10 min. Discard supernatant. Resuspend the sediment in 0.5
to 1.0 ml of sterile 0.05 M Tris-HC1 buffer (PH 7.5) and vortex for 30
seconds.
556 MICROBIOLOGY
z Smears are made from the sediment Microbiology
z Air dry the smear

z Fix smear by covering with cold acetone (-20°C) for 5-10 min
z Bring all the kit reagents to room temperature
z Cover the smear prepared from the specimen as above with 30µl of anti-
Pneumocystis MAB(reagent)
Notes
z Place the slide in a humid chamber (pertridish containing moist cotton) and
incubate for 30 min at room temperature
z Immerse the slide in PBS for 5 min
z Air dry the smear
z Add 3 drops of mounting fluid over the dried smear and cover with a cover
slip
z Examine the slide with fluorescence microscope under 40x or 100x
objectives
62.7 LABORATORY INTERPRETATIONS OF RESULTS

The slides are read using a fluorescence microscope in a dark room
62.7.1 ds DNA auto antibody detection

1. Read only single, well defined organisms in each field. Not all organisms
will appear optimal due to orientation on the slide, individual stages of
growth and microscope viewing hindrance
2. The kinetoplast is oriented toward the flagellum of the Crithidia luciliae.
A positive staining kinetoplast may appear as a solid fluorescent disk or
doughnut-shaped with more intense staining at the edges than in the middle.
The nucleus is larger and can fluoresce but it is not specific for ds DNA
auto antibodies. The nucleus may or may not show positive fluorescence
but do not use nuclear fluorescence as a criterion for the determination of
auto antibody
3. Five types of staining may result:
z No fluorescent staining of kinetoplast or nucleus. Interpretation: negative.
z No fluorescent staining of kinetoplast but positive fluorescence of
nucleus. Interpretation: negative for auto antibody.
z Fluorescent staining at the base of flagellum of organism. Interpretation:
negative. This is non-specific reaction.
MICROBIOLOGY 557
Microbiology z Flourescent staining of kinetoplast only. Interpretation: positive for auto

antibody. Report a positive reaction for a particular sample dilution only
if the kinetoplasts of the majority of organisms fluoresce.
z Fluorescent staining of kinetoplast and nucleus. Interpretation: positive
for auto antibody. Report a positive reaction at a particular sample
dilution only if the kinetoplasts of the majority of organisms fluoresce.
Notes
Fig. 62.5: ds DNA Ab positive-Kinetoplast of Crithidia luciliae

showing fluorescence
Fig. 62.6: ds DNA Negative-Kinetoplast of Crithidia luciliae

not showing any fluorescence
62.7.2 Detection of P jirovecii
Interpretation
The Pneumocystis organism are visible either as single or aggregates of extra
cellular thick walled cysts with bright apple green fluorescence in the foamy
alveolar exudate. The cysts aggregate may or may not be embedded in a brightly
stained extra cellular matrix. Individual cysts may show a comma shaped or
parentheses like structure. Cysts usually show peripheral green fluorescence.
Other developmental stages like mature sporozoites/trophozoites and precysts
may also be seen.
558 MICROBIOLOGY
Microbiology
Notes
Fig. 62.7: P jirovecii IF positive
62.8 QUALITY CONTROL

The quality control procedure for almost all IF tests are the same i. e. negative
and positive kit controls are used to assure the quality of performance of test.
The results of the negative and positive controls should always be correct, only
then the test results are taken as valid.
62.8.1 Quality control ds DNA auto antibody detection:

z Positive and negative controls must be included in each run to confirm
reproducibility, sensitivity and specificity of the test procedure
z The negative control serum should demonstrate no fluorescence of the
kinetoplast
z The positive control serum should demonstrate 3+ to 4+ fluorescence of
the kinetoplast. The kinoplast is oriented toward the flagellum of the
Crithidia luciliae. A positive staining kinoplast may appear as a solid
flourescence disk or doughnut-shaped with more intense staining at the
edges than in the middle .The nucleus is larger and can fluoresce but it is
not specific for anti-nDNA antibodies
z The positive and negative controls must demonstrate appropriate reactions
otherwise the run is considered invalid and must be repeated
62.8.2 Quality control detection of P jirovecii:

A known positive slide is stained along with the test slide every time the staining
is done and it should always give a positive result..
62.8.3 Safety precautions:

z All specimen used in this test should be considered potentially infectious.
Universal precautions (gloves) should be used for handling and disposal of
materials during and after use
MICROBIOLOGY 559
Microbiology z Use soap for routine hand washing

z Use biohazard waste disposal guidelines
62.9 APPLICATIONS
As has already been explained to you that out of the two IF techniques direct
and indirect, the indirect IF is most commonly used. IF is used in biology and
Notes medicine. Medical applications include Diagnostics and research. We have
given examples of detection of auto antibody to ds DNA (to diagnose auto
immune disorder) and detection of P jirovecii in BAL (to detect the infection
with Pneumocystis). You will find many applications of the IF technique in
medicine, biology and research.
Basically application of IF is to visualize antigens within cells using specific
antibodies as fluorescent probes and vice-versa. Various applications include:
z Resolution of details to the molecular level
z Study a cell population for viability (some fluorophores penetrate live cells
and not the dead cells as already explained under microscopy)
z Detect specific cells of interest in a specimen/material using FISH
techniques
z Viewing structural components of cells, bacteria, parasites, fungi and
bacteria
z Imaging the genetic material within a cell (DNA and RNA)
z Defining the spatial-temporal patterns of gene expression within cells

1. Auto fluorescent structures are ............. , ............. & .............
2. Exposure of fluorescence stained sample to light couasing photochemical
destruction is known as .............

z Immunofluorescence is one of the very common laboratory techniques used
in almost all disciplines of Biology including Medicine. Way back in 1942
Coons and Kaplan did some experiments and reported that the fluorescence
dyes can be conjugated with antibodies and these labeled antibody can be
used as probes to detect and locate the antigen specific to this antibody.
560 MICROBIOLOGY
z Direct immunofluorescence is used less frequently as the antibody against Microbiology
the molecule of interest is chemically conjugated to a fluorescent dye, so
for every antigen to be detected, the specific antibody will have to be
conjugated with FITC. In indirect fluorescence the antibody specific for the
molecule of interest (called the primary antibody) is unlabeled, and a second
anti-immunoglobulin antibody directed toward the constant portion of the
first antibody (called the secondary antibody) is tagged with the fluorescent
dye. Indirect fluorescence is used more commonly as the tagged secondary Notes
antibody can be used to detect many different antigens. However, the
primary antibody will have to be specific for the antigen to be detected.
z IF is defined as various techniques used for detecting an antigen or antibody
in a sample by coupling its specifically interactive antibody or antigen to
a fluorescent dye/compound, mixing with the sample, and then observing
the reaction under an ultraviolet-light fluorescence microscope.
z In immunofluorescence usually the fluorochrome like FITC is tagged to
antibody which in the test (the fluorescence antibody technique) will bind
to corresponding specific antigen/DNA/chemical present in the sample
(bacteria, virus, parasite, tissue, cells, etc.). The reaction is then read on
fluorescence microscope using blue light and appropriate filters to see the
bright green fluorescence in case of a positive result.
z Some of the fluorochromes can enter the living cells and others cannot. The
former type are used to differentiate the living and dead cells.
z Auto fluorescence interferes with the reading of test result in cases where
the specimen contains structures that auto fluoresce by causing unwanted
background weak signals.
z The method of the IF test will be as per the details given in the kit insert
of the test being performed. We have given examples of tests (detection
of autoantibody against ds DNA and detection of P jirovecii) for which the
requirements, method, interpretation, quality control have been detailed
above. Please go through the details to understand IF test.
z Basically application of IF is to visualize antigens within cells using specific
antibodies as fluorescent probes and vice-versa. Various applications
include:
z Resolution of details to the molecular level
z Study a cell population for viability (some fluorophores penetrate live
cells and not the dead cells as already explained under microscopy)
z Detect specific cells of interest in a specimen/material using FISH
techniques
MICROBIOLOGY 561
Microbiology z Viewing structural components of cells, bacteria, parasites, fungi and

bacteria
z Defining the spatial-temporal patterns of gene expression within cells
z Imaging the genetic material within a cell (DNA and RNA)
Notes TERMINAL QUESTIONS

1. What is immunofluorescence? Describe the different types of
immunofluorescence techniques.
2. Define IF and the basic requirements for IF.
3. Describe the principle of fluorescence and IF.
4. Define autofluorescence and photobleaching and enumerate the problems
these cause in IF test
5. Describe the materials required to perform IF test for detection of auto
antibody to ds DNA.
6. Describe how will you read and interpret an IF test.
7. Describe the various applications of IF.
62.1
1. Immunofluroescence
2. Fluorescent dyes
3. Blue light
4. Green light
62.2
1. Mitochondria, riboflavin & collagen
2. Photobleaching
562 MICROBIOLOGY
EIA and RIA MODULE
Microbiology
63
Notes
EIA AND RIA
63.1 INTRODUCTION
The ELISA, Enzyme linked Immunosorbent assay, also sometimes known as
EIA i.e. Enzyme Immuno Assay is a rapid test used for detecting and quantifying
antibodies or antigens in specimen against viruses, bacteria and other materials.
This method is used to detect/diagnose infectious, autoimmune and other
diseases.
ELISA is carried out on solid matrix, in 96 well microtitre plates or strips (12
wells or 8 wells each) made of polystyrene/commercially available coombs/
cartridges (Rapid ELISA). The protein antigen is affixed to any of above
mentioned surfaces, and then a specific antibody is applied over the surface so
that it can bind to the antigen. This antibody is linked to an enzyme, and in the
final step a substance/substrate specific for the enzyme is added that the enzyme
can convert to some detectable signal, most commonly a colour change.
Performing ELISA, like other antigen antibody reactions, involves at least one
antibody with specificity for a particular antigen. Depending upon whether we
want to detect antibody or antigen the type of ELISA will vary. For example the
antigen in a sample can be detected either by direct ELISA , sandwich ELISA
or competitive ELISA and antibody is usually detected by indirect ELISA. I
know it seems very confusing to you now but as we go along describing the
various types of ELISA technologies, each principle will become clear to you.
Another thing to note is that between each step of assay, whatever the ELISA
format is, the plate is washed with a mild detergent solution to remove any un
reacted proteins or antibodies. After the final wash step, the plate is developed
by adding an enzymatic substrate to produce a visible signal, which indicates
the quantity of antigen/antibody in the sample and the reaction is read either with
naked eye or with an ELISA Reader. The result is expressed as OD (Optic
Density) value (Reader) or titre.
MICROBIOLOGY 563
MODULE EIA and RIA
Microbiology Newer ELISA-like techniques have been developed which utilize fluorogenic,
electro chemi luminescent, and real-time PCR reporters to create quantifiable
signals. The new reporters are more sensitive. These newer techniques do not
use enzymes as in ELISA but use other reporter molecules as given above. We
will discuss ELISA in this chapter. Recently an ultrasensitive, enzyme-based
ELISA test using nanoparticles as a chromogenic reporter has been developed
which can detect attograms of analyte in the specimen and result can be read
Notes as colour signal with naked-eye.
OBJECTIVES
z define ELISA
z describe the history of ELISA
z discuss the process of ELISA
z describe the various types of ELISA
z describe the methods of various types of ELISA with examples
z practice “Internal quality control” for ELISA
z read the result of ELISA with naked eye and with ELISA Reader
z describe the various applications of ELISA
63.2 DEFINITION
Collins English Dictionary describes enzyme-linked immunosorbent assay as “
An immunological technique for accurately measuring the amount of a
substance, for example in a blood sample”.
American Heritage English Language Dictionary defines ELISA as “A sensitive
immunoassay that uses an enzyme linked to an antibody or antigen as a marker
for the detection of a specific protein, especially an antigen or antibody”. It is
often used as a diagnostic test to determine exposure to a particular infectious
agent, such as the AIDS virus, by identifying antibodies present in a blood
sample.
Random House Kernerman Webster’s College Dictionary defines ELISA as “A
diagnostic test for detecting exposure to an infectious agent, as the AIDS virus,
by combining a blood sample with antigen of the agent and probing with an
enzyme that causes a color change when antibody to the infection is present in
the sample”.
You see in all the above definitions the common thing is that ELISA is an
antigen-antibody reaction that uses enzyme/s and specific substrate wherein the
564 MICROBIOLOGY
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presence of unknown substance which may be an antigen or antibody or a protein Microbiology
in the specimen is detected by colour change at the end of the reaction/test. The
test is performed on solid phase –the polystyrene microtitre plates/strips and the
final result is mostly read on the ELISA Reader as OD value.
63.3 THE HISTORY OF ELISA

Before the development of the EIA/ELISA, the only option for conducting an Notes
immunoassay was Radioimmunoassay, a technique which used radioactively-
labeled antigens or antibodies for diagnostics before the advent of ELISA.
Rosalyn Sussman Yalow and Solomon Berson published a paper in 1960 on
Radioimmunoassay. Radioactivity was the reporter providing the signal which
indicated the presence of analyte being sought in the sample. However,
radioactivity posed a potential health threat, so safer reporter alternatives were
sought.
One such alternative reporter tested was enzyme peroxidase which reacted with
appropriate substrates (such as ABTS or 3, 3’, 5, 5’-Tetramethylbenzidine) to
produce a change in colour, which could be used as a signal. The next step in
history was the linking of an antibody /antigen to the enzyme. This linking
process was independently developed by two different scientists Stratis Avrameas
and G.B. Pierce. The next development was the idea of fixing the antigen/
antibody to prepare the immunosorbent surface as it is necessary to remove any
unbound antibody or antigen by washing. Wide and Porath developed the
technique and published the same in 1966.
Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton
Schuurs and Bauke van Weemen in The Netherlands used the knowledge
published by Wide and Porah to develop ELISA and EIA tests independently and
published the technologies in 1971. The clinical impact of EIA/ELISA as
immunoassays has been overwhelming. Perlmann, Schuurs, Engvall, and van
Weemen were honored with the German scientific award of the “Biochemische
Analytik” for their inventions in 1976. EIA/ELISA used the principles of
conventional radioimmunoassay, with the key difference that the antibodies are
labelled with an enzyme, rather than radioisotopes. Now other reporter
molecules like fluorogenic, electro chemi luminescent, and real-time PCR and
appropriate signal systems have been developed and available in Medicine for
diagnostics.
A well-established in vitro diagnostic industry has developed based on these
technologies and are marketing a huge number of EIAs/ELISAs.
63.4 PRINCIPLE AND PROCESS

As you know already ELISA is a biochemical technique used mainly in
infectious diseases and immunology to detect the presence of an antibody or an
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or antibody concentration in a sample.
63.4.1 Principle
ELISA combines the specificity of antibodies with the sensitivity of simple
enzyme assays, by using antibodies or antigens coupled to an easily assayed
enzyme that possesses a high turnover number.
Notes
Simply put for ELISA an antigen is adsorbed/fixed to the surface of a solid
matrix which may be a well on a micro titration plate/ well strip, beads or may
be a special type of paper made of Nitrocellulose. Then, a specific antibody is
applied over the surface so it can bind to the antigen. This antibody is linked
to an enzyme, and, in the final step, a substance containing the specific substrate
for the enzyme is added. The subsequent reaction produces a detectable signal,
a color change in the substrate.
ELISA technology has advanced a lot. To make this immunoassay more and
more sensitive as well as specific various advances have been made and now
we have different types of ELISAs. There are slight differences in the technology
though basically ELISA remains an antigen antibody reaction performed as wet
test using different reporter and signal systems.
63.4.1.1 Types of ELISAs

ELISAs can be divided into the following categories based on the principle used:
z Indirect
z Sandwich
z Competitive
z Antigen and antibody capture ELISA
63.4.1.2 Indirect ELISA

HIV antigens are attached covalently to the solid phase support allowing
corresponding/specific antibodies present in the specimen to bind, and these
bound antibodies are subsequently detected by enzyme labelled anti-human
immunoglobulin and specific substrate system. If the test specimen contained
the antibodies specific to the antigen fixed on the solid phase colour reaction
will take place. The colour reaction can be read with naked eye or with ELISA
Reader at the specified UV light using the special filters. The result is expressed
as OD (optical Density) value.
63.4.1.3 Sandwich ELISA

One of the most useful of the immunoassays is the two antibody “sandwich”
ELISA. This assay is used to determine the antigen concentration in unknown
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samples. This ELISA is fast and accurate, and if a purified antigen standard is Microbiology
available, the assay can determine the absolute amount of antigen in an unknown
sample.
To utilize this assay, one antibody (the “capture” antibody) is purified and bound
to a solid phase typically attached to the bottom of a plate well. Specimen
containing antigen is then added and allowed to complex with the bound
antibody. Unbound products are then removed with a wash, and a labeled second
antibody (the “detection” antibody) is allowed to bind to the antigen, thus Notes
completing the “sandwich”. The assay is then quantitated by measuring the
amount of labeled second antibody bound to the matrix, through the use of a
colorimetric substrate.
63.4.1.4 Competitive ELISA

In this assay the antibodies present in the specimen compete with the enzyme
conjugated antibodies in the reagent for binding to the antigen on the solid phase.
If the test specimen contains corresponding/specific antibodies, these will
compete with the labelled antibodies in the reagent for binding to antigen. So
that less or not labelled antibodies can attach to the solid phase. Hence, faint or
no colour is produced on addition of substrate if specimen contains antibodies
against the antigen on the solid phase.
63.4.1.5 Antigen and antibody capture ELISA

Antigen capture ELISA can be based on principle of indirect or competitive
ELISA, only difference being in the initial step of attaching antigen to the solid
phase. A monoclonal antibody directed against an antigen is bound to the solid
support. Next step is addition of antigen supplied as reagent in the test kit. This
antigen is captured by the monoclonal antibody bound to the solid phase. Test
specimen appropriately diluted is added next. Antibodies if present in the
specimen bind to the antigen on solid support. Remaining principle is same as
indirect ELISA Only advantage of antigen capture ELISA is that it is more
specific than indirect assay.

Match the following
1. Indirect ELISA (a) Determines antigen concentration
2. Sandwich ELISA (b) Antibodies compete to bind with antigen
3. Competitive ELISA (c) Captures monoclonal antibody
4. Antigen & Antibody (d) Detects bound antibodies
capture ELISA
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63.5 PROCESS OF ELISA
Although many variants of ELISA have been developed and used in different
situations, they all depend on the same basic elements:
z Coating/Capture: direct or indirect immobilization of antigens to the
surface of polystyrene microplate wells.
z Plate Blocking: addition of irrelevant protein or other molecule to cover
Notes all unsaturated surface-binding sites of the microplate wells.
z Probing/Detection: incubation with antigen-specific antibodies that affinity-
bind to the antigens.
z Signal Measurement: detection of the signal generated via the direct or
secondary tag on the specific antibody.
Basic steps are as shown below:
Fig. 63.1
In a typical assay designed to detect an antigen in a sample, either the antigen
is immobilized by direct adsorption or first antibody is adsorbed and then antigen
is adsorbed on the well surface of the ELISA plate. The plate is blocked with
albumin. The antigen is probed with a specific detection antibody. The detection
antibody may be directly labeled with a signal-generating enzyme or fluorophore
or it may be secondarily probed with an enzyme- or fluor-labeled secondary
antibody. For enzymatic detection, the appropriate enzyme substrate is added.
The signal observed is proportional to the amount of antigen in the sample. Every
step of test is followed by washing to remove the un-reacted reactants, only
specific (high-affinity) binding remains that causes the detection signal at the
final step.
63.6 METHODS OF ELISA

We will see now how the different types of ELISA are performed. We will take
example of HIV ELISAs.
63.6.1 Indirect ELISA

Always follow the instructions of the manufacturer of the kit which are given
in the kit literature and develop a protocol/SOP. One example is given below.
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Principle Microbiology
HIV antigens are attached covalently to the solid phase support allowing HIV
antibodies present in the specimen to bind, and these bound antibodies are
subsequently detected by enzyme labelled anti-human immunoglobulin and
specific substrate system. If the test specimen contained antibodies colour
reaction will take place.
Materials required: Notes
z Single / multichannel pipettes with disposable tips: 5-50ul , 50-200ul
z Incubator (37+20C)
z ELISA reader with or without washer
z Powderless disposable gloves
z Absorbent paper
z Deionised water
z Discard jar with hypochlorite solution
z Wash bottles
Protocol:
Add appropriate amount of diluted sample in various wells
↓
Incubate for required time at room temperature
↓
Empty plate and tap out residual liquid. Wash 3-5 times
↓
Appropriately diluted enzyme conjugate is added and incubated as specified
↓
Wash the plate (3-5 times) and tap out residual liquid
↓
Add appropriate amount of substrate solution to each well
↓
Incubate as specified
↓
Add required amount of stop solution Sodium hypochlorite solution (5.2%)
↓
Read plate with plate reader/read colour change with naked eye.
63.6.2 Sandwich ELISA

One of the most useful of the immunoassays is the two antibody “sandwich”
ELISA. This assay is used to determine the antigen concentration in unknown
samples. This ELISA is fast and accurate, and if a purified antigen standard is
available, the assay can determine the absolute amount of antigen in an unknown
sample.
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To utilize this assay, one antibody (the “capture” antibody) is purified and bound
to a solid phase typically attached to the bottom of a plate well. Antigen is then
added and allowed to complex with the bound antibody. Unbound products are
then removed with a wash, and a labeled second antibody (the “detection”
antibody) is allowed to bind to the antigen, thus completing the “sandwich”. The
assay is then quantitated by measuring the amount of labeled second antibody
Notes bound to the matrix, through the use of a colorimetric substrate.
Materials
As for Indirect ELISA test.
Protocol
The following is the general protocol for a sandwich ELISA. Follow manufacturers
instructions for precise steps.
Add required volume of sample containing antigen to the wells

↓
Incubate for required amount of time
↓
Wash the plate three to four times
↓
Add the labeled second antibody
↓
Incubate at room temperature for required amount of time
↓
Wash the plate 3-4 times
↓
Add substrate as indicated by manufacturer
↓
Read on the ELISA reader after suitable incubation time.
63.6.3 Competitive ELISA
Principle
In this assay the HIV-antibodies present in the specimen compete with the
enzyme conjugated antibodies in the reagent for binding to the antigen on the
solid phase. If the test specimen contains HIV antibodies, these will compete
with the labelled antibodies in the reagent for binding to antigen. So that less
or not labelled antibodies can attach to the solid phase. Hence, faint or no colour
is produced on addition of substrate if specimen contains HIV antibodies.
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Materials Microbiology
These are same as used in case of indirect / sandwich ELISA techniques
Protocol:
Add appropriate amount of standard/ sample solution to the wells
↓
Allow to incubate for required amount of time (as in kit insert) Notes
↓
Add appropriate amount of conjugate solution to the wells
↓
Wash the plate with wash solution 3-4 times
↓
Add substrate as indicated by manufacturer.
↓
After suggested incubation time has elapsed,
↓
Optical densities at target wavelengths can be measured on an ELISA reader
Fig. 62.2 Sandwich and Competitive ELISA techniques
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Microbiology 63.6.4 Antigen and antibody capture ELISA

Antigen capture ELISA can be based on principle of indirect or competitive
ELISA, only difference being in the initial step of attaching antigen to the solid
phase. A monoclonal antibody directed against an antigen is bound to the solid
support. Next step is addition of the standard antigen supplied as reagent in the
kit. This antigen is captured by the monoclonal antibody bound to the solid
phase. Test specimen appropriately diluted is added next. Antigen specific
Notes
antibodies if present in the specimen bind to antigen on solid support. Remaining
principle is same as indirect ELISA. Only advantage of antigen capture ELISA
is that it is more specific than indirect assays.
63.6.5 Enzyme Linked Fluorescent Assay (ELFA)

This is the new technology utilizing the fluorescence as the detection system
instead of the enzyme substrate in ELISA. The test requires a special equipment
which is an automated system (VIDAS and MINI VIDAS, Biomerieux). Here
also the specific instructions of the manufacturer are followed to perform the
test.
63.7 QUALITY CONTROL AND CONSIDERATIONS

WHILE PERFORMING ELISA
Flow chart for performing ELISA using quality control aliquot

Selection of ELISA kit Licensed Quality checked
Performance of ELISA test As per manufacturer’s guidelines Internal
controls are checked Inclusion of external
control indaily run
E-ratio of external contro Recorded O.D. value of external control
/cut off value (kit)
Daily run QC sample The OD value of the QC sample should
be within the specified OD range of the
QC sample.
63.8 APPLICATIONS OF ELISA

You now understand that ELISA is a highly sensitive and specific test. Since the
discovery of ELISA many advances have been made to make this assay more
and more sensitive and specific. ELISA is employed in Medicine to detect and
diagnose microbial infectious diseases, autoimmune diseases, detection of
antigen in a given sample.
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z ELISA can be applied to detect and measure antibody in serum against
viruses, bacteria, parasites
z ELISA has also been used in home pregnancy test (rapid ELISA)
z ELISA is used in food industry to detect potential food allergens such as
milk, peanuts, walnuts, almonds, and eggs.
z ELISA can also be used in toxicology as a rapid presumptive screen for
certain classes of drugs. Notes
z The ELISA was widely used in various areas such as immunology,

Biological Pharmacy, Diagnostic industry, and so on.

Match the following
1. Coating (a) Addition of irrelevant protein
2. Plate blocking (b) Incubation with antigen-specific antibodies
3. Signal measurement (c) Immobilization of antigens
4. Probing (d) Detection of signal generated by antibody

z The ELISA, Enzyme linked Immunosorbent assay, also sometimes known
as EIA i. e. Enzyme Immuno Assay is a rapid test used for detecting and
quantifying antibodies or antigens in specimen against viruses, bacteria and
other materials. This method is used to detect/diagnose infectious,
autoimmune and other diseases.
z ELISA is defined variously by different authors . The common definition
that emerges from all the definitions is that ELISA is an antigen-antibody
reaction that uses enzyme/s and specific substrate wherein the presence of
unknown substance which may be an antigen or antibody or protein in the
specimen is detected by colour change at the end of the reaction/test. The
test is performed on solid phase –the polystyrene microtitre plates/strips and
the final result is mostly read on the ELISA Reader as OD value.
z Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and
Anton Schuurs and Bauke van Weemen in The Netherlands used the
knowledge published by Wide and Porah to develop ELISA and EIA tests
independently and published the technologies in 1971.
MICROBIOLOGY 573
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Microbiology z ELISA combines the specificity of antibodies with the sensitivity of simple
enzyme assays, by using antibodies or antigens coupled to an easily assayed
enzyme that possesses a high turnover number. Simply put for ELISA an
antigen is adsorbed/fixed to the surface of a solid matrix which may be a
well on a micro titration plate/ well strip, beads or may be a special type
of paper made of Nitrocellulose. Then, a specific antibody is applied over
the surface so it can bind to the antigen. This antibody is linked to an
Notes enzyme, and, in the final step, a substance containing the specific substrate
for the enzyme is added. The subsequent reaction produces a detectable
signal, a color change in the substrate. ELISA/EIA can be divided into
various types based on the principle used: Indirect; Sandwich; Competitive;
Antigen and antibody capture ELISA.
z Applications of ELISAs include: Detection and measurement of antibody/
antigen in serum against viruses, bacteria, parasites; Home pregnancy test
(rapid ELISA); Detection of potential food allergens such as milk, peanuts,
walnuts, almonds, and eggs in food industry; As a rapid presumptive screen
in toxicology for certain classes of drugs; Immunology, Biological Pharmacy,
Diagnostic industry, and so on.
TERMINAL QUESTIONS
1. What is ELISA/EIA? How do you define ELISA?
2. Why and how was ELISA discovered?
3. Name different types of ELISAs based on technologies employed. Describe
in brief the principle of each type of ELISA
4. Briefly describe principle and method of indirect ELISA.
5. Briefly describe principle and method of sandwich ELISA.
6. Briefly describe principle and method of competitive ELISA.
7. Briefly describe the quality control process for ELISA.
8. Enumerate the different applications of ELISA.

63.1
1. (d) 2. (a) 3. (b) 4. (c)
63.2
1. (c) 2. (a) 3. (d) 4. (b)
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64
Notes
AUTOIMMUNITY AND
AUTOIMMUNE DISEASES
64.1 INTRODUCTION
Normally the function of immune system in our body is to recognize foreign
elements and to destroy these before they could harm us either by humoral
immune response (specific antibody formation) or cell mediated immune
response by activation and clonal expansion of T cells. Thus the immune system
defends the body against infections and certain other diseases by identifying,
attacking, and destroying germs and other foreign substances. Sometimes the
immune system makes a mistake and starts attacking the body’s own tissues or
organs. This is called autoimmunity. There are many autoimmune diseases one
example being type 1 diabetes in which the Islets cells (produce Insulin) in the
pancreas are destroyed by the immune system.
An autoimmune disease is a case of mistaken identity; it is an abnormal

condition in which the body reacts against constituents of its own tissues. The
result may be simple hypersensitivity reaction and or autoimmune disease when
the body begins attacking its own healthy tissues. We can say it is a case of
mistaken identity resulting in failure of the immune system to differentiate
between self and non self. About 5 % to 7 % of adults suffer from autoimmune
diseases and two thirds of these are females. Somehow left handed people are
more prone the reason for this is not known.
This failure to differentiate between self and non self may result due to some
extraneous environmental factors like some viral infections and exposure to
some mutagenic agents; can be due to the breakdown and failure of immune
regulation and due to some aberration in the genes. Whatever the reason the
result is autoimmune disease which may involve a particular organ when it is
called an organ specific disease (e.g. Addison’s disease involving Adrenal
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Microbiology glands) or it may involve particular cells/tissues all over the body when it is
called non-organ specific or disseminated disease (e.g. Rheumatoid arthritis).
OBJECTIVES
Notes After reading this lesson you will be able to:
z define autoimmunity
z describe the history of autoimmunity
z describe the various disease states caused by autoimmunity
z describe the causes of autoimmunity
z discuss the immunopathology of these diseases
z describe the diagnosis of autoimmune diseases
z describe the treatment of autoimmune diseases
History
A very famous, Nobel laureate Paul Ehrlich (1854-1915) coined the term
“Horror autotoxicus” to emphasize that body has innate aversion to immunological
self-destruction. “Horror autotoxicus” literally means the horror of self-toxicity.
However, as we now know, the immune system can upon occasion attack own
body and result is autoimmune disorders.
Scientists started talking about autoimmunity around 1900. By 1904 the

antibody nature of the autohemolysin responsible for cold hemoglobinuria was
described, and soon confirmed. However, the concept that autoimmunization
caused cold hemoglobinuria was not yet clear and was not accepted. It was only
during early 1960s that the concept of autoimmunization as cause of some
diseases was accepted. The publication of a monograph on autoimmune disease
in 1963, and surely by the consensus reached at a large international conference
published as proceedings in 1965 lead to acceptance of the state of autoimmunity.
The history of autoimmunity is far from over as autoimmunity is being
incriminated in aetiopathogenesis of more and more disease conditions.
64.2 DEFINITION
Autoimmunity refers to the body’s development of intolerance of the antigens
on its own cells i. e. there is an immune response to one’s own tissue antigens.
This type of body response results in a disease state characterized by a specific
576 MICROBIOLOGY
antibody or cell-mediated immune response against the body’s own tissues (auto Microbiology
antigens). So, we can say that autoimmunity is the breakdown of mechanisms
responsible for self tolerance and induction of an immune response against
components of self.
The immunological mechanism of the body is dependent on two major factors:

(1) the inactivation and rejection of foreign substances and (2) the ability to
differentiate between the body’s own antigens (‘self’) and foreign (‘non self’). Notes
It is not yet known exactly what causes the body to fail to recognize self proteins
as its own and to react to them as if they were foreign resulting in autoimmunity
and may be autoimmune diseases. Prominent examples include celiac disease,
diabetes mellitus type 1 (IDDM), sarcoidosis, systemic lupus erythematosus and
many others.

1. The function of immune system is to ................. & ................. foreign
bodies
2. Immune system attacking the body’s own tissue is known as .................
3. Autoimmune disease when involves a particular organ is called as .................
4. Auto immune disease when involves the whole body is called as .................
disease
64.3 AUTOIMMUNE DISEASE STATES

The autoimmune diseases can be divided into systemic, localized and haemolytic
disorders, depending on tissue/cells affected and the clinico-pathologic features
64.3.1 Systemic autoimmune diseases

These diseases are associated with auto antibodies to antigens which are not
tissue specific. One example can be polymyositis, here the tissue involved are
muscles, however the auto antibodies are found against the auto antigens
which are often ubiquitous “t-RNA synthetases”. Another example is rheumatoid
arthritis (RA). There is symmetric poly arthritis with muscle wasting and may
be associated with myositis, and vasculitis, etc. The specific marker (auto
antibody) found in blood in these patients is Rheumatoid Factor (RF) which
is usually 19 s IgM. RF is an antibody against Fc fragment of immunoglobulins.
Other systemic autoimmune diseases are polyarteritis nodosa, systemic lupus
erythematosus and Sjogren’s syndrome as shown in the figure.
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Notes
Fig. 64.1 Two types of autoimmune disease
So, let us recap, in case of systemic autoimmune diseases the incriminating

antigens and the autoimmunity are distributed in many tissues. The systemic
diseases are:
– Rheumatoid arthritis
– Systemic lupus erythematosus (SLE)
– Scleroderma
– Primary Sjogrens’s syndrome
– polymyositis
64.3.2 Organ specific or localized autoimmune diseases

As the name indicates in these cases the autoimmunity involves a particular
organ. One best studied organ is thyroid and examples are Hashimoto’s disease
which affects thyroid gland causing lymphadenoid goitre and the other is Graves
disease causing thyrotoxicosis. Anti thyroglobulin antibodies are produced in
578 MICROBIOLOGY
both these cases and these can be shown in sera of the patients by various tests. Microbiology
However, the pathology in the two is different and so are the resulting symptoms.
In Hashimoto’ goitre there is hypothyroidism and in Graves disease there is
hyperthyroidism. Another example is Addison’s disease in which the adrenal
glands are affected. There is lymphocytic infiltration of the adrenal glands and
production of antibodies directed against zona glomerulosa. Other diseases
include autoimmune disease of eyes, brain, skin and many others.
Notes
In organ specific autoimmune diseases the implicated antigens and the
autoimmunity are restricted to specific organs in the body
– Type I diabetes
– Goodpasture’s syndrome
– Multiple sclerosis
– Grave’s disease
– Hashimoto’ thyroiditis
– Myasthenia gravis
64.3.3 Haemolytic autoimmune diseases:

Auto antibodies are formed against RBCs leading to autoimmune haemolytic
anaemia; auto antibodies may form against platelets resulting in autoimmune
thrombocytopaenia; and formation of anti leucocyte antibodies resulting in
autoimmune leucopaenia and so on.
64.4 CAUSES OF AUTOIMMUNE DISEASES

Let us understand why normally immune response does not occur against our
own tissue antigens. This is due to “Tolerance to self antigens” which is acquired
by various mechanisms. Failure in immune recognition of self and injury of self
tissues (autoimmunity) results from a loss of self tolerance. Let us discuss the
mechanisms of self tolerance and how it is broken down to result in
autoimmunity.
64.4.1 Mechanisms of self tolerance

One mechanism is that the clones of lymphocytes which act against self antigens
are deleted. This is the “clonal deletion” theory. Clonal deletion is mediated by
ubiquitous self antigens. The second is inactivation of developing lymphocytes
so our immune system becomes self tolerant, no activation of immunity against
self antigens as specific lymphocytes are either deleted or inactivated. Clonal
inactivation can be mediated by tissue-specific antigens.
MICROBIOLOGY 579
Microbiology Peripheral T cell tolerance mechanisms:

These are explained below:
z Immunological Ignorance: Very few self proteins contain peptides that are
presented by a given MHC molecule at a level sufficient for T cell
activation, Autoreactive T cells are present but not normally activated.
z Suppressor or regulatory T cells: mediate active suppression of autoreactive
Notes cells
z Immunologically privileged sites: no lymphatic drainage or non-vascularized
areas; presence of immunosuppressive factors.
Peripheral B cell tolerance mechanisms

– Contact with soluble antigens: this leads to downregulation of surface IgM,
and so there is inhibition of signaling resulting in anergic (non reactive) cells
and so no immune response occurs against these soluble antigens, there is
tolerance to these antigens.
– Another mechanism of self tolerance is the Fas-mediated apoptosis
(programmed cell death) of anergic B cell following secondary encounter
with CD4 T cell

1. Systemic autoimmune diseases are associated with auto antibodies to
antigens are .............
2. Examples of systemic autoimmune diseases are ............., ............. &
.............
3. Example of organ specific diseases are .............,............. & .............
4. ............. theory is associated with self tolerance
64.4.2 Mechanisms of breakdown of self tolerance resulting in autoimmunity:

There may be loss of “Self Tolerance” germinal centers due to some genetic
aberration or some environmental factors.
64.4.2.1 MHC (Major Histocompatibility complex) molecules normally

should present the peptide (antigen) at optimal level to T cells for immune
response to occur. The level of autoantigenic peptide presented is determined
by polymorphic residues in MHC molecules that govern the affinity of peptide
binding.
580 MICROBIOLOGY
If the peptide is presented at levels too low to engage effector T cells, there will Microbiology
be tolerance to such self antigens and if peptide is presented at very high levels
then there is clonal deletion or anergy (tolerance).
Only a few peptides can act as autoantigens so there are a relatively few
autoimmune syndromes.
Individuals with a particular autoimmune disease tend to recognize the same

Notes
antigens with the same MHC.
Autoimmunity (breakdown of tolerance) arises most frequently to tissue-

specific antigens with only certain MHC molecules that present the peptide at
an intermediate level recognized by T cells without inducing tolerance.
64.4.2.2 Gene mutation/s leads to one or more autoimmune diseases.

Autoimmune diseases are associated with particular MHC genotypes. Sometimes
there may be more than one gene involved and the result is complex autoimmune
diseases.
64.4.2.3 Environmental factors which can cause breakdown of self tolerance

include pathogens (bacterial, viral and others), drugs, hormones, and toxins.
These are just a few ways that can trigger autoimmunity.
z Drugs
z Drug induced lupus
z Toxins
z Example: Toxic Oil Syndrome
z Hormones
z Example: Endometriosis and preeclampsia are both thought to be
autoimmune in nature
64.4.2.4 Complement deficiency: deficiencies in the classical complement
pathway renders patients more likely to develop immune complex diseases like:
z SLE
z RA
64.4.2.5 Molecular mimicry

Rheumatic fever is a classic example of molecular mimicry as shown in
picture below.
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Notes
Fig. 64.2
Let us enumerate causes of autoimmunity

z Lymphocytes abnormalities and polyclonal lymphocytes activation
z Cytokine dysregulation due to changes in lymphocytes
z Failure of central tolerance and peripheral tolerance
z Overcome of Ag released from hidden location
z Antigen modified/ generated by molecular changes in the body
z Molecular mimicry
z Alteration in Ag processing
z Infection
z Genetic factors.
64.5 IMMUNOPATHOLOGY OF AUTOIMMUNE

DISEASES
These are autoimmune diseases, so the pathology is immune response to self
antigens as explained above. Antibody mediated immune response by formation
of antibody to antigens on cell surface or in tissue matrix. Examples are
582 MICROBIOLOGY
glomerulonephritis (Ab to basement membrane); rheumatic myocarditis Microbiology
(molecular mimicry as described above), etc. Another mechanism is immune
complex mediated, Type III immune response to self antigen Examples are
Rheumatoid arthritis. The third mechanism is cell mediated (Type IV immune
response) immune response against brain componants, example is experimental
auto immune encephalomyelitis.
So you see type II, III and Type IV immune responses are involved in Notes
immunopathology of autoimmune diseases.
64.6 LE CELL
The LE cell was so termed because of its exclusive presence in the bone marrow
of 25 patients with confirmed or suspected SLE at the Mayo Clinic. LE cells
are polymorphonuclear cells which have phagocytosed the nuclear material of
other cells. LE cells are not usually found in peripheral blood. However,
sometimes LE cell phenomenon could form in the buffy coat of peripheral blood
after a period of]. LE cells are common in bone marrow but have also been found
in synovial fluid, cerebrospinal fluid and pericardial and pleural effusions from
patients with SLE.
Fig. 64.3: LE cells are formed in response to an auto immune factor

present in plasma in cases of SLE.
64.7 DIAGNOSIS OF AUTOIMMUNE DISEASES

It is very difficult to exactly diagnose autoimmune diseases. Clinical presentation
and physical examination are done to make a probable diagnosis. Laboratory
tests performed are of general nature and in case of organ specific disease
specific tests can be performed.
General tests:
z C Reactive Protein
z Autoantibody titers (anti DNA, anti phospholipids, etc)
MICROBIOLOGY 583
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z Erythrocyte sedimentation rate
Disease specific tests:

z Neurological exam – MS
z Fasting glucose – Diabetes
Notes
64.8 TREATMENT OF AUTOIMMUNITY
The general principle is to somehow stop the immune response to self antigens.
This can be achieved by the following methods:
z Immunosuppression (e.g., prednisone, cyclosporin A);
z Removal of thymus (some Myasthenia Gravis patients);
z Plasmapheresis (to remove Antigen+Antibody complexes);
z T-cell vaccination (to activate suppressing T cells so that immune response
to self antigens is suppressed??);
z Block MHC with similar peptide, so antigen processing is stopped;
z anti-CD4 monoclonal Antibody to inhibit immune response;
z anti-IL 2R monoclonal Antibody to inhibit immune response.

1. Common environmental factors causing autoimmunity diseases are .............,
............., ............. & .............
2. Disease like systemic lupus Erythromatosis are caused because of .............
deficiency
3. Rheumatic fever occurs because of .............
4. Common laboratory tests for diagnosis of Autoimmune diseases are

z An autoimmune disease is a case of mistaken identity; it is an abnormal
condition in which the body reacts against constituents of its own tissues.
z Autoimmunity is defined as the breakdown of mechanisms responsible for
self tolerance and induction of an immune response against components of
self.
584 MICROBIOLOGY
z The autoimmune diseases can be divided into systemic, localized and Microbiology
haemolytic disorders, depending on tissue/cells affected and the clinico-
pathologic features.
z Systemic autoimmune diseases are associated with auto antibodies to
antigens which are not tissue specific.
z Organ specific or localized autoimmune diseases are diseases in which
autoimmunity involves a particular organ.
Notes
z In organ specific autoimmune diseases the implicated antigens and the
autoimmunity are restricted to specific organs in the body
– Type I diabetes
– Goodpasture’s syndrome
– Multiple sclerosis
– Grave’s disease
– Hashimoto’ thyroiditis
– Myasthenia gravis
z Haemolytic autoimmune diseases involve formation of auto antibodies
against RBCs leading to autoimmune haemolytic anaemia; auto antibodies
may form against platelets resulting in autoimmune thrombocytopaenia; and
formation of anti leucocyte antibodies resulting in autoimmune leucopaenia
and so on.
z Mechanisms of self tolerance are one mechanism is that the clones of
lymphocytes which act against self antigens are deleted. This is the “clonal
deletion” theory. Clonal deletion is mediated by ubiquitous self antigens.
The second is inactivation of developing lymphocytes so our immune
system becomes self tolerant, no activation of immunity against self
antigens as specific lymphocytes are either deleted or inactivated. Clonal
inactivation can be mediated by tissue-specific antigens.
z The causes of autoimmunity are:
z Lymphocytes abnormalities and polyclonal lymphocytes activation;
z Cytokine dysregulation due to changes in lymphocytes;
z Failure of central tolerance and peripheral tolerance;
z Overcome of Ag released from hidden location;
z Antigen modified/ generated by molecular changes in the body;
z Molecular mimicry;
z Alteration in Ag processing;
z Infection;
MICROBIOLOGY 585
Microbiology z The autoimmune diseases can be detected by general tests for autoimmunity
and organ specific tests for autoimmunity. The general tests include
estimation of C Reactive Protein, Autoantibody titers (anti DNA, anti
phospholipids, etc), Rheumatoid Factor, and erythrocyte sedimentation rate.
Example of organ specific test is fasting glucose in Type 1 Diabetes.
z General principle for treatment of autoimmunity is to somehow stop the
immune response to self antigens. This can be achieved by:
Notes
immunosuppression, removal of thymus gland, plasmapheresis, T-cell
vaccination, block MHC with similar peptide, etc.
TERMINAL QUESTIONS
1. Define autoimmunity. What are autoimmune diseases?
2. Describe the various types of auto immune diseases.
3. Describe the causes of auto immunity.
4. Question: Enumerate the causes of autoimmune diseases.
5. Describe various immunopathology mechanisms of autoimmune diseases.
6. Write short note on LE cell.
7. Name the types of tests used to diagnose autoimmune diseases.
8. Briefly describe the principles and methods of treatment of autoimmune
diseases.
64.1
1. Recognize & destroy
2. Auto immunity
3. Organ specific disease
4. Non-organ specific
64.2
1. not tissue specific
2. Rheumatoid Arthritis, Systemic Lupus Erythematosus & Sjogren’s syndrome
586 MICROBIOLOGY
3. Hashimotos’ disease, Graves Disease & Type I diabetes Microbiology
4. Clonal deletion
64.3
1. Pathogens, drugs, hormones & toxins
2. Complement
Notes
3. Molecular mimicry
4. C-Reactive Protein, antibody Titres, Erthrocyte Sedimentation rate &
Rheumatoid factor
MICROBIOLOGY 587
MODULE Organ Transplantation
Microbiology
65
Notes
ORGAN TRANSPLANTATION
65.1 INTRODUCTION
Transplantation is the process of taking a graft which may be a cell, tissue or
an organ – from one site and placing it at another site in the same individual and
or taking a graft – cell, tissue or organ from one individual – the donor and
placing into another individual – the recipient. Graft can be Orthoptic i.e. the
graft is placed at normal anatomical location of the organ (liver, heart) in the
recipient or it can be Heterotopic i.e. the graft is located in different location
(kidney, pancreas). Transplantation is required when a tissue or an organ either
is congenitally non functional or becomes non functional due to injury or disease
to restore the functions. The transplanted organ or tissue is known by the name
of graft. Donor is the person who donates the tissue/organ to be grafted and
recipient is the person who receives the tissue or the organ (graft). The earliest
mention of patient’s own skin grafted to correct the severed nose is in Sushruta
Samhita (circa 800 BC).
First successful kidney transplant was an isograft, i.e. from the same species,
same genetic make up (identical twins). Starzl in Denver performed the First
liver transplant in1963. Since then transplantion has developed a long way and
now practically important different organs of the body are being transplanted to
help patients. Donors can be cadaveric (after death) or live donors. We will
discuss in brief the science and immunology of transplantation in this unit.
OBJECTIVES
z define transplantation
z describe the history of transplantation
588 MICROBIOLOGY
Organ Transplantation MODULE
z describe the various types of grafts Microbiology
z describe the immunologic basis of allograft rejection

z discuss the classification and effector mechanisms of allograft rejection
z describe basics of prevention and treatment of allograft rejection
z define xenotransplantation.
65.2 HISTORY OF TRANSPLANTATION Notes

The earliest mention of patient’s own skin grafted to correct the severed nose
is in Sushruta Samhita (circa 800 BC). Since then attempts have been made to
somehow restore the functionality of diseased/deformed organs by transplanting
tissues/organs from healthy donors. Many scientists working on various aspects
of transplantation and succeeding in one or the other aspect have been awarded
Nobel Prize. As mentioned above the kidney and liver transplantation was first
attempted in 1960s.
The details of work done by of some of the Nobel Prize awardees on
transplantation parameters are given below:
z Alexis Carrel (France) awarded Nobel Prize in Physiology or Medicine
1912 for his work on vascular suture and the transplantation of blood vessels
and organs.
z Peter Brian Medawar awarded Nobel Prize in Physiology or Medicine 1960
for the Discovery of acquired immunological tolerance; that the graft
reaction is an immunity phenomenon and in 1950s, induced immunological
tolerance to skin allografts in mice by neonatal injection of allogeneic cells.
z Joseph E. Murray Joseph E. Murray awarded Nobel Prize in Physiology or
Medicine 1980 and 1990 for his discoveries concerning organ transplantation
in the treatment of human disease; in 1954, the first successful human
kidney transplant was performed between twins in Boston; transplants were
possible in unrelated people if drugs were taken to suppress the body's
immune reaction Human transplantation antigens (HLA) ---- MHC.
z Gertrude B. Elion, George H. Hitchings were awarded Nobel Prize in
Physiology or Medicine in 1988 for discoveries of important principles for
drug treatment i.e. immunosuppressant drug (The first cytotoxic drugs)
---- azathioprine for preventing rejection of grafts.
Now a day’s transplantation of organs is one of the mainstream subjects of
medicine and many people are being helped by transplantation. Almost any one
of the important organs can be transplanted. Transplantation is much advanced
now. Today, kidney, pancreas, heart, lung, liver, bone marrow, and cornea
transplantations are performed among non-identical individuals with ever
increasing frequency and success.
MICROBIOLOGY 589
Microbiology

1. ..................is the process of taking a graft from donor to recipient
2. Graft placed at normal anatomical location of the organ is called ..................
3. Graft located in different location is called ..................
Notes 4. The transplanted organ or tissue is called as ..................
65.3 DEFINITION OF TERMS USED IN

TRANSPLANTATION
You may not be familiar with the terms used in transplantation science. These
are explained below.
Autograft: cell tissue taken from one site of body and transplanted to the
damaged site. Best example is skin grafting in a burns case.
Isograft: it is the term used when tissue/organ taken from one member of the
species is transplanted in another member of the same genetic makeup
(syngeneic) of the same species. Examples are liver, kidney transplantation done
between identical twins.
Allograft: it is the term used when tissue/organ taken from one member of the
species is transplanted in another member of the same species. There will be
genetic differences between recipient and donor.
Xenograft: Tissue or organ taken from one species is transplanted in another
species.
Donor: Person who donates the organ or tissue.
Recipient: Person who receives the tissue or organ from the donor.
MHC: Major histocompatibility complex.
HLA: Histocompatibility locus antigens
65.4 CLASSIFICATION OF GRAFTS

Grafts are classified according to the site, species and type of donor involved
in transplantation. Briefly it is described below.
65.4.1 According to species

z Autologous grafts also known as autografts:
Grafts transplanted from one part of the body to another in the same individual
z Syngeneic grafts also known as isografts:
590 MICROBIOLOGY
Grafts transplanted between two genetically identical individuals of the same Microbiology
species, e.g. between identical twins
z Allogeneic grafts also known as allografts:
Grafts transplanted between two genetically different individuals of the same
species e.g. kidney from son to father, etc.
z Xenogeneic grafts also known as xenografts:
Grafts transplanted between individuals e.g. organ from an animal species Notes
transplanted in another species.
65.4.2 According to site of grafting:

z Orthotopic:
Graft at normal anatomical location of the organ (liver, heart)
z Heterotopic:
Graft located in different location (kidney, pancreas)
65.4.3 According to source of graft:

z Live donor graft
The graft is from a living matched donor.
z Cadaveric (after death) donor graft
The graft is from a donor who has died due to some sickness, accident, etc.
Fig. 65.1: Types of grafts according to species
MICROBIOLOGY 591
Microbiology

1. Autograft a. Grafts transplanted between two genetically
different individuals of the same species
Notes 2. Allograft b. Grafts transplanted between two genetically
identical individuals
3. Xenograft c. Grafts transplanted from one part of the body
to another in the same individual
4. Isograft d. Grafts transplanted from dead donor
5. Cadaver e. Grafts transplanted from one species to another
species
65.5 IMMUNOLOGIC BASIS OF ALLOGRAFT

REJECTION
In earlier days when there was no understanding about transplantation, one of
the major problems in transplantation was the rejection of the graft. Gradually
after studies it was found that the grafts rejection is a kind of specific immune
response to the antigens in the grafted organ/tissue. The phenomenon was
specific and imparted immune memory in the recipient of graft if the graft from
the same donor was transplanted again the recipient will reject the graft faster
compared to first time. This was due to the immune memory of the first graft.
Grafts rejection can be of two types:
z First set rejection

z Second set rejection
65.5.1 First set rejection:

In initial set of experiments in experimental animals the skin graft was
transplanted from one animal to another animal of the same species. The graft
appeared to be accepted initially as it was vascularised and was normal
morphologically and functionally. However it was seen that by fourth day the
graft was invaded by lymphocytes, it was inflamed, blood vessels were occluded
by thrombi, ischemia (decreased vascularity) and necrosis (death of graft tissue)
set in and graft became shrinked looking like a scab and sloughed off by 10th
day. This is first set rejection.
592 MICROBIOLOGY
65.5.2 Second set rejection: Microbiology
A second graft transplanted from same donor in the same recipient results in
accelerated rejection. The same set of events as described above happens but at
an accelerated rate and graft is rejected by 6th day.
65.6 IMMUNOLOGIC BASIS OF GRAFT REJECTION

Transplantation antigens are the antigens which need to be taken into consideration Notes
for both the donor and the recipient to ensure successful transplantation. These
include Major histocompatibility antigens (MHC molecules), Minor
histocompatibility antigens (HLA antigens) and other alloantigens (ABO blood
group, and some tissue specific antigens, etc.). Mismatch of these antigens in
donor and recipient is a strong factor for graft rejection.
65.6.1 Mechanism of allograft rejection

z Cell-mediated Immunity: Recipient's T cell-mediated cellular immune
response against alloantigens on grafts (recognize the allogenetic MHC
molecules as foreign) lead to graft rejection.
z Humoral Immunity: Complements activation, antibody dependent cell
cytotoxicity, opsonization, etc. This is brought about by enhancing and
blocking antibody which results in hyperacute rejection of graft.
z Role of NK cells: NK cells get activated and help in graft rejection.
65.7 EFFECTOR MECHANISMS OF ALLOGRAFT

REJECTION
Allograft rejection can be of two types:
Host versus graft reaction (HVGR)
Graft versus host reaction/disease (GVHR/GVHD)
65.7.1 Host versus graft reaction (HVGR)

The host rejects the graft as seen in conventional organ transplantation. This may
be due to mismatch of MHC and other alloantigens. HVGR can be Hyperacute
rejection (occurs within minutes of transplantation), acute rejection (occurs
within days to 2 weeks after transplantation, 80-90% of cases occur within 1
month) and chronic rejection (develops months or years after acute rejection
reactions have subsided). Antibody against ABO blood type antigen; antibody
against VEC antigen and antibody against HLA antigen, either preformed or
formed after the graft due to mismatch/partial match is the basic cause of
rejection of graft.
MICROBIOLOGY 593
Microbiology 65.7.2 Graft versus host reaction/disease (GVHR/GVHD):

Here the graft is immunologically more powerful and so kind of starts reaction
against the host. Examples are bone marrow transplantation and immune cells
transplantation. In graft versus host disease (GVHD) the immunocompetent
cells in the graft react against the minor histocompatibility antigens and other
antigens which can damage the host.
Notes 65.8 PREVENTION AND TREATMENT OF ALLOGRAFT

REJECTION
Detailed laboratory work is extremely important that will ensure that the donor
and recipient match genetically and this will help to minimize the graft rejection.
The tests include tissue typing. The other modalities to minimize graft rejection
are imunosuppressive therapy and induction of immune tolerance.
65.8.1 Tissue typing includes:

z ABO and Rh blood typing
z Crossmatching (Preformed antibodies)
z HLA typing
– HLA-A and HLA-B
– HLA-DR
65.8.2 Immunosuppressive therapy

Various drugs are given to suppress the immune response of the host. These
include:
z Cyclosporine
z Azathioprine, Cyclophosphamide
z Ab against T cell surface molecules
z Anti-inflammatory agents like corticosteroids, etc.
65.8.3 Induction of immune tolerance by:

z inhibition of T cell activation by injecting soluble MHC molecules in the
recipient
z administration of anti-IL2R mAb
z administration of Th2 cytokines like anti-TNF-a, anti-IL-2?anti-IFN-g mAb
65.9 XENOTRANSPLANTATION
The demand for transplantation of organs damaged due to various diseases is
increasing day by day. The availability of organs is a big issue. Various scientists
594 MICROBIOLOGY
are trying to transplant organs from animal species in the humans (experimental Microbiology
stage). Such grafts are called xenografts. So xenotransplantation is defined as
the transplantation of organ from one species to another species e. g. pig heart
in a human being and so on. So far there has been no success.

1. ................., ................. & ................. antigens are factors for graft rejection
2. Types of graft rejection are ................. & .................
3. Types of Allograft rejection are ................. & .................
4. Transplanting organs from animal species to human is known as .................

z Transplantation is the process of taking a graft which may be a cell, tissue
or organs – from one site and placing it at another site in the same individual
and or taking a graft – cell, tissue or organs from one individual – the donor
and placing into another individual – the recipient. Transplantation is
required when a tissue or an organ either is congenitally non functional or
becomes non functional due to injury or disease to restore the functions.
The earliest mention of patient’s own skin grafted to correct the severed
nose is in Sushruta Samhita (circa 800 BC).
z The earliest mention of patient’s own skin grafted to correct the severed
nose is in Sushruta Samhita (circa 800 BC). Since then attempts have been
made to somehow restore the functionality of diseased/deformed organs by
transplanting tissues/organs from healthy donors. Many scientists working
on various aspects of transplantation and succeeding in one or the other
aspect have been awarded Nobel Prize. As mentioned above the kidney and
liver transplantation was first attempted in 1960s. many scientists in
medicine were awarded Nobel prize for their contributions in transplantation.
z Now a day’s transplantation of organs is one of the mainstream subjects
of medicine and many people are being helped by transplantation.
z Grafts are classified according to the site, species and type of donor involved
in transplantation. According to species can be autografts: grafts transplanted
from one part of the body to another in the same individual; isografts: grafts
transplanted between two genetically identical individuals of the same
MICROBIOLOGY 595
Microbiology species, e.g. between identical twins; allografts: grafts transplanted between
two genetically different individuals of the same species e. g. kidney from
son to father, etc. xenografts: grafts transplanted between individuals e. g.
organ from an animal species transplanted in another species; on basis
of site of grafting: orthotopic: graft at normal anatomical location and
heterotopic: graft located in different location and finally according to
source of graft: live donor graft: the graft is from a living matched donorand
Notes
z Grafts rejection can be of two types:
First set rejection
Second set rejection
z Transplanted graft maybe rejected that is due to the immunological
mechanism. Transplantation antigens are the antigens which need to be taken
into consideration for both the donor and the recipient to ensure successful
transplantation. These include Major histocompatibility antigens (MHC
molecules), Minor histocompatibility antigens (HLA antigens) and other
alloantigens (ABO blood group, and some tissue specific antigens, etc.).
Mismatch of these antigens in donor and recipient is a strong factor for graft
rejection. Cell-mediated Immunity, humoral immunity and NK cells play
a role in graft rejection.
z Detailed laboratory work is extremely important that will ensure that the
donor and recipient match genetically and this will help to minimize the graft
rejection. The tests include tissue typing. The other modalities to minimize
graft rejection are imunosuppressive therapy and induction of immune
tolerance.
65.1
1. Transplantation
2. Orthoptic
3. Heterotopic
4. Graft
65.2
1. (c)
2. (a)
596 MICROBIOLOGY
3. (e) Microbiology
4. (b)
5. (d)
65.3
1. Major Histocompatibility antigens, minor histocompatibility antigens and
alloantigens Notes
2. First set & second set rejection
3. Host versus graft reaction and Graft versus Host reation/disease
4. Xenotransplantation
MICROBIOLOGY 597

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Morphology and Classification of Bacteria MODULE

Fig. 1.1: Prokaryote Cell

Fig. 1.2: Eukaryote Cell

Difference between Prokaryotic and Eukaryotic Cells

Character Prokaryotes Eukaryotes

Nucleus Absent. No nuclear Present with nuclear

Membrane-bound Absent Present. Includes

Chromosome (DNA) Single coiled chromosome Multiple linear

Mitotic division Absent Present

Ribosomes 70S. Free in cytoplasm 80S. Both free in cytoplasm

Flagella when present consist of consist of 9+2 arrangement

Cytoplasmic membrane Eubacteria= Fatty acids Fatty acids joined to

Mitochondria Absent Present

Lysosomes Absent Present

Golgi apparatus Absent Present

Endoplasmic Reticulum Absent Present

1 micron (μ) or micrometer (μm) – one thousandth of a

Microbiology 1.3.2 Microscopy

The types of microscope are

Notes (i) Light or optical microscope

Light or optical microscope

Phase contrast microscope

Dark field / Dark ground microscope

1.3.3 Stained Preparations

Microbiology Differential Stains

INTEXT QUESTIONS 1.2

1.4 SHAPE OF THE BACTERIA

Fig. 1.3: Shapes of bacteria.

INTEXT QUESTION 1.3

Fig. 1.4: Arrangement of Cocci.

INTEXT QUESTIONS 1.4

Cell wall Notes

Microbiology stains. It may be demonstrated by microdissection, reaction with specific

Characteristics Gram Positive Gram Negative

Cytoplasmic membrane is present immediately beneath the cell wall, found in

Microbiology phosphates and other substances. Volutin granules are polymetaphosphates

Fig. 1.7: Flagella.

Various types of mobility is observed because of the presence of the flagella as

INTEXT QUESTIONS 1.5

Microbiology Characteristics of Bacteria Cell Structures

Structure Functions(s) Predominant chemical

Flagella Swimming movement Protein

Common pili or fimbriae Attachment to surfaces; Protein

Capsules (includes “slime Attachment to surfaces; Usually polysaccharide;

Gram-positive bacteria confers rigidity and shape on Peptidoglycan (murein)

Gram-negative bacteria confers rigidity and shape; Peptidoglycan (murein)

Plasma membrane Permeability barrier; Phospholipid and protein

Ribosomes Sites of translation (protein RNA and protein

Inclusions Often reserves of nutrients; Highly variable;

Chromosome Genetic material of cell DNA

Plasmid Extrachromosomal genetic DNA

Fig. 1.8: Spere

1.6 GROWTH AND MULTIPLICATION OF BACTERIA

1.6.1 Bacterial Growth Curve

(i) Lag phase

Microbiology (ii) Log or exponential phase

(iii) Stationary phase

(iv) Phase of decline

Fig. 1.8: The growth curve of bacteria showing different phases

1.7 FACTORS THAT AFFECT THE GROWTH OF

Most bacterial of medical importance are facultative anaerobes. Anaerobic

The lowest temperature that kills a bacterium under standard conditions in a

Microbiology Moisture and Drying