MICROBIOLOGY

FMGE - LAST MINUTE REVISION

Dr. Priyanka Sachdev

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Dr Priyanka Sachdev
Unacedemy code SACHDEV10
 GENERAL
MICROBIOLOGY
DR.PRIYANKA SACHDEV
HISTORICAL ASPECT
Micro-organism not meeting the criteria of KOCH’s
POSTULATES:
MICROSCOPES
 STUDY OF BACTERIA

Unstained (Wet) Preparations Stained Preparations

 A. Unstained (Wet) Preparations

Unstained preparations are examined mainly

1.For bacterial motility (e.g. hanging drop preparation)

2.For demonstration of spirochaetes (e.g. dark ground
microscopy)
 B. Stained Preparations

• Structural detail of bacteria cannot be seen under light

microscope due to lack of contrast.
• Hence it is necessary to use staining methods to produce
colour contrast.
 Common Staining Techniques

1.Simple stains
2.Negative staining
3.Impregnation methods
4.Differential stains
 1. Simple stains

• They provide the colour contrast, but impart the same

colour to all the bacteria in a smear.

• Basic dyes such as methylene blue or basic fuchsin are

used as simple stains.
 2. Negative staining
• In negative staining, the background is stained, while the
structure to be demonstrated is not stained
• Unstained bacteria stand out in contrast against stained
background

• Indian ink and nigrosin dyes, are commonly used for

negative staining.
• This is particularly useful for Bacterial capsule and very
slender bacteria e.g. spirochetes
 3. Impregnation methods

• Bacterial cells and structures that are too thin to be

seen under the light microscope
• So they are thickened by impregnation of silver on the
surface to make them visible

• e.g. demonstration of bacterial flagella and

spirochaetes (Treponema, Borrelia, Leptospira)
 Common Staining Techniques

1.Simple stains
2.Negative staining
3.Impregnation methods
4.Differential stains
 4. Differential stains

• They impart different colours to different bacteria or

bacterial structures.

• The most commonly employed differential stains are →

1. Gram stain
2. Acid-fast stain
3. Albert stain
1.Gram stain
2.Acid-fast stain
3.Albert stain
 GRAM STAIN

• It is the most widely used stain in bacteriology.

• The stain was originally devised by the histologist Christian

Gram (1884) as a technique of staining bacteria in tissues.
METHOD
 REMEMBER
• Gentian violet is used as primary stain
• Ethanol is used as decolorizing agent.
• Carbol fuchsin is used for counterstaining
 INTERPRETATION

• 1. Gram positive → Resist decolourization and retain

primary stain, appear violet

• 2. Gram negative → Decolourised by organic solvents,

and therefore, take the counterstain, appearing red
Most cocci are Gram Positive except:

1. Neisseria
2. Moraxella
Most bacilli are Gram Negative except:

A- Actinomyces
B- Bacillus
C- Clostridium
D- Diptheria Corynebacterium
L-Listeria
M- Mycobacterium
 Poorly Gram Staining Organisms
• 1. Mycobacterium -high lipid content in cell wall(mycolic
acid) -acid fast staining
• 2. Mycoplasma - no cell wall
• 3. Treponema pallidum -too thin to see - Darkfield
Microscopy
• 4. Chlamydia – intracellular very small - Giemsa stain
• 5. Ricketsiae - intracellular,very small Cytoplasmic inclusion
bodies - Giemsa
• 6. Legionella pneumo - poor uptake of counterstain - Silver
stain
 Principle

• Why do grant-positive organisms retain primary stain,

but not gram negative organisms????
 Structure of cell wall
• In gram-positive bacteria there is a thick layer of
peptidoglycan (cell wall) just outside the bacterial
cytoplasmic membrane.

• This thick wall traps the primary stain (crystal violet) which
is not decolorised by ethanol.
• In gram-negative bacteria this peptidoglycan (cell wall)
layer is thin and just outside this peptidoglycan layer is
outer cell membrane (which is not present in gram
positive bacteria).
• This outer cell membrane is partially dissolved by
ethanol, thus washing out the crystal violet and allowing
the counterstain to take.
 REMEMBER

• The integrity of the cell wall is essential for a positive

stain.

• The Gram positive bacteria become Gram negative

when cell wall is damaged.
1.Gram stain
2.Acid-fast stain
3.Albert stain
ACID-FAST STAIN (ZIEHL-NEELSEN
STAIN)

• The acid-fast stain was discovered by Ehrlich

• Subsequently modified by Ziehl and Neelsen

• Staining of Mycobacteria (usually tubercle and lepra

bacilli) is done by this technique.
METHOD
 REMEMBER

• Concentrated carbol fuchsin is used as primary stain

• 20% sulphuric acid / 5% sulphuric acid is used as

decolorizing agent.

• Methylene blue is used for counterstaining

 INTERPRETATION
• Acid-fast organisms resist dccolourisation with acids and
appear red (colour of carbol fuchsin) in blue (colour of
methylene blue) background of pus cells and epithelial
cells.

• Non acid fast organism → decolorisation occurs.So takes

counter stain and appears blue
Principle
 Cell wall of mycobacterium is impermeable to stains

But mycobacterium can be stained by phenolic stain ie. Carbol fuschin

Because Cell wall of mycobacterium tuberculosis contains high content of lipids

specially Mycolic acid (a wax)
Carbol fuschin is soluble in mycolic acid

So cell wall becomes permeable to carbol fuschin

Further heating increases the penetration

Now other bacteria except mycobacterium are decolorized by acid→ So

mycobacterium is called as acid fast bacteria
• Acid-fastness is due to the high content of lipids, fatty
acids (mycolic acid) found in the cell wall of
mycobacterium.
 Modified Ziehl Neelsen stain
(Modified Acid -fast stain)

• There are some modification in any of the reagent.

 1) Use of alcohol as secondary
decolorizer :
• After primary decolorization, 95% alcohol is used to further
decolorize the smear.

➢M tuberculos is resist decolorization with both (i.e acid fast

as well as alcohol fast)
➢Saprophytic mycobacteria are decolorized by alcohol (ie.
they are only acid fast)
 2) Use of acid -alcohol as decolorizer:
• Instead of 20% sulfuric acid, 3% acid in 95 % alcohol is
used as decolorizer.

• This also differentiate tubercle bacilli from saprophytic

mycobacteria
3) Modification in percentage of
sulfuric acid:
Modified ZN stain is used for
 REMEMBER
• M. leprae is less acid fast than M. tuberculosis.

• Hence 5% sulfuric acid instead of 20% is employed for

decolourisation after staining with carbolfuschin
Compare with gram stain
1.Gram stain
2.Acid-fast stain
3.Albert stain
 ALBERTS STAIN

• Staining of Corynebacteria (Corynebacterium diphtheriae

and other corynebacteria is done by this technique.
 Method
• 1. The smear is heated gently by flaming the slide from
underneath. It will fix the smear.
• 2. Cover the smear with Albert I (Albert's stain) for 5
minutes.
• 3. Drain off the whole stain without washing.
• 4. Pour Albert II (iodine solution) over the smear so as to
cover it completely, leave it for 2 minutes.
• 5. Drain off the Albert II solution without washing.
• 6. Blot dry the smear with the help of filter paper.
 Microscopic Examination of the
Smear

• Corynebacterium diphtheriae appear as green coloured

bacilli with bluish black metachromatic granules

• These bacilli are arranged in Chinese letter or cuneiform

arrangement.
Polymetaphosphate (volutin) granules
• Some bacteria contain granules composed of
polymetaphosphate.

• They were first described in spirillum volutans, so they were

called as Volutin granules.

• These granules are also known as Babes Ernst granules or

polar bodies or metachromatic granules.
• Volutin granules
• Babes Ernst granules
• Polar bodies
• Metachromatic granules.
• These granules stain reddish violet with methylene
blue or toluidine blue

• These granules are strongly basophilic.

• In the granules, there is stored phosphate in the form of
linear chains of inorganic phosphate.

• The phosphate is incorporated into nucleic acid during the

synthesis of the latter.

• When nucleic acid synthesis is prevented by starvation , the

granules accumulate in the cytoplasm.
• These granules represent intracellular phosphate reserve
when nucleic acid syntheis does not occur.

• So, volutin metachromatic granules are most frequent in cells

grown under conditions of nutritional deficiency (starvation)
and tend to disappear when the deficient nutrients are
supplied.
Volutin metachromatic granules are common in –

• i) Corynebacteria diptheriae
• ii) Mycobacteria
• iii) Gardenella vaginalis
• iv) Spirillum voluants
• v) Agrobacterim tumefaciens
 BIPOLAR STAINING
(SAFETYPIN APPEARANCE)
• Some bacteria display a safety pin appearance due to the
accumulation of dye at the poles of the cells.

• This characteristic is called bipolar staining.

MNEMONICS
Motile with peritrichous flagella:

Cute Baby SLEePing

Clostridia all except cl.perfringens & cl.tetany
Bacillus except b.antrax
Salmonella except salmonella gallenarum -
pullorum
Listeria monocytogen
E.coli
Proteus
 Motile with polar flagella:

Very Protective Solution HCL is polar

Vibrio
Pseudomonas
Spirochetes
H.pylori
Campylobacter
Legionella
 Anaerobes
Obligate or strict anaerobes

• They grow only in anaerobic environment and can

not tolerate aerobic conditions.
Can't Breathe Air

Clostridia
Bacteroides
Actinomyces
Catalase positive organisms
Oxidase positive organisms
Urease positive organisms
Important Bacterial Tests
HYP
Important Skin Tests
Intracellular Organisms
Pigment Producing Bacteria
Bacterias Producing Biofilms in vivo
Bacterias With special temperature
Bacteria and their alternate names
Virulance factors
Basis of Typing / Classification
HACEK Group of Organisms
CULTURE MEDIA
Dr. PRIYANKA SACHDEV
 INTRODUCTION
• A culture medium is a liquid or gel designed to support
the growth of microorganism or cell.

• Culture media are required to grow the organisms from

infected material to identify the causative agent

• A culture medium may be liquid or solid.

• Inoculum: Suspension of microorganisms

• Inoculation: Introduction of microbes into culture medium

• Colony: visible growth of microbes on the surface of a solid

Culture Media
• A colony is a population of cells arising from a single cell or
spore or from a group of attached cells A colony is often called
a colony-forming unit (CFU)
 Basic constituents
• 1. Water
• 2. Electrolyte
• 3. Peptone
• 4. Meat extract
• 5. Blood or serum
• 6. Agar
• 1. Water: Source of hydrogen and oxygen.

• 2. Electrolyte: Sodium chloride or other electrolytes.

 3. Peptone:
• It is a complex mixture of partially digested proteins.
• It is obtained from lean meat or other protein material
such as heart muscle, casein or fibrin, usually by digestion
with proteolytic enzymes.
• Special brands of peptone such as neopeptone, proteose
peptone are available for special uses
• 4. Meat extract: It is available commercially as ‘Lab-
lemco’. It contains protein degradation products,
inorganic salts, carbohydrates and growth factors.

• 5. Blood or serum: These are used for enriching culture

media. Usually 5-10% defibrinated sheep blood is used.
 6. Agar:
• It is prepared from sea weed (Algae— geladium species) .
• It contains mainly long-chain polysaccharide, a small amount
of protein-like material and a variety of inorganic salts.
• It is available either in powder form.
• It is used in concentration of 2-3%.
• It melts at 98°C and usually solidifies at 42°C.
• Agar does not provide any nutrition to the bacteria but acts
as a solidifying agent only.
• NewZealand agar has twice the jellifying capacity than that of
Japanese agar.
 Agar is preferred to gelatin as a
solidifying agent in culture media→

• This is because gelatin melts at relatively low

temperature (< 35 C), while agar melts at around 98° ).

• Therefore, when gelatin is incubated at the normal

temperature for bacterial growth, i e. at 37° C, it melts,
while agar remains solid.
 Basic constituents
• 1. Water
• 2. Electrolyte
• 3. Peptone
• 4. Meat extract
• 5. Blood or serum
• 6. Agar
 TYPES OF MEDIA
1.Based on physical state

2.On the basis of presence of molecular oxygen and

reducing substances in the media

3.Based on nutritional factors

 Based on physical state

1.Liquid media
2.Semisolid media
3.Solid media
 On the basis of presence of oxygen
and reducing substances in the media

1.Aerobic media
2.Anaerobic media
 Based on nutritional factors
1. Simple media
2. Complex media
3. Synthetic media
4. Special media

(a) Enriched media

(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(i) Transport media
(g) Sugar media
TYPES OF MEDIA
 Simple Media
This is simple with no added ingredients.

1.Nutrient broth and peptone water (liquid) is an

example of simple medium.
• It contains peptone water and meat extract 1%.

2. Nutrient agar (solid) → When 2-3% agar is added to

nutrient broth, it becomes Nutrient agar
• It is the simplest and most common medium in routine
diagnostic laboratories.
 Complex Media

• These have added ingredients for special purpose or

for bringing out certain characteristics or providing
special nutrients required for the growth of the
bacterium under study

• All media other than simple media are complex

 Synthetic Media
• These are prepared from pure chemicals and the exact
composition of the medium is known.
• These are used for special studies such as metabolic
requirements.

• Dubo’s medium with tween 80 is one example of a

synthetic medium.
 Based on nutritional factors
1. Simple media
2. Complex media
3. Synthetic media
4. Special media

(a) Enriched media

(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(i) Transport media
(g) Sugar media
Special Media
 Based on nutritional factors
1. Simple media
2. Complex media
3. Synthetic media
4. Special media

(a) Enriched media

(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(i) Transport media
(g) Sugar media
 (a) Enriched Media

• When basal medium is added with some nutrients such

as blood, serum or egg, it is called enriched medium.

• They are used to grow bacteria which are more exacting

in their nutritional needs.
• eg:
1. Blood agar - Blood is added to nutrient agar. It may be used for growing
a number of bacteria but one specific example is Streptococcus which
requires blood for its growth.

2. Chocolate agar - It is a heated blood agar used for isolation of Neisseria

and Haemophilus influenza

3. Loeffler’s serum slope - Serum is added for enriching the medium. This
medium is used for grouping Corynebacterium diphtherias.
 Based on nutritional factors
1. Simple media
2. Complex media
3. Synthetic media
4. Special media

(a) Enriched media

(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(i) Transport media
(g) Sugar media
 (b) Enrichment Media
• In mixed cultures or in materials containing more than
one bacterium, the bacterium to be isolated is often
overgrown by the unwanted bacteria
• In such situations, substances which have a
stimulating effect on the bacteria to be grown or an
inhibitory effect on those to be suppressed are
incorporated in the liquid medium.
• If such substances are added to a liquid medium, the
result is an absolute increase in the numbers of the
wanted bacterium relative to the other bacteria.
• Such media arc called enrichment media,
 Example
1.Tetrathionate broth where the tetrathionate inhibits
coliforms while allowing typhoid-paratyphoid bacilli
(salmonella) to grow freely,

2.Selenite F broth for dysentery Bacilli

3.Alkaline peptone water for vibrio cholerae

 Based on nutritional factors
1. Simple media
2. Complex media
3. Synthetic media
4. Special media

(a) Enriched media

(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(i) Transport media
(g) Sugar media
 (c) Selective Media

• Substances which have a stimulating effect on the

bacteria to be grown or an inhibitory effect on those to
be suppressed are incorporated in the solid medium.
 Based on nutritional factors
1. Simple media
2. Complex media
3. Synthetic media
4. Special media

(a) Enriched media

(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(i) Transport media
(g) Sugar media
 (d) Indicator Media

• Media contain an indicator which changes colour

when a bacterium grows in them.
 Example

1.Incorporation of sulphite in Wilson and Blair

medium. S. typhi reduces sulphite to sulphide in the
presence of glucose and the colonies of S. typhi have
a black metallic sheen

2.Potassium tellurite in McLeod's medium is reduced

to metallic tellurium by the diphtheria bacillus to
produce black colonies
 Based on nutritional factors
1. Simple media
2. Complex media
3. Synthetic media
4. Special media

(a) Enriched media

(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(i) Transport media
(g) Sugar media
 (e) Differential media

• A medium which has substances incorporated in it,

enabling it to bring out differing characteristics of
bacteria and thus helping to distinguish between them,
is called a differential medium.
 EXAMPLES
1. Mac conkey’s media

• which contains peptone, lactose, agar, sodium

taurocholate and neutral red.
1.The lactose -fermenter (LF)— pink coloured colonies
2.Non-lactose fermenters (NLF) produce colourless or
pale colonies.
2. XLD
3. TCBS
 Based on nutritional factors
1. Simple media
2. Complex media
3. Synthetic media
4. Special media

(a) Enriched media

(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(i) Transport media
(g) Sugar media
 (f) Transport Media
• These are used in the case of delicate organisms (e.g.
gonococci) which may not survive the time taken for
transit or may be overgrown by nonpathogenic
bacteria (e.g. cholera organisms).

• For transport of specimens to the laboratory, special

media are devised and these are termed transport
media.
 Based on nutritional factors
1. Simple media
2. Complex media
3. Synthetic media
4. Special media

(a) Enriched media

(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(i) Transport media
(g) Sugar media
REMEMBER
Colony Appearance on Culture
Important Growth Factors
HYP
HYP
NUMERICALLY NAMED DISEASES
HYP
IMP. FEVERS
 Antigen Antibody
reactions
The only fight with a good cause

Dr. PRIYANKA SACHDEV , MD

Dr. PRIYANKA SACHDEV

 IMMUNITY

Cell mediated Humoral

Dr. PRIYANKA SACHDEV

Cell mediated immunity
 Antigen is identified by Antigen presenting cells (APC)

Engulfed and degraded into antigenic peptides within the APC

The degraded peptides then form complexes with class I or class II

MHC molecules

Transported to the surface membrane of the cell (antigen

processing)

They are displayed on the surface(antigen presentation)

Presented to CD4 T cells

Dr. PRIYANKA SACHDEV
Humoral immunity
 Antigen directly activates B cells

formation of plasma cells

Plasma cells forms antibodies

Ag combines with Ab

Ag- Ab complex is formed

Macrophage phagocyte this Ag- Ab complex

Dr. PRIYANKA SACHDEV
ANTIGEN

Dr. PRIYANKA SACHDEV

• Immunogen
• Antigen

• Immunogenicity
• Antigenicity
Dr. PRIYANKA SACHDEV
• Immunogen is a substance that induce an immune
response.

• Antigen is a substance that reacts with products of

immune response ie. with antibody or antigen binding
receptor on lymphocytes.

All immunogens are antigen but all antigens are not

immunogen

Dr. PRIYANKA SACHDEV

Immunogenicity is the ability to induce a
humoral and/or cell-mediated immune
response.

Antigenicity is the ability of an antigen to react

with products of immune response ie.antibody
or antigen-binding receptors on lymphocytes.

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
 TYPES OF ANTIGEN

1. Complete Ag

2. Incomplete Ag/ Haptans

Dr. PRIYANKA SACHDEV

• Complete Ag→ immunogenic as well as antigenic
➢ ie.they can induce Ab formation as well as can react with Ab

• Incomplete Ag/ Haptans→ not immunogenic but antigenic

➢ ie. they can NOT induce Ab formation but can react with Ab
➢Molecular weight < 100 KD
➢They are not immunogenic but they became immunogenic
when linked to large carrier proteins (protein in nature)
➢2 types → Simple and Complex

Dr. PRIYANKA SACHDEV

 ADJUVANT

• A substance, different from antigen but when mixed with an

antigen, it increases immunogenicity of that antigen

Eg:
• Alum (Aluminium potassium sulphate), Aluminium hydroxide
are commonly used with human vaccines.
• Bordetella pertussis acts as a good adjuvantfor diphtheria and
tetanus toxoid in triple vaccine (DPT vaccine)
• Freunds adjuvant is used with a suspension of killed tubercle
bacilli.
Dr. PRIYANKA SACHDEV
Dr. PRIYANKA SACHDEV
• EPITOPE
• PARATOPE

Dr. PRIYANKA SACHDEV

 EPITOPE / antigenic determinants
• Whole antigen molecule does not induces immune response,
only a part of it where antibody can bind & induce immune
response. These regions of the antigen are called epitope.

• Epitope is immunologically active regions of an antigen that

binds to specific receptors on lymphocytes or antibodies.
• It is also called antigenic determinants.

• It is smallest unit of antigenicity

Dr. PRIYANKA SACHDEV
epitope
Epitope vs Pa ratope

Paratope

Ep¡¡¡¡¡

Dr. PRIYANKA SACHDEV

EPITOPES

Dr. PRIYANKA SACHDEV

 Types
Epitopes are 2 types:

• Linear / Sequential epitope: formed by adjacent

amino acid residues.

• Conformational epitope: formed by amino acid but

not in sequence ( spatially juxtaposed in the folded
protein)
T cell identify linear or sequential epitope

B cell identify conformational epitope

• EPITOPE
• PARATOPE

Dr. PRIYANKA SACHDEV

 Paratope

• It is combining area of the antibody molecule,

corresponding to the epitope

• Specific antigen determinants on paratope are called

IDIOTOPES

Dr. PRIYANKA SACHDEV

epitope
Epitope vs Pa ratope

Paratope

Ep¡¡¡¡¡

Dr. PRIYANKA SACHDEV

EPITOPES

Dr. PRIYANKA SACHDEV

• Epitope and paratope determine specificity which is
hallmark of immunological reaction.

Dr. PRIYANKA SACHDEV

 Superantigen
• Superantigens are potent activators of T-lymphocytes

• Superantigens stimulate very large numbers of T cells, without relation

to their epitope specificity.

• For superantigens, antigen specificity of TCR is not required.

• This leads to an excessive and dysregulated immune response with

release of cytokines IL -1, IL - 2, TNF - a and IF - y.

• Superantigens are capable of activating up to 20% of the peripheral T-

cellpool, where as conventional antigens activate < 1 in 10,000
ANTIBODIES
• Most of the antibody are gamma globulin (but equine
antitoxin is beta or alpha globulins).

• Ig constitute 20-25% of total serum proteins

Dr. PRIYANKA SACHDEV

 TYPES
5 types

GAMDE
1. IgG
2. IgA
3. IgM
4. IgD
5. IgE
Dr. PRIYANKA SACHDEV
STRUCTURE

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
Dr. PRIYANKA SACHDEV
• Ig (glycoprotein) consist of two pairs of polypeptide
chains

➢ 2 Heavy chains → 2H
➢ 2 Light chains chains → 2L

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
• VL = Variable domain of light chain.
• CL = Constant domain of light chain.
• VH = Variable domain of heavy chain.
• CH = Constant domain of heavy chain.

• S-S = Disulphide bond

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
Highly variable zones or Hypervariable regions or Hot spots.

• 3 in L chain
• 4 in H chain

• involved in the formation of antigen-binding sites

• Sites on the hypervariable regions which make actual contact
with the epitope
• They are called complementarity determining regions (CDR)
Dr. PRIYANKA SACHDEV
 H → heavy chain
• Molecular weight 50,000
• 1 variable (VH) and 3 domains in constant region (CH1, CH2, CH3)

• H chain are structurally and antigenically different for each class and are
designated by Greek letter
1. γ→ IgG
2. α→ IgA
3. μ→ IgM
4. δ→ IgD
5. ε → IgE
Dr. PRIYANKA SACHDEV
Heavy chains are of 5 types. They are;

ロ Gamma (у) - IgG

ロ Alpha (a) - lgA
ロ Mu (p) - lgM
ロ Del1a(õ) - IgD
ロ Epsilon (s) - IgE

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
 L → Light chain

• molecular weight of 25,000

• 1 variable (VL) and 1 constant domain (CL)
• L chain is similar in all classes of Ig
• 2 types of L chain are Kappa and lambda
• 1 molecule of Ig may have either kappa or lambda chains
but never both

• Kappa and Lambda occur in ratio of about K : λ = 2 : 1

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
There are two types of light chains, named as,

ロ Kappa (k) chain – K type (60%)

ロ Lambda (L) chain - L tуре (40%)

Dr. PRIYANKA SACHDEV

Constant region = Carboxyterminus ( C terminus) = Fc
• contains only heavy chain
• determines Ig biological properties of antibody molecule

Variable region = Aminoterminus( A terminus) = Fab = Antigen binding

region
• contains both Heavy and Light chains
• determines immunological specificity of antibody molecule.

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
 IMMUNE RESPONSE
• Primary response
• Secondary response

Dr. PRIYANKA SACHDEV

 The Primary Response
• When an individual encounters an antigen for the first
time,

• antibody to that antigen is detectable in the serum after

days or weeks (slow)

• The first antibodies formed are IgM, followed by IgG

• IgM levels tend to decline sooner than IgG levels.

Dr. PRIYANKA SACHDEV
 The Secondary Response
• If second encounter with the same antigen (or a closely
related “cross-reacting” antigen) months or years after the
primary response,

• The antibody response is more rapid and rises to higher

levels than during the primary response.

IgG is produced

• This is due to the persistence of “memory cells”.

Dr. PRIYANKA SACHDEV
 Valency of antibody

• The valency of antibody refers to the number of

antigenic determinants that an individual antibody
molecule can bind.

• The valency of all antibodies is at least two

• It can be more in some instances like IgA and IgM

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
Dr. PRIYANKA SACHDEV
 Antibody

Ig G

Ig A
;
〝 〝
~

lg M
Ig D
IgE.

Dr. PRIYANKA SACHDEV

 IgM Valancy
• Though the theoretical/Actual valency of IgM is ten, this
is observed only with small haptens.

• With larger antigens, the effective valency falls to five,

probably due to steric hindrance

• Thus, IgM is pentavalent (effective valency 5)

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
 TYPES
5 types
GAMDE
• IgG
• IgA
• IgM
• IgD
• IgE

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
 TYPES
5 types
GAMDE
• IgG
• IgA
• IgM
• IgD
• IgE
 IgG
• General purpose antibody
• It has four subclasses G1 > G2 > G3 > G4
• IgG1 is 65% of the total IgG.
• Maximum concentration
• Maximum half life
• Minimum molecular wt
• Half life and concentration of Ig : IgG 23d > IgA 6d > IgM 5d
> IgD > IgE
• Predominant antibody in secondary immune response
• It readily crosses the placenta and therefore is the most
abundant immunoglobulin in newborns.
• Eg of IgG –

• -Ab against Rh factor (Anti Rh D)

• – (LATS) long acting thyroid stimulator Ab in Grave’s
disease
• – SLE, GB Syndrome, syphilis
 TYPES
5 types
GAMDE
• IgG
• IgA
• IgM
• IgD
• IgE
 IgA
• Occur in two forms →
1. Serum IgA (monomer)
2.Secretory IgA (dimer joined by J chain) → present on
respiratory/intestinal mucosa and in secretions such as
milk, saliva and tears and in other secretions of the
respiratory, intestinal and genital tracts.
• Each secretory IgA molecule (dimer)consists of two
H2L2 units and one J chain and one secretory
component.

• J chain is synthesized by plasma cells

• Secretory piece is synthesized by mucosal or
glandular epithelial cells
 TYPES

5 types
GAMDE
• IgG
• IgA
• IgM
• IgD
• IgE
 IgM
• IgM is produced in the primary immune response.
• Earliest Ig to be synthesized by fetus (begin at 20 week of age)
• At 20th weeks Peyer’s patches and lymphoid cells in spleen, and
lymph nodes are developed so fetus has IgM, IgD, IgG
(transplacentally) but not IgA and IgE.
• five H2L2 and 1 J chain (pentamer)
• Heaviest immunoglobulin.
• Maximum molecular wt., sedimentation coefficient,
• Maximum intravascular distribution
• It has highest avidity among all Ig.
• Actual valency of 10
• Effective valency 5
• Though the theoretical/Actual valency of IgM is ten,
this is observed only with small haptens.

• With larger antigens, the effective valency falls to five,

probably due to steric hindrance

• Thus, IgM is pentavalent (effective valency 5)

Eg
– Ab against ABO
– Biological false positive Ab in syphilis
– Rheumatoid factor
– Ab to typhoid O Ag (endotoxin).
 TYPES
5 types
GAMDE
• IgG
• IgA
• IgM
• IgD
• IgE
 IgD

• IgD acts as an antigen receptor

when present on the surface of certain B lymphocytes
 TYPES
5 types
GAMDE
• IgG
• IgA
• IgM
• IgD
• IgE
 IgE / Reagin antibody

• Mostly extravascular
• It mediates Type I hypersensitivity and Prausnitz
kustner (PK) reaction.
• maximum carbohydrate content
• Heat labile
 IMP
• – Sedimentation coefficient - max. IgM
• – Molecular weight - max. IgM, min - IgG
• – Serum concentration, Half life in days, Daily production
(mg/kg) – G > A > M > D > E
• – Intravascular distribution (%) - max IgM, min - IgA
• – Carbohydrate (%) - max IgE
• – Placental transport - only IgG
• – Present in milk - IgG and IgA
• – Selective secretion by seromucinous gland - IgA
• – Heat labile : only IgE
• – J chain - IgA and IgM
Abnormal Ig
 Bence Jones Protein (BJP)

• Monoclonal Ig consist of light chain

• Found typically in multiple myeloma.

• Identified in urine by its characteristic property of

coagulation when heated to 50 C but redissolving at 70 C.
ii. Myeloma (M) protein
• Monoclonal Ab seen in multiple myeloma (IgG, IgA, IgD, IgE) and
waldenstrom’s macroglobulinemia (IgM).

iii. Cryo globulinemia

• Formation of gel or precipitate on cooling the serum, which redissolves
on warming.
• Most cryoglobulin consist of either IgG, IgM or mixed precipitates.
Ag-Ab REACTIONS
 Ag-Ab REACTIONS
1. Precipitation
2. Aglutination
3. Nutralisation
4. Complement fixation test
5. Opsonisation
6. Immunofluorescence (IF)
7. Radioimmunoassay (RIA)
8. Enzyme Linked Immunosorbent Assay (EIA/ELISA)
9. Chemiluminescence Assay (CLIA)
10. Immunochromatography
11. Immunoblotting Dr. PRIYANKA SACHDEV
 Precipitation Reactions:
• When a soluble antigen combines with its antibody in the
presence of electrolytes (NaCl) at a suitable temperature
and pH, the antigen-antibody complex forms an insoluble
precipitate.→ Precipitation
• The precipitate usually sediments at the bottom of tube

• When instead of sedimenting, the precipitate remains

suspended as floccules, the reaction is known as
flocculation
Dr. PRIYANKA SACHDEV
Dr. PRIYANKA SACHDEV
¡Y

Dr. PRIYANKA SACHDEV

Mug-n.
«mn.-:

z… ы
m…
…” mmm….

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
antigen antibody

epitopes
polyclonal antiserum

Dr. PRIYANKA SACHDEV

• It is very sensitive in detection of antigens

• It is relatively less sensitive for the detection of

antibodies
 Mechanism of precipitation
• Marrack proposed the lattice hypothesis for formation of
precipitate
• According to this concept →

• Multivalent antigens combine with Bivalent antibodies

• precipitation occurs only when a large lattice is formed ie Ag

and Ab are in proper proportion

• This occurs in the zone of equivalence (Zone phenomenon)

Dr. PRIYANKA SACHDEV
antigen antibody

epitopes
polyclonal antiserum

Dr. PRIYANKA SACHDEV

 Zone phenomenon
• When the antibodies and antigens are in proper ratio →
precipitation reactions normally occur.

• When there is excess amount of either of two → no

visible precipitate is formed.

Dr. PRIYANKA SACHDEV

 Zone phenomenon
Seen in agglutination and precipitation
consist of 3 parts :

• i. Prozone = Ab excess = weak or absent precipitation

reaction
• ii. Zone of equivalence = Proper proportion = peak
amount of precipitation
• iii. Post zone = Ag excess = weak or absent precipitation
reaction
Dr. PRIYANKA SACHDEV
Dr. PRIYANKA SACHDEV
 serial dilution of
antiserum

zone of equivalence —
visible precipitation

standard antigen
solution

precipitin rings

Dr. PRIYANKA SACHDEV

 Types
It is of following types :

a. Ring test
b. Slide test
c. Tube test
d. Immunodiffusion (ppt in gel) – 4 types
e. Electro immunodiffusion/ electrophoresis
– 2 types
 Ring Test
• A column of antiserum is taken in a narrow tube.
• Layering the antigen solution over it

• A ring of precipitate forms at the junction of the two

liquids.

• Eg. Ascoli s thermoprecipitin test

• Grouping of streptococci by the Lanccficld technique
 Types
It is of following types :

a. Ring test
b. Slide test
c. Tube test
d. Immunodiffusion (ppt in gel) – 4 types
e. Electro immunodiffusion/
electrophoresis – 2 types
 Slide test
• When a drop of the appropriate antiserum is added to
uniform suspension of a soluble antigen on a slide,
precipitation takes place.

• Eg. VDRL test for syphilis

 Types
It is of following types :

a. Ring test
b. Slide test
c. Tube test
d. Immunodiffusion (ppt in gel) – 4 types
e. Electro immunodiffusion/ electrophoresis
– 2 types
 Tube test
A set of test tubes is prepared by adding an constant antigen
solution

serial dilution of the antiserum is added.

Precipitation ring is a visible in the tubes that have an antigen-

antibody ratio within the equivalence zone

This highest dilution with a visible ring is called titer of the

antibodies.
The titer is the reciprocal of the highest dilution showing a positive
result, expressed as a whole number.
Eg. Kahn test for syphilis
 Types
It is of following types :

a. Ring test
b. Slide test
c. Tube test
d. Immunodiffusion (ppt in gel) – 4 types
e. Electro immunodiffusion/
electrophoresis – 2 types
Immunodiffusion (Precipitation in
• several advantages →
gel)
1. The reaction is visible as a distinct band of precipitation, which is stable and
can be stained for preservation
2. As each antigen- antibody reaction gives rise to a line of precipitation, the
number of different antigens in the reacting mixture can be readily observed
3. Immunodiffusion also indicates identity, cross-reaction and nonidentity
between different antigens.

• Immunodiffusion is usually performed in a soft (1%) agar or agarose gel

 4 Types
• 1. Single diffusion in one dimension (Oudin procedure)

• 2. Double diffusion in one dimension (Oakley- Fultborpe

procedure)

• 3. Single diffusion in two dimensions (Radial

immunodiffusion)

• 4. Double diffusion in two dimensions (Ouchterlony

procedure
 Types
It is of following types :

a. Ring test
b. Slide test
c. Tube test
d. Immunodiffusion (ppt in gel) – 4 types
e. Electro immunodiffusion/
electrophoresis – 2 types
 Electroimmunodiffusion

• Immunodiffusion can be speeded up if antigen and

antibody are driven by electricity

• It is combination of electrophoresis and diffusion.

• Performed on agar or agarose gel on a slide,

•Antigen well

•Antibody trough
 The test serum is placed in the antigen well

Electrophoresed for about an hour.

Antibody against human serum is then placed in the trough

Electrophoretic separation of a composite antigen (such as serum) into its

constituent proteins

Immunodiffusion against its antiserum

Separate precipitin lines (Indicating reaction between each individual protein with
its antibody)

Enables identification and approximate quantitation of the various proteins

present in the Ag
• 2 types →

1. Counterimmunoelectrophoresis (CIE or
CIEP)
2. Rocket electrophoresis
2. Rocket electrophoresis
 Quantitative estimation of antigens.

The antiserum to the antigen to be quantitated is incorporated in agarose

The antigen, in increasing concentrations, is placed in wells punched in the set

gel.

The antigen is then electrophoresed into the antibody containing agarose

The pattern of immunoprecipitation resembles a rocket and hence the name.

The length of these rocket like structures corresponds to the concentration of

the antigen
 Types
It is of following types :

a. Ring test
b. Slide test
c. Tube test
d. Immunodiffusion (ppt in gel) – 4 types
e. Electro immunodiffusion/
electrophoresis – 2 types
 Ag-Ab REACTIONS
1. Precipitation
2. Aglutination
3. Nutralisation
4. Complement fixation test
5. Opsonisation
6. Immunofluorescence (IF)
7. Radioimmunoassay (RIA)
8. Enzyme Linked Immunosorbent Assay
(EIA/ELISA)
9. Chemiluminescence Assay (CLIA)
10. Immunochromatography
11. Immunoblotting Dr. PRIYANKA SACHDEV
 Agglutination reactions

• The reaction of particulate antigens with antibodies in

presence of electrolytes to form a large interlocking
aggregates (lattices) is called precipitation reaction.

Dr. PRIYANKA SACHDEV

Dr. PRIYANKA SACHDEV
 Zone phenomenon
• When the antibodies and antigens are in proper ratio
→ Agglutination reactions normally occur.

• When there is excess amount of either of two → no

visible agglutinate is formed.

Dr. PRIYANKA SACHDEV

 Zone phenomenon
Seen in agglutination and precipitation
consist of 3 parts :

• i. Prozone = Ab excess = weak or absent precipitation reaction

• ii. Zone of equivalence = Proper proportion = peak amount of
precipitation
• iii. Post zone = Ag excess = weak or absent precipitation
reaction Dr. PRIYANKA SACHDEV
Dr. PRIYANKA SACHDEV
 Types
It is of following types :

a. Slide test
b. Tube test
c. Antiglobulin (Coombs) test
d. Passive agglutination test
e. Reverse Passive agglutination test
 SLIDE TEST
• When a drop of the appropriate antiserum is added to
uniform suspension of a particulate antigen on a slide,
agglutination takes place.

• It is essential to have on the same slide a control consisting

of the antigen suspension in saline, without the antiserum,
to ensure that the antigen is not autoagglutinable.

• Eg. blood grouping and cross matching

 Types
It is of following types :

a. Slide test
b. Tube test
c. Antiglobulin (Coombs) test
d. Passive agglutination test
e. Reverse Passive agglutination test
 Tube test
• When a fixed volume of a particulate antigen
suspension is added to an equal volume of serial
dilutions of an antiserum in test tubes, the
agglutination titre of the serum can be estimated.

• Eg. Widal test, Brucellosis, Weil-Felix reaction, Paul

Bunnel test, cold agglutination and Streptococcus MG
test.
 Types
It is of following types :

a. Slide test
b. Tube test
c. Antiglobulin (Coombs) test
d. Passive agglutination test
e.Reverse Passive agglutination test
 The antiglobulin (Coombs) test:

• Devised by Coombs, Mourant and Race (1945) for the

detection of anti-Rh antibodies that do not agglutinate Rh
positive erythrocytes in saline.

Dr. PRIYANKA SACHDEV

Rh incompibility

Dr. PRIYANKA SACHDEV

Principle of antiglobulin (Coombs)
test
When antisera containing incomplete anti-Rh
antibodies are mixed with Rh positive red cells,

the antibody coats the surface of the erythrocytes,

but they are not agglutinated

When such erythrocytes coated with anti Rh antibody
globulin

Mixed with a rabbit serum containing antibody against

human gammaglobulin (antiglobulin or Coombs serum)

the cells are agglutinated

Dr. PRIYANKA SACHDEV
Dr. PRIYANKA SACHDEV
脳 e… 喇 ¿Ram

Dr. PRIYANKA SACHDEV

 Types
1. Direct
2. Indirect

Dr. PRIYANKA SACHDEV

 Direct
• The sensitisation of the erythrocytes with incomplete Rh
antibodies takes place in vivo

• eg in infant of HDN

Dr. PRIYANKA SACHDEV

 Occurs in 1 step
• 1. RBC of infants (already sensitized with Rh antibody in
vivo )are mixed with a drop of Coombs serum,

• agglutination results.

Dr. PRIYANKA SACHDEV

 INDIRECT
• Sensitisation of red cells with the antibody globulin is
performed in vitro.

• Eg. In mother of HDN

Dr. PRIYANKA SACHDEV

 Occurs in 2 steps
• 1 . Rh Antibodies of mother are sensitized with RBC in vitro
• 2. Sensitised RBC are mixed with Coombs serum

• Agglutination takes place

Dr. PRIYANKA SACHDEV

REVISION

Dr. PRIYANKA SACHDEV

 Types
It is of following types :

a. Slide test
b. Tube test
c. Antiglobulin (Coombs) test
d. Passive agglutination test
e. Reverse Passive agglutination test
 Passive agglutination test:
• The only difference between the requirements for the
precipitation and agglutination tests is the physical
nature of the Ag.

• By attaching soluble antigens the surface of carrier

particles, it is possible to convert precipitation tests
into agglutination tests

• Such tests arc known as passive agglutination tests.

• The commonly used carrier particles are red cells,
latex particles →
1. Latex agglutination
2. Haemagglutination

• Eg. Rose-Waaler test

 Types
It is of following types :

a. Slide test
b. Tube test
c. Antiglobulin (Coombs) test
d. Passive agglutination test
e. Reverse Passive agglutination test
 Reverse Passive agglutination test
• When instead of the antigen, the antibody is
adsorbed to carrier particles in tests for estimation
of antigens, the technique is known as reversed
passive agglutination.
 Types

It is of following types :

a. Slide test
b. Tube test
c. Antiglobulin (Coombs) test
d. Passive agglutination test
e. Reverse Passive agglutination test
 Ag-Ab REACTIONS
1. Precipitation
2. Aglutination
3. Nutralisation
4. Complement fixation test
5. Opsonisation
6. Immunofluorescence (IF)
7. Radioimmunoassay (RIA)
8. Enzyme Linked Immunosorbent Assay
(EIA/ELISA)
9. Chemiluminescence Assay (CLIA)
10. Immunochromatography
11. Immunoblotting
 COMPLEMENT FIXATION TEST
• Antigen/antibody complexes can be measured by
their ability to fix complement

• Because only an antigen/antibody complex will

"consume" complement if it is present, whereas
free antigens or antibodies do not.
• Used to test antibodies for a particular antigen

• Antigen may be soluble or particulate

• Source of complement is guinea pig serum

• Free complement lyse Sheep RBC

• Eg. Wassermann reaction; coaglutinating

complement adsorption
 Ag-Ab REACTIONS
1. Precipitation
2. Aglutination
3. Nutralisation
4. Complement fixation test
5. Opsonisation
6. Immunofluorescence (IF)
7. Radioimmunoassay (RIA)
8. Enzyme Linked Immunosorbent Assay
(EIA/ELISA)
9. Chemiluminescence Assay (CLIA)
10. Immunochromatography
11. Immunoblotting
 Ag-Ab REACTIONS
1. Precipitation
2. Aglutination
3. Nutralisation
4. Complement fixation test
5. Opsonisation
6. Immunofluorescence (IF)
7. Radioimmunoassay (RIA)
8. Enzyme Linked Immunosorbent Assay
(EIA/ELISA)
9. Chemiluminescence Assay (CLIA)
10. Immunochromatography
11. Immunoblotting Dr. PRIYANKA SACHDEV
Enzyme Linked Immunosorbent Assay (ELISA)
ENZYME IMMUNOASSAYS (EIA)
Dr. PRIYANKA SACHDEV
ELISA is the method of choice in big laboratories as large number
of samples can be tested together using the 96 well microtiter
plate. But it is not preferred in small laboratories:

q It is economical, takes 2-3 hours

q ELISA is the most sensitive immunoassay, thus is the preferred
screening test at blood banks and tertiary care sites.
• ELISA can be used for detection of antigen or antibody

• The principle of ELISA is almost same as that of

immunofluorescence, the only difference being, an
enzyme is used instead of fluorescent dye. The enzyme
acts on substrate to produce a colour in a positive test.

• It is very sensitive as radioimmunoassay.

• It requires only microlitre quantities of test reagents.
Dr. PRIYANKA SACHDEV
Dr. PRIYANKA SACHDEV
 TYPES

1. SANDWICH ELISA
2. INDIRECT ELISA
3. COMPETITIVE ELISA

Dr. PRIYANKA SACHDEV

 SANDWICH ELISA

• For antigen detection

Dr. PRIYANKA SACHDEV

 Wells of microtitre plate are coated with specific antibody
against the antigen to be detected

Specimens to be tested are added in coated wells.

If antigen is present in specimen, it binds to coated antibody.

Antibody against Ag conjugated with an enzyme is added.

This conjugated antibody binds to an antigen already attached to coated antibody.

Substrate is added
In case of binding (positive result), an enzyme acts on substrate to produce colour
Dr. PRIYANKA SACHDEV
• Intensity of which can be read by spectrophotometer
or ELISA reader.

• Colour detection can also be seen by naked eye.

Dr. PRIYANKA SACHDEV

• The color intensity of the sample caused by the end
product is measured with a spectrophotometer.

• The amount of color produced (measured as

absorbance) is directly proportional to the amount of
enzyme, which in turn is directly proportional to the
captured antigen.
Dr. PRIYANKA SACHDEV
• At every step of ELISA test, incubation and washing is
done to wash off unbound reagents.

Dr. PRIYANKA SACHDEV

 TYPES

1. SANDWICH ELISA
2. INDIRECT ELISA
3. COMPETITIVE ELISA

Dr. PRIYANKA SACHDEV

 INDIRECT ELISA
• For antibody detection

Dr. PRIYANKA SACHDEV

 Wells of microtitre plate are coated with antigen.

Sera to be tested are added in these coated wells.

If antibody is present in specimen, it binds to coated antigen.

To detect this antigen-antibody reaction, a goat antihuman immunoglobulin

antibody (Ab against Ab) conjugated with an enzyme is added.

Enzyme conjugated antihuman immunoglobulin binds to antibody

To detect this binding, a substrate is added and enzyme acts on substrate to

produce colour in a positive reaction.
• At every step of ELISA test, incubation and washing is
done to wash off unbound reagents.

Dr. PRIYANKA SACHDEV

 TYPES

1. SANDWICH ELISA
2. INDIRECT ELISA
3. COMPETITIVE ELISA

Dr. PRIYANKA SACHDEV

 COMPETITIVE ELISA

• for detection of HIV antibodies.

• Competition occurs between two antibodies for same

antigen.
Dr. PRIYANKA SACHDEV
• Positive result shows no colour
• Negative result shows appearance of colour

Dr. PRIYANKA SACHDEV

 The microtitre plate wells are coated with HIV antigen.

Sera to be tested is added to these wells and incubated at

37°C and then washed.
 If antibodies are present, antigen-antibody reaction occurs.

To detect this reaction, enzyme labelled specific HIV antibodies are

added.

There is no antigen left for these antibodies to act.

These antibodies remain free and washed off during washing.

Substrate is added but there is no enzyme to act on it.

Therefore, positive results show no colour

 If serum to be tested is negative for antibodies,

antigen is there to combine with enzyme conjugated antibodies

enzyme acts on substrate

produce colour

Dr. PRIYANKA SACHDEV

• Substrates are specific for each enzyme.

• The enzyme are→

1. horseradish peroxidase
2. alkaline phosphatase

Dr. PRIYANKA SACHDEV

• o-phenyl-diamine dihydrochloride for horseradish
peroxidase
• p-nitrophenyl phosphate for alkaline phosphatase

Dr. PRIYANKA SACHDEV

 Uses of ELISA
It has been uscu iul detection of antigens and antibodies of
various microorganisms

• (i) Detection of HIV antibodies in serum

• (ii) Detection of mycobacterial antibodies in tuberculosis
• (Hi) Detection of rotavirus in faeces
• (iv) Detection of hepatitis B markers in serum.
• (v) Detection of enterotoxin of Escherichia coli in faeces.
 Ag-Ab REACTIONS
1. Precipitation
2. Aglutination
3. Nutralisation
4. Complement fixation test
5. Opsonisation
6. Immunofluorescence (IF)
7. Radioimmunoassay (RIA)
8. Enzyme Linked Immunosorbent Assay (EIA/ELISA)
9. Chemiluminescence Assay (CLIA)
10. Immunochromatography
11. Immunoblotting Dr. PRIYANKA SACHDEV
Dr. Priyanka Sachdev
q MD (Tata Memorial Hospital)

❏ Consultant Histopathologist

❏ Educating MBBS students

❏ Teaching experience >10 years

ICONIC
ICONIC
SACHDEV10 SACHDEV10
MICROBIOLOGY
LAST MINUTE REVISION
 Parasitology

Dr. PRIYANKA SACHDEV , MD

 PARASITE
• Parasites are organisms that obtain food & shelter by living
on or within another organism (host), and harm the host
 Medical Parasitology
• Protozoa (unicellular organisms)

• Helminths / Metazoa (multicellular organisms)

 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

 Amoeba

Pathogenic Nonpathogenic Free living

Entamoeba histolytica Entamoeba coli Naegleria fowleri

Entamoeba gingivalis Acanthamoeba
Entamoeba dispar Balamuthia M.
Entamoeba hartmanni
Endolimax nana
Iodamoeba buetschlii
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

 Flagellates

Intestinal,oral ,genital Haemoflagellates

Giardia lamblia Leishmania

Trichomonas Trypanosomes
Chilomastix mesnili
Enteromonas hominis
Retortamonas intestinalis
Dientamoeba fragilis
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

Sporozoans

Plasmodium
Babesia
Toxoplasma gondii
Sarcocystis
Isospora belli
Cyclospora cayetanensis
Cryptosporidium parvum
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

 Cilliates

Balantidium coli
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

Microspora

Microsporidia
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

Three major groups of parasites related to human
beings are: -

• 1. Protozoa → Belong to kingdom protista

• 2. Helminths (Metazoa) → Belong to kingdom
animalia.
• 3. Arthropodes → Belong to Kingdom animalia
REMEMBER
MOST COMMON IN PARASITOLOGY
Infective Stage
LARGEST & SMALLEST IN
PARASITOLOGY
• Largest protozoa→ Balantidum coli

• Largest helminth (largest worm) → T. saginata (beef

tapeworm)
• Largest helminth (largest worm) → T. saginata (beef
tapeworm)

• Largest cestode → T. saginata (beef tapeworm)

• Largest trematode infecting man → Fasciolopsis buski
• Largest Nematode → Ascaris
• Smallest Nematode → Trichinella
• Smallest intestinal amoeba → Dientamoeba fragilis

• Only protozoan parasite found in small intestine of

man → Giardia lamblia

• Only ciliate protozoan parasite of man →

Balantidum coli
• Largest liver fluke → F. Hepatica

• Smallest tapeworm found in human intestine → H.

nana
• Parthenogenic worm (female is able to produce
fertile eggs → Strongyloides stercoralis
 Parasites exhibiting antigenic variations
GTP
•Giardia
•Trypanosoma
•Plasmodia
 Parasites exhibiting acid fastness
"sporas"

• Spore of Microspora
• oocystss of cyclospora, isospora, cryptospora
Parasites causing malabsorption
Parasites causing Anemia
 HOST

• Host is defined as an organism that harbours the

parasite and provides shelter and nourishment to
parasite
 TYPES

• i) Definitive host
• ii) Intermediate host
• The definations of these different types of host differ
according to the type of parasite that infects them.
For protozoan parasites
 Definifive host
• The host in which the parasite undergoes sexual
reproduction is called the definitive host.

• Eg. in malarial parasites, sexual reproduction takes

place in mosquitoes, thus, mosquitoes are the
definitive hosts.
 Intermediate host

• The host in which the asexual stages of a parasite are

found is called the intermediate host.

• Eg. in malarial parasites, asexual multiplication takes

place in human beings. Thus, humans are the
intermediate hosts.
For helminthic parasites
 Definitive host
That harbors the adult form of the parasite
 Intermediate host
That harbors the larval form of the parasite.
 REMEMBER
• For protozoan parasites
1. Definitive host
2. Intermediate host

• For helminthic parasites

1. Definitive host
2. Intermediate host
 Paratenic host (carrier or transport
host)

• Where the parasite remains viable without further

development.

• The host in which larval stage of a parasite survives

but does not develop further, is called paratenic host.
 Reservior host

• It is a host that harbours the parasite and acts as an

important source of infection to other susceptible
hosts.
LIFE CYCLE OF HUMAN
PARASITES
 Simple / Direct life cycle:
• When a parasite requires only single host to complete
its development, it is called as direct life cycle

• There is no intermediate host in these parasites

• e.g. Entamoeba histolytica requires only a human host

to complete its life cycle.
 Complex / Indirect life cycle:

• When a parasite requires 2 or more species of host to

complete its development, the life cycle is called as
indirect life cycle

• e.g. malarial parasite requires both human host and

mosquito to complete its life cycle.
Simple -> 1 host
Complex
2 hosts
3 hosts
 REMEMBER
• For most of the parasites human acts as definitve
host
uman acts as intermediate
(secondary) host
Human acts as intermediate
(secondary) host
CLINICAL POINTERS IN SOME
PARASITIC DISEASE
PARASITIC TESTS
ANTIGEN DETECTION IN PARASITIC DISEASE
PROTOZOA
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

 Amoeba

Pathogenic Nonpathogenic Free living

Entamoeba histolytica Entamoeba coli Naegleria fowleri

Entamoeba gingivalis Acanthamoeba
Entamoeba dispar Balamuthia M.
Entamoeba hartmanni
Endolimax nana
Iodamoeba buetschlii
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

 Flagellates

Intestinal,oral ,genital Haemoflagellates

Giardia lamblia Leishmania

Trichomonas Trypanosomes
Chilomastix mesnili
Enteromonas hominis
Retortamonas intestinalis
Dientamoeba fragilis
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

Sporozoans

Plasmodium
Babesia
Toxoplasma gondii
Sarcocystis
Isospora belli
Cyclospora cayetanensis
Cryptosporidium parvum
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

 Cilliates

Balantidium coli
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

Microspora

Microsporidia
 Protista

Protozoa

Sarcomastigophora Apicomplexa Ciliophora Microspora

Amoeba Flagellates sporozoans Cilliates Microsporidia

 Cystic stage is NOT seen in:
• Infective stage in almost all protozoa is cyst. Here are
exceptions (Infective stage is trophozoite for these
protozoa):-
HYP
 PLASMODIUM
• In 1880, Charles Laveran discovered the malarial parasite.

• The life cycle of malarial parasite was described by A.

Bignami, B. Grass and G. Bastianelli in 1898.

• Ronald Ross in India demonstrated that mosquitoes of

Anopheles species are vectors of malarial parasites. In 1902,
he was awarded Nobel prize for this discovery.

• Trager and Jensen were first to culture malarial parasites in-

vitro in 1976.
• Malarial parasites infecting humans belong to four
species of Genus Plasmodium:

1. P. vivax
2. P. falciparum
3. P. malariae
4. P ovale
• In India, P vivax and P falciparum are very common.
 Morphology

1. Early trophozoite or ring form

2. Trophozoite
3. Schizont
4. Merozoites
5. Gametocyte
• (i) Female gametocyte (Macro- gametocyte)
• (ii) Male gametocyte (Micro gametocyte)
 Modes of infection
• Bite of infected mosquito (Female anopheles
mosquito )
 Life Cycle
• The malarial parasites pass their life cycle in two hosts

1. Man (intermediate host)

2. Female anopheles mosquito (definitive host).
 Human cycle
• The sporozoite is the infective form of the malarial
parasite.

• These sporozoites are present in the salivary gland of

female anopheles mosquitoes.
• Bite of infected mosquito

• Sprozoites are introduced directly into the blood

circulation of man
• Thus, human cycle starts and it comprises of following
stages:
1. Pre-erythrocytic schizogony
2. Erythrocytic schizogony
3. Gametogony
4. Exo-erythrocytic schizogony
Pre-erythrocytic schizogony
 Sprozoites in the blood circulation of man

Within one hour, all the sporozoites leave the blood stream

Enter into liver parenchymal cells

They undergo multiple nuclear division and develop into schizont

Schizont contains 20,000-50,000 merozoites

Liver cells rupture

Release merozoites into the blood stream

Erythrocytic schizogony
The merozoites released from pre-erythrocytic schizogony penetrate RBC

They pass through the stages of trophozoite, schizont and merozoite

RBC rupture to release the merozoites which attack new red blood cells
and continue their erythrocytic schizogony

In RBC parasite causes digestion of haemoglobin into malarial pigment

Clinical attack of malaria

 Gametogony
Some merozoites of erythrocytic schizogony

Develop into male gametocytes(microgametocytes) and

female gametocytes (macrogametocytes)
Exo-erythrocytic schizogony
 P vivax and P ovale

Some sporozoites, on entering into liver cells, do not undergo asexual multiplication

Enter into a resting (dormant) phase.

Hypnozoite

After some period (usually up to 2 years), hypnozoites reactivate to become schizonts

Release merozoites.

Merozoites attack RBC

Responsible for relapse of malaria

 REMEMBER
• Exo-erythrocytic schizogony is absent in P. falciparum

• Therefore, relapses do not occur in malaria caused by

P falciparum.
 Mosquito cycle

• The sexual cycle of malarial parasite actually starts in

the human host by the formation of gametocytes
which are then transferred to mosquito for further
development.
• A female anopheles during its blood meal from the
patient, ingests both the asexual and sexual forms of
parasite
 The mosquito is now capable of transmitting the infection to
man

In the midgut of the mosquito

1 microgametocyte develops into 4 to 8 thread like filamentous

structures named microgametes by the process of
exflagellation

1 macrogametocyte develops into 1 macrogamete

 Fertilisation occurs

Zygote

Zygote lengthens and matures into an ookinete

Ookinete develops into an oocyst

As oocyst matures, a large number of sporozoites develop inside it

oocyst ruptures

Releases sporozoites in the body cavity of the mosquito (special predilection for
salivary glands)
 Laboratory Diagnosis
1. Microscopic examination of peripheral blood smear
2. Fluorescent microscopy
3. Quantitative buffy coat (QBC) test
4. Serological tests
5. Rapid diagnostic tests
6. Molecular methods
 Microscopic examination
of peripheral blood smear

• Thick and thin smears of the blood are prepared on the

same slide or two different slides.

• Blood for smear should be collected a few hours after

the height of febrile paroxysm because the parasites are
most abundant during this period.
1. For thick smear, take a drop of the blood on the slide and
spread it in an area of 1 cm square.

2. For thin smear, take a drop of the blood at one corner of

the slide and spread it with another slide so that tail
formation occurs at another end.

3. Both thick and thin peripheral blood smears are stained

with Giemsa or Leishman stain.
4. These smears are examined under oil immersion lens.
 Fluorescent microscopy

• Blood smear is prepared on a slide and is stained with

acridine orange.

• The stained slide is examined with a fluorescent

microscope.
• Nuclear DNA of malarial parasite is stained green and
cytoplasmic RNA red.
 Quantitative buffy coat (QBC)
test
• Blood is collected in QBC tube coated with
fluorescent dye and is subjected to centrifugation.
• After centrifugation, the buffy coat in the tube is
examined directly under a fluorescent microscope.
• Acridine orange- stained malarial parasites appear
brilliant green.
• QBC is more sensitive method than the thick blood
smear. It can detect 3—4 parasites/pl of blood.
 Serological tests

• Indirect immunofluorescence test (IFAT), indirect

haemagglutination assay (IHA) and enzyme linked
immunosorbent assay (ELISA) are the serological tests
used for the diagnosis of malaria.
 Rapid diagnostic tests

• Rapid diagnostic tests (RDTs) are based on the detection

of antigens using immunochromatographic methods.

• In these tests, a dipstick or teststrip, containing

monoclonal antibodies directed against the parasitic
antigens, is used.
• These tests take about 15 minutes and thus are rapid.
1. Kits available detect histidine-rich protein (HRP-II) of P.
falciparum.
• The test is commercially available as the ‘Parasite F’ test.

2. Parasite lactate dehydrogenase (pLDH) of all four

Plasmodium species that infect humans can also be detected
using rapid tests.

3. A dipstic test for detecting species specific LDH is also

available.
 Molecular methods
DNA probes
• These are sensitive and specific diagnostic methods
for the diagnosis of malaria. It can detect even a low
parasitemia i.e. < 10 parasites/pl

• Polymerase chain reaction (PCR)

 Laboratory Diagnosis
1. Microscopic examination of peripheral blood smear
2. Fluorescent microscopy
3. Quantitative buffy coat (QBC) test
4. Serological tests
5. Rapid diagnostic tests
6. Molecular methods
 Treatment
1. Chloroquine is used for treatment of acute malaria.

2. Mefloquine is active against chloroquine resistant

strains.

3. Artemisinin and its derivatives are the newer

antimalarial drugs used for mefloquine-resistant
and multidrug resistant cases of falciparum malaria.
Treatment of uncomplicated
malaria:
 1. P. Vivax
• P. Vivax cases should be treated with chloroquine for
three days and primaquine for fourteen days.

• Primaquine is used to prevent relapse but is

contraindicated in pregnant women, infants and
individuals with G6PD deficienc
 2. P. falciparum
• 2. P. falciparum uncomplicated cases should be
treated with Artesunate/Artemisinin combination
therapy (ACT), i.e. artesunate 3 days + sulphadoxine –
pyrimethamine on Day-1, accompanied by a single
dose of primaquine on Day-2
Severe malaria
 Helminths / Metazoa
• Helminths are multicellular bilaterally symmetrical
metazoa.

• They are elongated, flat or round in shape.

 CLASSIFICATION
1. Phylum Platyhelminthes
2. Phylum Nemathelmithes
 Helminths / Metazoa

Platyhelminthes Nemathelmithes

Cestodes Trematoda Nematodes

Tapeworms Flukes Round worms
 Life cycle
• Cestodes complete their life cycle in two hosts

• Exception is H nana which have only one host.

• Trematodes complete their life cycle in three hosts
(one definitive and two intermediate).
• Nematodes complete their cycle in one host

• Exception is Dracunculus medinensis and filarial

nematode which have two hosts.
 Treatment
• Praziquantal is the DOC for all cystode and
trematode infestations

• Exception
1. Cysticercosis & hydatid disease (DOC is
albendazole)
2. Fasciola hepatica (DOC is Bithionol).
• Albendazole is the DOC for all nematode infestations

Exceptions
1. Enterobius (DOC is mebendazole)
2. Filarial parasites (DOC is DEC)
3. Onchocerca and strongyloides (DOC is ivermectin)
4. Angiostrongylus cantonensis (DOC is
thiabendazole)
5. Dracunculus (DOC is metronidazole).
Cestodes: Tapeworms
 Cestodes: Tapeworms
• Cestodes are segmented
• Dorsoventrally compressed and resemble a
measuring tape.

• Hence, they are called tapeworms.

• Their size varies from a few millimetres to several
metres in length.
• Introduction

• Morphology

• MOT

• Life cycle

• Pathogenesis

• Lab diagnosis

• Treatment

• Prevention
 Nematodes

• Nematodes are helminths which belong to Phylum

Nemathelminthes
• They resemble the common earthworm.
• They are elongated, bilaterally symmetrical, cylindrical,
unsegmented worms with tapering ends.
• The name ‘nematode’ means thread-like, from nema
meaning thread.
• The digestive system consists of the oesophagus,
intestine, rectum, mouth and subterminal anus.

• Excretory and nervous systems are rudimentary

• Circulatrory system is absent.

• The male is generally smaller than female.
• The sexes are separate (diecious).

• The male reproductive system consists of testis, vas

deferens, seminal vesicle and ejaculatory duct which
opens into the cloaca.
• The female reproductive system consists of the ovary,
oviduct, seminal receptacle, uterus and vagina.
 Life Cycle

• Nematodes pass their life cycle in one host

• Exception Filarioidea and Dracunculoidea which

require two hosts.
Classification based on habitat
Classification based on
they Lay Egg or Larva
 Oviparous

• Most of the nematodes are oviparous, i.e following

fertilization, the female worms produce eggs that
take some time to hatch out to form larvae in the
environment.
• Eggs with segmented ovum—Hookworm species
• Eggs with unsegmented ovum—Ascaris species
• Eggs with unsegmented ovum with mucus plug at
both the poles—Trichuris species
• Eggs containing larva that takes some time to hatch
out—Enterobius species.
 Viviparous

• Female worms directly give birth to larvae; there is no

egg stage.

• Eg. Filarial worm, Trichinella species, Dracunculus

species.
 Ovoviviparous

• Female worms lay eggs containing larvae that

immediately hatch out.

• Eg. Strongyloides species

• Introduction

• Morphology

• MOT

• Life cycle

• Pathogenesis

• Lab diagnosis

• Treatment

• Prevention
 FILARIAL PARASITES

• Filariasis is a parasitic disease, that is caused by filarial

nematode worms.
• Eight species of filarial worms infect humans, who
are the definitive hosts

1. Wuchereria bancrofti,
2. Brugia malayi,
3. Loa loa
4. Onchocerca volvulus
5. Mansonella ozzardi,
6. M. perstans,
7. M. streptocerca
8. Brugia timori
 Classification
• According to the habitat of the adult worm, human
filarial infections are classified as follows:
Lymphatic filariasis
• Wuchereria bancrofti
• Brugia malayi
• Brugia timori

Subcutaneous filariasis
• Loa loa
• Onchocerca volvulus
• Mansonella streptocerca

Serous cavity filariasis

• Mansonella perstans
• Mansonella ozzardi
• The female worms are viviparous giving birth to
larvae known as microfilariae.

1. In some species, the microfilariae retain their

egg membranes which envelop them as sheath
known as ‘sheathed’ microfilariae.
2. Some other species of filarial nematodes
rupture their egg membranes and come out as
‘unsheathed’ microfilariae.
1. Sheathed microfilariae
• Wuchereria bancrofti
• Brugia malayi
• Loa loa

2. Unsheathed microfilariae
• Mansonella streptocerca
• Mansonella ozzardi
• Mansonella streptocerca
• Oncocerca volvulus
 Microfilarial periodicity:
• It is defined as the time when most of the
microfilariae are found in the peripheral blood
 Nocturnal periodicity
(night time, between 9 pm and 2 am):

• Wuchereria
• Brugia
 Diurnal periodicity
(day time):

• Loa loa
 Sub-periodic
• (present throughout; with slight increase in the
afternoon):

• Rarely Wuchereria and Brugia can be sub-periodic

 Non periodic
(Any time):
• Mansonella
• Onchocerca
TREMATODS
 Trematodes

• Trematodes are leaf-like unsegmented flat worms.

• They are also named flukes.

• The most characteristic external structures are two
suckers — the oral sucker and the ventral
(acetabulum) sucker.

• They are hermaphrodite (monoecious) except the

schistosomes in which sexes are separate.
• The body is covered by an integument which often
bears spines.

• They do not have body cavity, circulatory or

respiratory organs.
• The alimentary system consists of a mouth surrounded by
the oral sucker, a muscular pharynx and the oesophagus
which bifurcates anterior to the ventral sucker into two
blind caeca.
• The alimentary canal therefore appears as an inverted ‘Y’

• The anus is absent.

• The excretory system consists of flame cells and collecting
tubules which lead to a median bladder opening at the
posterior end of the body.
• The nervous system is rudimentary and consists of a
group of paired ganglion cells.
• The reproductive system is well developed.
• They are hermaphroditic (having both male and
female structures) except schistosomes in which sexes
are separate.
• The genital organs lie between the two branches of
the intestine.
• Trematodes (except schistosomes) are oviparous and
lay eggs which are operculated.
Classification according to the
habitat
HYP
REMEMBER
VIROLOGY
Dr. PRIYANKA SACHDEV , MD
GENERAL PROPERTIES OF VIRUSES
• Viruses are the smallest obligate intracellular infective
agents

• They contain only one type of nucleic acid (DNA or RNA)

as their genome ,never both

• They are resistant to antibiotics and sensitive to

interferons
• They have no metabolic activity outside the living cells

• They do not possess a cellular organization like ribosomes , ER,

mitochondria etc.

• They lack the enzymes necessary for protein and nucleic acid
synthesis.

• They are dependent for replication on the synthetic machinery of

host cells → so, they cannot grow in cell free culture media /
inanimate media.
• Viral genome (nucleic acid) diverts the host’s metabolism to
synthesise a number of virus specific macromolecules required for
the production of virus progeny.

• The extracellular infections virus particle is called the virion

• They multiply by a complex process and not by binary fission.

• With few exceptions, viruses are very heat labile

 MORPHOLOGY OF VIRUSES
• Size
• Structure
• Symmetry
• Shape
 Size
• Viruses are much smaller than other organisms.
• The extracellular infectious virus particle is called the
virion
• The size of viruses ranges from 20 to 300 nm in
diameter.
 REMEMBER
• Smallest virus → Parovovirus
• Largest virus → Poxvirus

• Smallest DNA virus → Parovovirus

• Largest DNA virus → Poxvirus

• Smallest RNA virus → Picornavirus

• Largest RNA virus → Paramyxovirus
 Measuring the Size of Viruses
1. Collodion membrane filters →In earlier days the virus
particles were measured by passing them through the
collodion membrane filters of different pore sizes (gradocol
membranes).

2. Rate of sedimentation→ Now With the development of

ultracentrifuge, the virus size could be calculated from the
rate of sedimentation of virus in the ultracentrifuge

3. Electron microscopy
 Structure of virus
• The virion consists of →

1. A nucleic acid core (genome)

1. Surrounded by a protein coat (capsid)

• The capsid together with the enclosed nucleic acid is
known as the nucleocapsid

3. Envelop→Some of the viruses also have envelop

A nucleic acid core (genome)
 CLASSIFICATION OF VIRUSES
• Based on nueclic acid →

1.DNA viruses
2.RNA viruses
DNA viruses
 HHAPPPy
Hepadna
Herpes
Adeno
Parvo
Papova
Pox
 REMEMBER
• All DNA viruses have double stranded DNA, except
Parvovirus which has single stranded DNA
DNA viruses
RNA viruses
 REMEMBER
• All RNA viruses have single stranded RNA, except
reoviridae which has double stranded RNA.
 Segmented nucleic acid
Some viruses have segmented nucleic acid (genome).
These are (Mnemonic BORA) :-

• 1. Bunyaviridae: 3 segments of single stranded RNA.

• 2. Orthomyxoviridae (influenza): 8 segments of single
stranded RNA.
• 3. Reoviridae (rotavirus, reovirus): 10-12 segments of
double stranded RNA.
• 4. Arenaviridae: 2 segments of single stranded RNA.
 Structure of virus
• The virion consists of →

1. A nucleic acid core (genome)

1. Surrounded by a protein coat (capsid)

• The capsid together with the enclosed nucleic acid is
known as the nucleocapsid

3. Envelop→Some of the viruses also have envelop

 Capsid
• The capsid is composed of a large number of protein
subunits (polypeptides) which are known as
capsomers
 Functions of capsid→
1. Forming an impenetrable shell around the nucleic
acid core,So protect it
2. To introduce viral genome into the host cells by
adsorbing readily to cell surfaces.
3. Provides the structural symmetry to the virus
particle.
4. Antigenic
 Envelope
• Certain viruses also contain envelope that surrounds the
nucleic acid.

It is lipoprotein in nature.
➢The lipid is largely of host cell origin
➢The protein is virus coded
 Functions of envelop→
1. Enveloped viruses are susceptible to the action of
lipid solvents like ether and chloroform.

2. Envelopes confer antigenic, biological and

chemical properties on viruses
 REMEMBER
• Non-enveloped DNA viruses → PAP
1. Papova
2. Adeno
3. Parvo

• Non-enveloped RNA viruses → PCR

1. Picorna
2. Calci
3. Reo
SUMMERY
 Peplomers:
• Protein subunits are exposed as projectile spikes on
the surface of the envelope. These structures are
called peplomers

• Examples of peplomers →
1. Hemagglutinin
2. Neuraminidase
 HAEMAGGLUTININ

• Peplomers on the envelope which can agglutinate

erythrocytes of different species.

• Haemagglutination of influenza virus has been studied

extensively.
The viral haemagglutinin (glycoprotein) has special affinity
for a different glycoprotein located on the surface of
erythrocyte

When erythrocytes are added to serial dilutions of viral

suspension

The virus and ertythrocytes adhere to each other

Haemagglutination
Procedure of Viral Haemagglutination
Test
Serial dilutions of a viral suspension taken

Fixed amount of erythrocytes are added to all

The highest dilution that produces haemagglutination

Haemagglutinaton titre (HA units)

• This test provides a simple and rapid method for
detection of viruses

• The haemagglutination reaction is specifically inhibited

by the antibody to the virus.

• The haemagglutination inhibition test (HI) is routinely

used for detecting antiviral antibody in diagnosis and
research.
Haemagglutinate human erythrocytes at 37°C

RIPE Mango→
Reo
Influenza
Para-influenza
Entero and some cox and ECHO
Mumps
 Peplomers:
• Protein subunits are exposed as projectile spikes on
the surface of the envelope. These structures are
called peplomers

• Examples of peplomers →
1. Hemagglutinin
2. Neuraminidase
 Neuraminidase
• Another Peplomers on the envelope of virus.

• It acts on the receptors on erythrocytes and destroys

them.
• It is known as Receptor destroying enzyme (RDE)
• Destruction of surface receptors results in the reversal
of haemagglutination and the release of viruses from
the surface of erythrocyte.

• This process is known as elution.

 REMEMBER

• Elution is found only in the myxoviruses that possess

neuraminidase.
 MORPHOLOGY OF VIRUSES
• Size
• Structure
• Symmetry
• Shape
 Symmetry

• 3 types of symmetry are determined by the

arrangement of capsid around the nucleic acid core →

• (i) Icosahedral (cubical) symmetry

• (ii) Helical symmetry
• iii) Complex symmetry
i) Icosahedral (cubical) symmetry
• An icosahedron is a polygon with 12 vertices or corners
and 20 facets in the shape of equilateral triangular
faces

• Icosahedral symmetry has a rigid structure.

• 1. Icosahedral DNA viruses:
• All DNA viruse except Poxvirus→ Parvovirus,
Papovavirus, Adenovirus, Hepadnavirus (HBV), Herpes
virus.

• 2. Icosahedral RNA viruses:

• All non enveloped RNA virus→ Picornavirus, Calcivirus,
Reovirus,
 (ii) Helical symmetry
• The nucleic acid and the capsomers are wound
together to form a helical or spiral tube

• Examples → All enveloped RNA viruses.

 (iii) Complex symmetry:
• Some viruses do not show either icosahedral or helical
symmetry due to the complexity of their structures

• Example → Poxvirus
 REMEMBER
• All DNA viruses (whether enveloped or nonenveloped)
have icosahedral symmetry except poxvirus which has
complex symmetry
• Pox virus have complex symmetry
• No DNA virus has helical symmetry.

• All non enveloped RNA viruses have icosahedral

symmetry
• All enveloped RNA viruses have helical symmetry
 MORPHOLOGY OF VIRUSES
• Size
• Structure
• Symmetry
• Shape
 Shape of virus
• Varies in different groups

• Pox virus is brick-shaped

• Rabies virus is bullet-shaped
• Tobacco mosaic virus is rod-shaped
• Adenovirus is Space vehicle shaped
SUMMARY
 REPLICATION OF VIRUSES
Due to lack of biosynthetic enzymes

viruses replicate by taking over the biochemical machinery

of the host cell

Synthesise virus specific macromolecules required for the

production of virus progeny.
 Six sequential phases

• 1. Adsorption
• 2. Penetration
• 3. Uncoating
• 4. Biosynthesis
• 5. Maturation and
• 6. Release
 1. Adsorption or Attachment
• The viruses come in contact with the cells by random
collision

• Adsorption or attachment is mediated by the binding of

virus surface structures, known as ligands, to the
receptors on cell surface.
• In influenza virus, the haemagglutinin (a surface
glycoprotein) binds specifically to sialic acid residue of
glycoprotein receptor sites on the surface of
respiratory epithelium.

• In human Immunodeficiency virus (HIV), attachment

is between the viral surface glycoprotein gp 120 and
the CD4 receptor on host cells.
 Six sequential phases

• 1. Adsorption
• 2. Penetration
• 3. Uncoating
• 4. Biosynthesis
• 5. Maturation and
• 6. Release
 2. Penetration
➢After attachment, the virus particles engulfed by a
mechanism resembling phagocytosis, a process known
as viropexis

➢Alternatively, in case of the enveloped viruses, the

envelope may fuse with the plasma membrane of the
host cell releasing the nucleocapsid into the
cytoplasm.
 Six sequential phases

• 1. Adsorption
• 2. Penetration
• 3. Uncoating
• 4. Biosynthesis
• 5. Maturation and
• 6. Release
 3. Uncoating

• Stripping the virus of its outer layers and capsid to

release the nucleic acid into the cell.
 Six sequential phases

• 1. Adsorption
• 2. Penetration
• 3. Uncoating
• 4. Biosynthesis
• 5. Maturation and
• 6. Release
 4. Biosynthesis
• After uncoating, the viral genome directs the
biosynthetic machinery of the host cell→

1. To shut down the normal cellular metabolism

2. To production of viral components.
➢Nucleic acid genome of most DNA viruses is
synthesised in the host cell nucleus (exept poxviruses)

➢Nucleic acid genome of most RNA viruses is

synthesised in the cytoplasm (except orthomyxoviruses
,retroviruses)

➢Viral protein is synthesised only in the cytoplasm

 Biosynthesis consists of the
following steps
1. Transcription of messenger RNA (mRNA) from viral nucleic
acid.

2. Translation of the mRNA into ‘early proteins’ or

‘nonstructural proteins’ → These are enzymes which
initiate and maintain synthesis of virus components and also
induce shutdown of host protein and nucleic acid synthesis.

3. Replication of viral nucleic acid

4. Synthesis of ‘late proteins’ or ‘structural proteins’ which
constitute daughter virion capsids.
a) Double stranded DNA viruses
• This double stranded viral DNA acts as a template for
its replication, and also for transcribing into mRNA
which are translated into viral proteins.
b) Single stranded DNA viruses
stranded DNA viruses

A complementary strand is first synthesised,

Producing double stranded ‘replicative forms’.

This double stranded viral DNA acts as a template for its

replication, and also for transcribing into mRNA which are
translated into viral proteins.
 c) Double stranded RNA viruses

• The double stranded RNA is transcribed to mRNA by

viral polymerases
 d) Single stranded RNA viruses

• 3 types →

1. Plus strand / positive sense RNA viruses.

2. Negative strand / minus sense RNA viruses
3. Retroviruses
 Plus strand / positive sense RNA
viruses

• The viral RNA itself act as the mRNA and is translated

directly into viral proteins in the host cell cytoplasm
 RNA viruses with positive strand
RNA

1. Picomaviridae
2. Togaviridae
3. Coronaviridae
4. Calciviridae
5. Flaviviridae
 MNEMONIC
• CALL PICO and FLAVa flav.
• There's a RETRO TOGA party with lots of CORONAs
 Negative strand / minus sense RNA
viruses
• Virus RNA is not able to begin translation immediately.
• Negative strand RNA first transcribed into positive
strand RNA by viral RNA dependent RNA polymerase
• Then translated into viral proteins
 Retroviruses
• They have reverse transcriptase which causes
transcription to occur in reverse fashion i.e.
• synthesis of DNA from RNA (normally in transcription,
RNA is synthesized from DNA).
• This DNA is transcripted into RNA.
 Six sequential phases

• 1. Adsorption
• 2. Penetration
• 3. Uncoating
• 4. Biosynthesis
• 5. Maturation
• 6. Release
 5. Maturation
• The viral nucleic acid and capsid polypeptide assemble
together to form the daughter virions.

• The assembly takes place in either the nucleus (herpes

and adenoviruses) or cytoplasm (picorna and pox
viruses).
 Six sequential phases

• 1. Adsorption
• 2. Penetration
• 3. Uncoating
• 4. Biosynthesis
• 5. Maturation and
• 6. Release
 6. Release
• Enveloped viruses are released by a process of
budding from the cell membrane

• The host cell may be affected (lysis) or may not

affected
 Six sequential phases

• 1. Adsorption
• 2. Penetration
• 3. Uncoating
• 4. Biosynthesis
• 5. Maturation and
• 6. Release
 ECLIPSE PHASE
• From the stage of penetration of virus into the host
cell till the appearance of first infectious virus progeny
particle, the virus cannot be demonstrated inside the
host cell.

• This period is known as eclipse phase

• The duration of eclipse phase is about 15 to 30 minutes

for bacteriophages and 15-30 hours for animal viruses.
 Abnormal Replicative Cycles
Von magnus phenomenon

• When cells are infected with a high dose of influenza

virus
• There is defective assembly during replication
• The produced virus has high hemagglutinin titre but low
infectivity
 CULTIVATION OF VIRUSES
• As viruses are obligate intracellular parasites they
cannot be grown on any inanimate culture medium

• Viruses multiply only in living cells

 3 methods →

• A. Animal inoculation
• B. Embryonated egg inoculation
• C. Tissue culture
 VIRUS CULTIVATION

Animal inoculation
Embryonated egg inoculation
Tissue culture

Mice CAM Organ culture

Allantoic cavity Explant culture
Amniotic cavity Cell culture
Yolk sac

Primary Diploid Continuous

 A. Animal Inoculation
• Mice are most widely employed animals.

• Mice may be inoculated by several routes—intracerebral,

subcutaneous, intraperitoneal or intranasal.

• The growth of the virus in inoculated animals may be indicated

by death, disease or visible lesions.

• Other animals used are guinea pigs, rabbits and ferrets.

 VIRUS CULTIVATION

Animal inoculation
Embryonated egg inoculation
Tissue culture

Mice CAM Organ culture

Allantoic cavity Explant culture
Amniotic cavity Cell culture
Yolk sac

Primary Diploid Continuous

 B. Embryonated Egg Inoculation

• Goodpasture first used embryonated hen’s egg (7 to 12

days old) for cultivation of viruses.
 1. Chorioallantoic Membrane (CAM)

• CAM is inoculated mainly for growing poxviruses.

• It produces visible lesions (pocks)
• Each pock is derived from a single virion.
• So Pock counting indicates the number of viruses present
in the inoculum.
• Pocks produced by different viruses have different
morphology
 2. Allantoic Cavity
• Inoculation into the allantoic cavity provides a rich
yield of influenza virus and and rabies virus for
vaccine production

• Duck’s eggs being bigger, provide a better yield of

rabies virus and were used for the preparation of the
inactivated non- neural rabies vaccine.
 3. Amniotic Sac

• Inoculation into the amniotic sac is mainly used for

the primary isolation of the influenza virus
 4. Yolk sac Inoculation

• It is inoculated for the cultivation of some viruses and

certain bacteria (chlamydia and rickettsiae)
 REMEMBER
a) Chorioallantoic Membrane→ HSV Vaccinia, Small pox,
Monkey pox, Cow pox Camel pox.

b) Amniotic Sac → Influenza, mumps

c) Allantoic Cavity → Influenza, Some paramyxoviruses

d) Yolk Sac → Some viruses, Chlamydia, Rickettsia

 VIRUS CULTIVATION

Animal inoculation
Embryonated egg inoculation
Tissue culture

Mice CAM Organ culture

Allantoic cavity Explant culture
Amniotic cavity Cell culture
Yolk sac

Primary Diploid Continuous

 C. Tissue Culture

Three types of tissue cultures are available:

• 1. Organ culture
• 2. Explant culture
• 3. Cell culture
 Organ Culture
• Small bits of organs are maintained in tissue culture
growth medium.

• Organ cultures are useful for the isolation of highly

specialized viruses of certain organs
• e.g. tracheal ring culture for the isolation of the
coronavirus, a respiratory pathogen.
 2. Explant Culture

• Fragments of minced tissue can be grown as

‘explants’

• This method is rarely done nowadays

 3. Cell Culture
• This is the type of culture routinely employed for diagnostic
virology.

1. Tissues are dissociated into the component cells by the

action of proteolytic enzymes such as trypsin.
2. The dissociated cells are washed, counted and suspended
in a growth medium.
3. The cell suspension is distributed in glass or plastic
bottles, tubes or petri dishes.
4. On incubation, the cells adhere to glass or plastic surface
(wall of test tube) and divide to form a confluent
monolayer sheet of cells within a period of one week.
 Cell culture types
3 types on the basis of their number of generations
through which they can be maintained:

• (i) Primary cell culture

• (ii) Diploid cell strains
• (iii) Continuous-eell lines.
 (i) Primary cell cultures
• These are normal cells freshly taken from the organs
of animal or human being and cultured.

• They are capable of very limited growth in culture 5-

10 divisions at the most.

• They are commonly employed for the primary

isolation of viruses
 (ii) Diploid cell strains
• These are cells of a single type that contain the same
number of chromosomes as the parent cells and are diploid.
• They can be subcultured for a limited number by growth
factors

• After about 50 serial subcultures they undergo ‘senescence’

and the cell strain is lost.
• They are commonly employed for the primary isolation of
viruses and their cultivation for vaccine production.
 (iii) Continuous cell lines
• These are cells of a single type that are capable of indefinite
growth in vitro.

• They are usually derived from cancerous tissue.

• They can be j serially cultivated indefinitely, therefore, they are

termed continuous cell lines.
• These cell lines may be maintained by serial subcultures or stored
in the cold (-70°C) for use when necessary.

• Used for vaccine manufacture

 VIRUS CULTIVATION

Animal inoculation
Embryonated egg inoculation
Tissue culture

Mice CAM Organ culture

Allantoic cavity Explant culture
Amniotic cavity Cell culture
Yolk sac

Primary Diploid Continuous

 VIRAL ASSAYS
• A. Total Particle Count
• B. Infectious Virions Count
 A. Total Particle Count

1. Electron Microscopy
• The virus particles can be counted directly under the
electron microscope.

2. Haemagglutination
• With haemagglutinating viruses, quantitation is done
by the determination of haemagglutination titres.
 B. Infectious Virions Assay

Quantitative assays
 Quantitative Infectivity Assay

• This assay measures the number of viable

infectious virus particles in a suspension

• (i) Plaque assay

• (i) Pock assay
 (i) Plaque assay
1. A viral suspension is inoculated into confluent monolayer
of cultured cells in a bottle or petri dish.

2. Each infectious virus particle gives rise to a localised

focus of infected cells, called a plaque.

3. Plaques can be seen with the naked eye.

4. Each plaque indicates an infectious virus, therefore, the

plaque titre is the infectivity titre.
 (ii) Pock assay
1. Each pock on CAM arises from a single virus
particle.

2. Viruses that form pocks on CAM can be assayed

by counting the number of pocks which
corresponds with the number of viruses present
in the inoculum.

3. This is known as pock assay.

 Inclusion bodies:
• Inclusion bodies are structures with distinct size, shape,
location and staining properties that can be demonstrated
in virus infected cells under the light microscope.

• They typically represent sites of viral multiplication in a

bacterium or a eukaryotic cell

• Usually consist of viral capsid proteins

• The appearance of inclusion bodies is the most
characteristic histological feature in virus-infected
cells.
• They may be →

➢Acidophilic (stained by eosin) or basophilic (stained

by hematoxylin),
➢single or multiple
➢Large or small
➢Round or irregular.
 TYPES
1. Intracytoplasmic inclusion bodies
2. Intranuclear inclusion bodies
3. Intranuclear and intracytoplasmic inclusion bodies
 Intracytoplasmic inclusion bodies
Those viruses that have cytoplasmic assembly (mainly RNA
viruses) yield cytoplasmic inclusions.

1. Rabies virus (Negri bodies)

2. Vaccinia (Guarnieri bodies)
3. Variola (Paschen bodies)
4. Fowlpox (Bollinger bodies)
5. Molluscum contagiosum (Henderson-Patterson bodies )
 Intranuclear inclusion bodies
In general, those viruses that are assembled in the
nucleus (usually DNA viruses) produce intranuclear
inclusions.

• Intranuclear inclusion bodies were classified into 2

types by Cowdry
1. Cowdry type A
2. Cowdry type B
Cowdry type A →
• Herpes simplex virus (Lipschutz bodies)
• Varicella zoster virus
• Torres bodies in Yellow fever

Cowdry type B →
• Polio virus
• adenovirus
 Intranuclear and intracytoplasmic
inclusion bodies:

• Measles virus (Warthin–Finkeldey bodies)

• Cytomegalovirus (Owl's eye appearance)
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