Medical Microbiology

By Dawit Admasu(B.Sc., M.Sc.)

1
 Course outline
 Day two

• Introduction to microbiology (definition, biological

principles and classification of microorganisms)
• The microbial cell structure(2hrs)
 Day three

• Introduction to microbial genetics (2hrs)

• Classification of bacteria (1hrs)

• The growth, survival and death of microorganisms(1hrs)

Cont’d…

• Basic science lab

• Microbial charts and atlas
• Basic microbiology equipments and reagents
• Demonstration of basic staining techniques
Cont’d…

• Cultivation of microorganisms(1hr)
• Normal microbial flora(1hr)
• Overview of virology (4hrs)
• Overview mycology(4hrs)
Cont’d…

• Basic Science Lab

• Preparing cultivation media for microorganisms
• Anti microbial susceptibility testing
• Computer simulations of properties of viruses
• Computer simulations of properties of fungi
Cont’d…

• Principles of infection prevention (2 hrs0

• Principle of disinfection and Sterilization (2 hrs)
• Overview of Immune system (4hours)
• Diseases of the immune system, autoimmune diseases
and immunodeficiency states (4hrs)
• Principles of diagnostic medical microbiology(2hours)
Introduction to Microbiology

7
 Objectives for general bacteriology
 After the end of this session, you should be able to

 Define medical microbiology

 Classify microorganisms

 Characterize cellular / anatomical structure of

microorganisms
 Explain bacterial growth, multiplication and death

 Bacterial physiology and nutrition

 differentiate the genetic makeup of microorganisms

 apply d/t mechanisms of disinfection and sterilization

 Introduction to microbiology
• Microbiology is a subject which deals with living
organisms that are individually too small to be seen with
the naked eye
• These organisms include
– bacteria
– fungi
– algae and
– protozoa

9
Introduction…

• It also includes viruses, which are microscopic but not

cellular.
• It deals about their reproduction, physiology, and
participation in the process of nature, helpful and harmful
relationship with other living things
 The Beginnings of Microbiology
• The debate concerning the origin of life is perhaps the
single most important factor that led to the development
of microbiology as a science and paved the way for the
discovery of microorganisms
• Man kind has always been affected by diseases-
believed to be visitations by the gods and meant to
punish evil doers.
• Hippocratus, father of medicine, observed that ill health
resulted due to changes in air, winds, water, climate,
food, nature of soil and habits of people. 11
Cont’d…
• Fracastorius (1500 G.C.) proposed that the agents of
communicable disease were living germs, that could be
transmitted by direct contact with humans and animals, and
indirectly by objects ; but no proof because of lacking
experimental evidence.

• Antony Van Leeuwenhoek (1632-1723 G.C.), Dutch scientist

father of Microbiology, observed “animalcules” using his simple
microscope with one lens.

• The first who properly described the different shapes of bacteria.

• Question raised - where did they originate?

12
The Beginnings…
– Although Leeuwenhoek was not concerned about the
origin of micro-organism; many other scientists were
searching for an explanation for spontaneous appearance
of living things from decaying meat, stagnating ponds,
fermenting grains and infected wounds.

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The Beginnings…
• On the bases of this observation, two major theories were
formulated.
1. Theory of Abiogenesis
2. Theory of Biogenesis
1. Theory of Abiogenesis deals with the theory of spontaneous
generation; stating that living things originated
“spontaneously” from non-living things.
• Aristotle (384-322 BC) is the founder of a theory
spontaneous generation.
• He observed spontaneous existence of fishes from dried
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ponds, when the pond was filled with rain.
The Beginnings…
2. Biogenesis: - States that life comes from pre existing life
Francesco Redi (1626-1697): He is the scientist who first
tried to set an experiment to disprove spontaneous
generation.

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The Beginnings…
Francisco Redi
• Introduced experimental procedure to disproof
spontaneous generation
• Utilized jars containing meat some were covered, some
were not.
• Maggots appeared in uncovered jars and conclude that
maggots did not emerge spontaneously but from the eggs
laid on the meat by the fly.
• The controversy on spontaneous generation took 200
years. 16
The Beginnings…

John Needham (1749)

• Performed experiments similar to Redi’s on the origin of
life in microscopic organisms
• Introduced the first culture medium for microbial growth.
• Utilized infusion broth prepared by boiling meat, grain,
etc. to extract nutrients.
• Broth put in flasks, some were sealed with corks, and
some were not.

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 The Beginnings…

• All flasks became cloudy, result different from Redi’s

experiment.
• He suggested that life originate spontaneously from
nonliving matters
• The spontaneous generation opponents didn’t accept
his conclusion, they said it could be due to entrance
organisms from air or flasks, improper seal.
The Beginnings…
Lazzaro Spallanzani (1776)
• Repeated Needham’s experiments to disproof
spontaneous generation in microscopic life.
• Boiled broth after placing in flasks.
• Sealed flasks by plugging with solid stopper.
• Results more consistent with Redi’s.
• Occasionally sealed flask  cloudy.
• Not accepted by spontaneous generation supporters,
because they said that heating may have destroyed,
degraded “vital force” and air was not allowed to enter.19
The Beginnings…

Louis pasture (1822- 1895) was the scientist who

disproved the theory of abiogenesis once and for all.
• He Performed experiment to disprove theory of
spontaneous generation by designing a large curved
flask/swan-necked (pasture goose neck flask) and
placed a sterile infusion broths. Flasks remained sterile
unless tilted or neck broken.
In ‘A’ air freely moved through the tube, but dust particles were trapped in the curved portion of the flask and no microbial growth was observed.
.

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Therefore, Pasteur proved that microorganisms entered to
the broth with the air and micro organisms did not evolve
spontaneously 22
Cont’d…
• Then scientists tried to confirm the causative agent of
disease
• Until relatively recently the fact that many kinds diseases
are related to microorganisms was unknown
• Before the time of Pasteur, effective treatments for many
diseases were discovered by trial and error, but the
causes of the diseases were unknown
• Germ theory of disease holds that microorganisms are
responsible for infectious disease

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Cont’d…
• This theory was a difficult concept for many people to
accept at that time.
• But gradually scientists accumulated the information
needed to support the new germ theory
• It is now a fundamental part of modern medicine and
clinical microbiology, leading to such important
innovations as antibiotics and hygienic practice

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Foundation of modern medicine
• Germ theory of disease is the single most important
contribution to medical science and practice
 Joseph Lister

• Connected and applied Pasteur’s work to develop and

popularize the  chemical inhibition of infection during surgery
• Washed surgical wounds with phenol
• Lister is considered to be the father of antiseptic surgery

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Cont’d…

 Robert Koch (1843-1910)

• He proofed that bacteria actually cause disease in 1876

• Koch discovered rod-shaped bacteria now known as
Bacillus anthracis in the blood of cattle that had died of
anthrax
• He is the father of Bacteriology

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Koch…

• He cultured the bacteria on nutrients and then injected

samples of the culture into healthy animals

• When these animals became sick and died, Koch isolated

the bacteria in their blood and compared them with the
bacteria originally isolated

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Koch…

• He found that the two sets of blood cultures contained

the same bacteria

• Koch thus established a sequence of experimental

steps for directly relating a specific microbe to a

specific disease.

• These steps are known today as Koch's postulates

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 Koch's postulates
• They are summarized as follows:

1. The same pathogen must be present in every case of

the disease
2. The pathogen must be isolated from the diseased host
and grown in pure culture
3. The pathogen from the pure culture must cause the
disease when it is inoculated into a healthy, susceptible
laboratory animal
4. The pathogen must be isolated from the inoculated
animal and must be shown to be the original organism 29
Koch’s postulate…

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 Exceptions to Koch's Postulates
• Although Koch's postulates are useful in determining the
causative agent of most bacterial diseases, there are some
exceptions.

A. Some microbes have unique culture requirements

 For example, the bacterium T. pallidum is known to cause
syphilis, but virulent strains have never been cultured on
artificial media
 The causative agent of leprosy, M. leprae, has also almost
impossible to cultivate on artificial media.
 Moreover, many rickettsia and viral pathogens cannot be
cultured on artificial media because they multiply only within
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Exceptions to Koch's Postulates…
B. All disease is not cause by microorganisms
 E.g. Diabetes, scurvy.

C. Some infectious diseases are not as clear-cut and

provide another exception to Koch's postulates
 For example, nephritis (inflammation of the kidneys)
can involve any of several different pathogens, all of
which cause the same signs and symptoms. Thus, it
is often difficult to know which particular
microorganism is causing a disease.

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Exceptions to Koch's Postulates…
D. Many pathogens are species specific
 E.g. Brucella abortus cause abortion in animals but not
in humans
E. Certain diseases develop only when an opportunistic
pathogen invades immuno-compromised host

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Exceptions to Koch's Postulates…
F. some pathogens can cause several disease conditions
 M. tuberculosis, for example, is implicated in diseases
of the lungs, skin, bones, and internal organs
 S. pyogenes can cause sore throat, scarlet fever, skin
infections, and osteomyelitis (inflammation of bone),
among other diseases
• When clinical signs and symptoms are used together
with laboratory methods, these infections can usually be
distinguished from infections of the same organs by
other pathogens 34
Exceptions to Koch's Postulates…
G. Ethical considerations may also impose an exception to
Koch's postulates
 Human experiments with untreatable diseases are not
acceptable today
For example, some agents that cause disease in humans
have no other known host. An example is HIV, the cause of
AIDS.

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 Pathological Mechanisms of
Bacterial Infections
1. Bacteria-mediated
Pathogenesis
2. Host-mediated
Pathogenesis

Adopted from Samuel Baron “Medical Microbiology”

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1. Tissue destruction
a. By-product of bacterial growth (acids, gases)
b. Degradative enzymes
breaks down tissues promotes spread of bacteria
– Phospholipase
– Protease- destroy protein
– Hyaluronidase
– DNAse

37
Cont’d…
2 Toxins - Bacterial products that destroy tissues or trigger

destructive biological activities which comes from:-

 Cell wall components

 degradative enzymes

 specific binding proteins

Two types of toxin

A. Exotoxin

Protein produced by both G+ and some G-ve bacteria

Commonly secreted by living cell 38

Cont’d…
B. Endotoxin
 Produced by only Gram negative bacteria
 The lipid A portion of LPS has endotoxin activity
 Released when the cell is lyzed
 Activates blood clotting proteins that blocks capillaries
e.g. caspase-11 which activate TF

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Cont’d…
3. Siderophores
• iron in blood is bound either to hemoglobin in
erythrocytes or to transferrin in plasma and lactoferrin in
milk and other secretions.
• Some bacteria has receptors for eukaryotic iron-binding
proteins called Siderophores
• compete for iron effectively

40
Cont’d…

Immuno-pathogenesis

 When there is large amount of endotoxin, there is over

production of cytokines which in turn over stimulation of

neutrophils, macrophages, and complement that causes

tissue damage at the site of infection

 Anti-M proteins antibodies cross reacts with heart tissue

and causes rheumatic fever

 Deposition of Immune complexes on gromeruli causes

kidney damage 41
 Microbial defense against host immunologic
clearance
Avoids antibacterial defense by:

 evading recognition and killing by phagocytic cell

 Inactivating/evading the complement systems and Ab

 Growing inside the cells/antigenic variation

Capsule

Shields from immune and phagocytic responses

Poor immunogens (carbohydrate)

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Cont’d…
Capsule of S. pyogens
Made of hyaluronic acid (mimic host tissue)
Masking (unrecognizing by host immune system
Capsule
Hard to be grasped by phagocytes
Protect destruction within phagolysosomes
S. pneumonia, B. anthracis, K. pneumonia

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Biofilm

Prevent entrance of Ab and complement in to the

bacteria.

Bacteria evades from antibody by:-

 Intracellular growth (Mycobacteria, Francisellae,

brucellae, chlamydiae etc.,)

 Antigenic variation (N. gonorrhoea)

 Inactivation of antibody or complement

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Cont’d…
• N.gonorrhoeae produce protease that degrade IgA
• S. Pyogens degrades C5a and inhibit chemotaxis WBC
to the site of infections or
• Some produce enzymes which kill phagocytes

– streptolysin, alpha toxin

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Cont’d…
• Gram negative bacteria has long O antigen which

prevent deposition of complement

• N. gonorhoae and S. aureus produced protein which

coats their body and prevent deposition of Ab.

• S. aureus produce coagulase which convert fibrinogen

to fibrin( clot) and hide inside the clot.

• MTB promote formation of granuloma and hide for the

patient life time and waits for decline of resistance to

grow. 47
Cont’d…
• Protein A
 coats the surface of S. aurues
 has affinity to Fc portion of Ig
 prevent antibody mediated immune clearance of the
organism

48
 Structure and Function of Prokaryotic Cells
• Prokaryotic cells have three architectural regions

1. appendages (proteins attached to the cell surface) in the form of

flagella and Pili

2. a cell envelope consisting of

 capsule,

 cell wall and

 plasma membrane

3. a cytoplasmic region that contains

 cell genome (DNA) and

 ribosomes and
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 various sorts of inclusions
Bacterial Structure

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 1. Bacterial Pili /fimbriae
• thin, hair-like appendages
• Short protein fibers
• Found among gram –ve and
some g +ve bacteria
• Protrude from surface of cell
• used for attachment to other
cells and to host cell or tissue for
pathogens
• Conjugation pili
– attachment between cells
– for exchange of genetic
material

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  Joins bacterial cells for DNA transfer (conjugation)

 sex pili attach male to female bacteria

during conjugation
Results in one way transfer of DNA
from donor (male) cell to recipient
(female) cell through conjugative (sex)
pilus

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 2. Bacterial Flagella
• Much longer than cell
• It is thread like appendages
• Organ of locomotion
– Provide motility
• made of protein subunit
called flagellin
• Flagellins are immunogenic
(H-antigen)

53
Flagellar arrangements
• Different species of bacteria have different numbers
and arrangements of flagella
• Useful in identifying and classifying bacteria

A. Monotrichous – single flagellum

at one end- V. cholera
B. Lophotrichous – small bunches
arising from one end of cell
C. Amphitrichous – flagella at both
ends of cell
D. Peritrichous – flagella dispersed
over surface of cell---E. choli
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Importance of Flagella
 Attachment to surfaces (epithelial tissue)
 Motility
 as a virulence factor
 Diagnostic value and Identification

antigenic nature
arrangement and number

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 3. Glycocalyx
• Coating of bacteria, external to the cell wall
• Made of sugars and/or proteins
• 2 types
1. capsule - highly organized, tightly attached
2. slime layer - loosely organized and attached, non
uniform in density and thickness

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 Functions
• Attachment to other bacteria or host tissues
• inhibits killing by WBCs and detergents
• Receptor
• Some produce biofilm that protects from
antibiotics and host defense ( tooth plaques, S.
mutans)
• Capsule and slime layer -unnecessary for growth
but need for survival
• important determinants of virulence
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 4. Cell wall

 Thick and relatively rigid layer (peptidoglycan) which made

up of protein and sugar
• Components are unique to bacteria
• Contain strong activator of immune response
• Structural components and their function used for
classification
• Crucial for growth and division

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Cell wall of Gram positive bacteria
• Thick peptidoglycan
• Peptidoglycan contains: TA and LTA
• LTA anchored to the cell membrane
• TA and LTA antigenic (used for classification)

- used as attachment organ (virulent factor)

- Initiate host response

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Cell wall of Gram positive bacteria

60
Structure of Cell walls of bacteria

61
Cell wall of gram negative
• Contain thin peptidoglycan
• Contain outer membrane but has no TA/LTA
• Contain periplasmic space containing:-
 variety of hydrolytic enzyme which are used for
metabolism/virulence factors (phosphatase, Beta
lactamase, lipase, protease, collagenase etc.)
 Transport and binding proteins

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Gram negative cell wall

Lipopolysaccharide

Porin proteins

Outer membrane
Periplasmic space
Peptidoglycan
Cytoplasmic membrane

Cytoplasm

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Cytoplasmic membrane

• Lipid bilayer
• Semipermeable barrier

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 The acid fast cell wall
• Resemble Gram positive cell wall
• Acid-fast bacteria have a cell wall with a relatively
impermeable cell wall containing a waxy lipid called
mycolic acid
• Difficult to stain
• Mycolic acids confer resistance to desiccation, most
antibiotics and Phagocytosis
Contributes to pathogenicity

e.g. Complex cell wall structure of Mycobacteria

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Ribosomes
• Made of 60% ribosomal RNA & 40% protein
• consist of 2 subunits: large & small  70’S
• Prokaryotic differ from eukaryotic ribosomes in size &
number of proteins
• Site of protein synthesis
• Target of some drugs

66
Plasmids
• small circular, double-stranded DNA
• free or integrated into the chromosome
• duplicated and passed on to offspring
• not essential to bacterial growth & metabolism
• may encode genes for antibiotic resistance, enzymes &
toxins
• used in genetic engineering- readily manipulated &
transferred from cell to cell

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 Spore
• Multilayered structure

• Made by some member of gram

positive bacteria

• Made in response of harsh

environmental condition (depletion

of nutrients)

• withstand extreme heat, drying,

freezing, radiation & chemicals

• Not a means of reproduction but for

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survival
• Location the spore is important for classification
 Central, Subterminal, Terminal
• Used for quality control of heat sterilization equipment
spores produced by bacillus stearothermophilus
-Endospore formers
are a serious concern to food industries
 Cause health problems: anthrax, tetanus, gas
gangrene e.g. Bacillus and Clostridium

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70
Classification of microorganisms
• Despite their complexity and variety, all living cells can

be classified into two groups, prokaryotes and

eukaryotes, based on certain structural and functional

characteristics

• In general, prokaryotes are structurally simpler and

smaller than eukaryotes.

• Prokaryotes and eukaryotes are chemically similar, in the

sense that they both contain nucleic acids, proteins,

lipids, and carbohydrates 71

Classification…
• They use the same kinds of chemical reactions to
metabolize food, build proteins, and store energy
• It is primarily the structure of cell walls and membranes,
and the absence of organelles (specialized cellular
structures that have specific functions), that distinguish
prokaryotes from eukaryotes

72
 prokaryotes
• Their DNA is not enclosed within a membrane and is
usually a singular circularly arranged chromosome
– (Some bacteria, such as Vibrio cholerae, have two
chromosomes, and some bacteria have a linearly
arranged chromosome).

73
Prokaryote…
• They lack membrane-enclosed organelles
• Their cell walls almost always contain the complex
polysaccharide peptidoglycan
• They usually divide by binary fission

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 Eukaryotes
• Their DNA is found in the cell's nucleus, which is
separated from the cytoplasm by a nuclear membrane,
and the DNA is found in multiple chromosomes.
• Their DNA is consistently associated with chromosomal
proteins called histones and with non histones.

75
Eukaryotes…

• They have a number of membrane-enclosed organelles,

including mitochondria, endoplasmic reticulum, Golgi
complex, lysosomes, and sometimes chloroplasts.
• Their cell walls, when present, are chemically simple
• Cell division usually involves mitosis, in which
chromosomes replicate and an identical set is distributed
into each of two nuclei

76
 Comparison features of prokaryotic and eukaryotic cells
Characteris
Prokaryotes Eukaryotes
tic
Typical
bacteria protozoan, fungi, plants, animals
organisms
nucleoid
Type of
region; no real real nucleus with double membrane
nucleus
nucleus
circular linear molecules (chromosomes)
DNA
(usually) with histone proteins
RNA-/
coupled in RNA-synthesis inside the nucleus
protein-
cytoplasm protein synthesis in cytoplasm
synthesis
Ribosomes 50s+30s=70s 60s+40s=80s

77
 Comparison of features….
Characterist
Prokaryotes Eukaryotes
ic
Cytoplasmati Very few highly structured by
c structure structures endomembranes and a cytoskeleton

one to several thousand (though

Mitochondria None
some lack mitochondria)
Chloroplasts None in algae and plants
usually single single cells, higher multicellular
Organization
cells organisms with specialized cells

Binary fission
Mitosis
Cell division (simple
Meiosis
division)
78
Bacterial morphology
 Bacteria are differentiated into major categories

based on microscopic observation of their

morphological features

1. Shape

2. Size

3. Cell and flagella arrangement

4. Staining Characteristics

5. Presence of spores, capsule

79
 Bacterial shapes and arrangement
 Morphologically bacteria can resemble:
1. Cocci (Singular: Coccus)
2. Bacilli (Singular: Bacillus)
3. Spirilla (Singular: Spirillum)

80
Morphological features of bacteria

81
 1. Cocci:
• are round or oval bacteria
• may form pairs, chains, or irregular groups (grape like)

– cocci in pairs (diplococci), e.g. meningococci and gonococci

– cocci in chains (streptococci), e.g. Streptococcus pyogenes
– cocci in irregular groups (staphylococci)
• Gram reaction: Staphylococci and streptococci are Gram
positive, where as diplococci can be Gram positive or Gram
negative

82
2. Rods (bacilli):
• are stick-like bacteria with rounded, tapered (fusiform),

square, or swollen ends

• short rods with rounded ends are often called coccobacilli

• Gram reaction: Many rods are Gram negative such as the
large group of Enterobacteria
• Gram positive rods include Clostridium species,
Corynebacterium species, Bacillus anthracis, and Listeria
monocytogenes
83
4. Spirochaetes:
• are flexible, coiled, motile organisms.
• They progress by rapid body movements
• Most are not easily stained by the Gram method

84
• Spirochaetes are divided into three main groups:

–Treponemes: thin delicate spirochaetes with regular tight

coils, e.g.Treponema pallidum and Treponema pertenue

– Borreliae: are large spirochaetes with irregular open coils,

E.g. Borrelia duttoni and Borrelia vincent

– Leptospires: are thin spirochaetes with many tightly

packed coils that are difficult to distinguish.
 leptospire of medical importance is Leptospira
interrogans
85
Basic classification of medically important bacteria

86
Microscopic morphology and staining reactions
THE GRAM STAIN:
– A staining method used to classify different bacteria
– It is one of the differential stains
– Used for the identification of pathogens in clinical
Specimens and cultures by their gram reactions, and
morphology
Differences in Gram reaction is attributed to the difference
in cell wall structure

87
 Reagents used in Gram-stain:
1. Crystal violet/Gentian violet: primary stain
2. Gram’s lodine/Lugol’s iodine: this serve as mordent
3. Acetone-alcohol: as decolorizer
4. Safranin: as counter stain

88
 Basic Gram Stain Method
Recommended for general bacteriology use
• Flood smear with Crystal Violet - 30 - 60 seconds then
rinse with tap water

• Flood with Iodine solution - 30 - 60 seconds then rinse as

above

• Hold slide at a 45 degree angle & run the acetone/alcohol

mixture over the slide watching the spill off, when the spill off
is no longer blue, rinse immediately.

• Flood slide with counterstain - 30 seconds then rinse & allow

to air dry (may be carefullyPage
blotted)
 89
 Advantages of the Gram Stain
• Rapid procedure
• Cost effective
• Accessible
• Effective screening technique
• Reliable
• Semi-quantitative
• Provides culture clues

Page  90
 Examination & Interpretation
For smears prepared from clinical specimens
• 105 organisms/ml of un-centrifuged fluid (104 centrifuged)
organisms are required to be visible on slide
• At lower concentrations, organism will not be revealed even
if culture is positive
• Organisms seen on Gram stain, but fail to grow in culture
– fastidious (require specific media for growth)
– anaerobic bacteria
– patient has received antibiotics
Page  91
 Reporting of Gram staining:
• The laboratory report should include:

– Gram Reaction

– morphology

– intracellularity or extracellularity

• Examples: Gram-positive diplococci consistent with

Streptococcus pneumoniae
• Small Gram-negative coccobacilli, consistent with
Haemophilus
• Gram-negative intracellular diplococci, consistent with
92
 Ziehl- Neelson Staining Reactions (AFB Staining)

• Used for diagnosis of Mycobacterium species

• Mycobacterium, unlike other bacteria, doesn’t stain by
Gram-technique
• This is due to the difference in the cell composition of
Mycobacterium species from other bacteria

93
 Cell Wall Structure of Mycobacterium species:

• It deserves special attention because it is unique among

prokaryotes, and it is a major determinant of virulence for the
bacterium
• The cell wall complex contains peptidoglycan, but otherwise it
is composed of complex lipids
• Over 60% of the Mycobacterium cell wall is lipid
• MTB is not classified as either Gram-positive or Gram-
negative because it does not have the chemical
characteristics of either

94
 AFB Staining…

• Acid-fast staining method for M. tuberculosis is the Ziehl-

Neelsen stain
• When this method is used, Smear is fixed, stained with
carbol-fuchsin (a pink dye), and decolorized with acid-alcohol
• The smear is counterstained with methylene-blue or certain
other dyes
• Acid-fast bacilli appear pink in a contrasting background

95
 AFB Staining…
• Reagents used in AFB:
– Carbol fuchsin- used as primary stain
– Acid- alcohol- as decolorizer
– Methylene blue or malachite green for staining back
ground
• Reporting system: Grading of AFB

96
ZN Stained Smear

97
 Reporting: Grading of ZN Smears
WHO scale ZN 1000x, AFB count
• Negative 0 AFB / 100 HPF
• Scanty 1-9 AFB / 100 F
• 1+ 10-99 AFB / 100 F
• 2+ 1-10 AFB / 1 F on average
• 3+ >10 AFB / 1 F on average

98
 Question

• Describe the following

 Gram positive bacteria
 Gram negative bacteria
 Acid fast bacilli

99
 Summery
• If you have any questions and/ or comments

• Thank you

100