Bacteriology

Basics
Morphology, Classification, Staining
Methods
Dr.T.V.Rao MD
 Introduction:
Microorganisms several classes of living
beings
Based on the organization of their cellular
structures, all living cells can be divided into
two groups: eukaryotic and prokaryotic
Eukaryotic cell types - Animals, plants, fungi,
protozoans, and algae
Prokaryotic cell types - bacteria & blue green
algae

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 Antioni van Leeuwenhoek
Leeuwenhoek is called
"the inventor of the
microscope"
Created a simple
microscope that could
magnify to about 275x, and
published drawings of
microorganisms in 1683
Could reach magnifications
of over 200x with simple
ground lenses

Dr.T.V.Rao MD 3
How a Microscope Works with..
Ocular Lens Objective Lens
(Magnifies Image) (Gathers Light,
Magnifies
And Focuses Image
Body Tube Inside Body Tube)

Dr.T.V.Rao MD
(Image Focuses)

Bending Light: The objective (bottom) convex lens

magnifies and focuses (bends) the image inside the
body tube and the ocular convex (top) lens of a
4
microscope magnifies it (again).
 The Light Microscope
many types
bright-field microscope
dark-field microscope

Dr.T.V.Rao MD
phase-contrast microscope
fluorescence microscopes
compound microscopes
image formed by action of 2 lenses 5
 The Compound Microscope
The Optical System
Objective Lens: the lens closest to the
specimen; usually several objectives are
mounted on a revolving nosepiece.
Parafocal: when the microscope is focused with one

Dr.T.V.Rao MD
objective in place, another objective can be rotated
into place and the specimen remains very nearly in
correct focus.
Eyepiece or Ocular Lens: the lens closest to the
eye.
Monocular: a microscope having only one eyepiece
6
Binocular: a microscope having two eyepieces.
 Phase Contrast Microscopy
light rays through objects of different
change in phase, not intensity
special ring-shaped condenser diaphragm
special glass disc in objective
change phase differences to intensity
differences
can view transparent
objects as dark on light
background (without staining)
Right; human brain glial
cells Dr.T.V.Rao MD 7
 The Bright-Field Microscope
Produces a dark image against a
brighter background
Has several objective lenses

Dr.T.V.Rao MD
par focal microscopes remain in focus
when objectives are changed
total magnification
product of the magnifications of the
ocular lens and the objective lens 8
 Fluorescence Microscopy
Illuminate specimen with UV visible fluorescence
(filter removes harmful UV)
View auto-fluorescent objects (e.g., chloroplasts)
Stain with specific fluorescent dyes, which absorb in
region 230-350 nm & emit orange, yellow or
greenish light
Images appear coloured against a dark background

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Schematic of typical animal (eukaryotic) cell, showing subcellular
components.

Organelles: (1) nucleolus (2) nucleus (3) ribosome (4) vesicle

(5) rough endoplasmic reticulum (ER) (6) Golgi apparatus (7) Cytoskeleton
(8) smooth ER (9) mitochondria (10) vacuole
(11) cytoplasm (12) lysosome (13) centrioles

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 Background Information
Prokaryotes

Prokaryotes represent two domains,

bacteria and archaea.

Archaea live in Earths extreme

environments.

Bacteria are the most abundant and

diversified organisms on Earth.
Dr.T.V.Rao MD 11
 Eukaryotes have organelles
Much larger; more complex than prokaryotes
Processes compartmentalized into organelles
Nucleus
Protein synthesis (ribosomes, RER, Golgi)
Mitochondria; chloroplasts
Lysosomes
Plasma membranes have different modifications
Cytoskeleton

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 Differences between prokaryotic & eukaryotic cells
Character Prokaryotes Eukaryotes

Nucleus Nuclear Absent Present

membrane
Nucleolus Absent Present

Chromosome One circular One or more paired

and linear
Cell division Binary fission Mitosis

Cytoplasmic Structure and fluid phospholipid fluid phospholipid

membrane Composition bilayer, lacks sterols bilayer containing
sterols
Function Incapable of Capable of
endocytosis endocytosis and
(phagocytosis and exocytosis
pinocytosis) and
exocytosis
Dr.T.V.Rao MD 13
 Differences between prokaryotic & eukaryotic
cells
Character Prokaryotes Eukaryotes

Cytoplasm Mitochondria Absent Present

Lysosomes Absent Present

Golgi apparatus Absent Present

Endoplasmic Absent Present

reticulum

Vacuoles Absent Present

Ribosomes 70 S 80 S
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 Differences between prokaryotic &
eukaryotic cells

Character Prokaryotes Eukaryotes

Cell Wall Present Animals & Protozoans

Absent
Plants, Fungi & Algae -
Present
Composition Peptidoglycan Cellulose or chitin
complex
carbohydrate
Locomotor Flagella Flagella/ Cilia
organelles

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 Prokaryotic Cells
Much smaller (microns) and more simple
than eukaryotes
Prokaryotes are molecules surrounded by
a membrane and cell wall.
They lack a true nucleus and dont have
membrane bound organelles like
mitochondria, etc.
Large surface-to-volume ratio : nutrients
can easily and rapidly reach any part of
the cells interior
Dr.T.V.Rao MD 16
 Size of Bacteria
Unit of measurement in
bacteriology is the micron
(micrometre, m)
Bacteria of medical importance
0.2 1.5 m in diameter
3 5 m in length
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 Eukaryotic cell Prokaryotic cell
(e.g. animal) Gram + Flagellum
Rough endoplasmic Cell membrane Nucleoid Cell wall
reticulum
Nucleus

Gram -
Pili

Granule
Capsule
Cytoplasm
Cell (inner) membrane Outer membrane
Mitochondria
Ribosomes
Dr.T.V.Rao MD
Cell wall 18
 Shapes of Bacteria
Cocci spherical/ oval shaped major groups
Bacilli rod shaped
Vibrios comma shaped
Spirilla rigid spiral forms
Spirochetes flexible spiral forms
Actinomycetes branching filamentous
bacteria
Mycoplasmas lack cell wall
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Bacteria Have One of Three Cellular
Shapes
Rods (bacilli)

Coccoid-Shaped

Spirilla

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 Arrangement of bacteria: Cocci
Coccus

Cocci in pair Diplococcus

Tetrad groups of four

Cocci in chain - Streptococci

Cocci in cluster - Staphylococci Sarcina groups of eight

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Reproduction
Prokaryotic cell division is
binary fission.
Single DNA molecule that
first replicates.
Attaches each copy to a
different part of the cell
membrane.
Cell begins to pull apart.
Following cytokinesis, there
are then two cells of
identical genetic
composition.

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Arrangement of bacteria: Bacilli

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Other shapes of bacteria

Comma shaped
Spirilla

Spirochetes

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Anatomy of a Bacterial Cell

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Anatomy of A Bacterial Cell
Outer layer two components:
1. Rigid cell wall
2. Cytoplasmic (Cell/ Plasma) membrane
present beneath cell wall
Cytoplasm cytoplasmic inclusions,
ribosomes, mesosomes and nucleus
Additional structures plasmid, slime
layer, capsule, flagella, fimbriae (pili),
spores
Dr.T.V.Rao MD 26
 Structure of Bacteria
All cells have 3 main components:
DNA (nucleoid)
genetic instructions
surrounding membrane (cytoplasmic
membrane)
limits access to the cells interior
cytoplasm, between the DNA and the
membrane
where all metabolic reactions occur
especially protein synthesis, which occurs
on the ribosomes
Bacteria also often have these features:
cell wall
resists osmotic pressure
flagella
movement
pili
attachment
capsule
protection and biofilms Dr.T.V.Rao MD 27
 Typical shapes of bacteria

Most bacteria retain a particular shape; a few

are pleiomorphic
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Typical prokaryotic structures

Working from the outside in

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Characteristic grouping (or not grouping)

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The Cell Envelope

Gram Positive Gram Negative

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 GRAM POSITIVE
Lipoteichoic acid Peptidoglycan-teichoic acid

Cytoplasmic membrane

Cytoplasm

GRAM NEGATIVE Porin

Lipopolysaccharide

Outer Membrane Braun lipoprotein

Inner (cytoplasmic) membrane

Cytoplasm
Dr.T.V.Rao MD 32
Gram-positive and gram-negative bacteria
 Difference Between
Gram-Negative
Gram-Negative Bacteria
and Gram-Positive Bacteria
Gram-Positive Bacteria

More complex cell wall. Simple cell wall.

Thin peptidoglycan celll wall layer. Thick peptidoglycan celll wall layer.

Outer lipopolysaccharide wall layer. No outer lipopolysaccharide wall layer.

Retain safranin. Retain crystal violet/iodine.

Appear pink/red. Appear blue/purple.

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 Cell Envelope
The cell envelope is all the
layers from the cell
membrane outward,
including the cell wall, the
periplasmic space, the outer
membrane, and the capsule.
All free-living bacteria
have a cell wall
periplasmic space and
outer membrane are
found in Gram-negatives
the capsule is only found
in some strains
Dr.T.V.Rao MD 35
Structure & Function
of Cell Components
 CELL WALL
Outermost layer, encloses cytoplasm
1. Confers shape and rigidity
2. 10 - 25 nm thick

1. Composed of complex polysaccharides

(peptidoglycan/ mucopeptide) - formed by N
acetyl glucosamine (NAG) & N acetyl muramic
acid (NAM) alternating in chains,
held by peptide chains.
Dr.T.V.Rao MD 37
 Cell Wall
Cell wall
4. Carries bacterial antigens important in
virulence & immunity
5. Chemical nature of the cell wall helps to divide
bacteria into two broad groups Gram positive
& Gram negative
6. Gram +ve bacteria have simpler chemical nature
than Gram ve bacteria.
7. Several antibiotics may interfere with cell wall
synthesis e.g. Penicillin, Cephalosporins

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 Transport Across the Cell Membrane
Basic rule: things spontaneously
move from high concentration
to low concentration (downhill).
This process is called diffusion.
Getting many molecules into the
cell is simply a matter of opening
up a protein channel of the proper
size and shape. The molecules
then move into the cell by
diffusing down the concentration
gradient. Passive transport, or
facilitated diffusion.

To get things to move from low

to high (uphill), you need to add
energy: the molecules must be
pumped into the cell. Pumps
are driven by ATP energy.
Dr.T.V.Rao MD 39
Active transport.
 Outer Membrane
Gram negative bacteria
major permeability barrier
space between inner and outer
membrane
Periplasmic space
store degradative enzymes
Gram positive bacteria
no Periplasmic space
Dr.T.V.Rao MD 40
Gram positive cell wall

The Gram-positive cell wall is composed of a thick, multilayered

peptidoglycan sheath outside of the cytoplasmic membrane. Teichoic
acids are linked to and embedded in the peptidoglycan, and
lipoteichoic acids extend into the cytoplasmic membrane

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 Gram negative cell wall

The Gram-negative cell wall is composed of an outer membrane

linked to thin, mainly single-layered peptidoglycan by lipoproteins.
The peptidoglycan is located within the periplasmic space that is
created between the outer and inner membranes. The outer
membrane includes porins, which allow the passage of small
hydrophilic molecules across the membrane, and
lipopolysaccharide molecules that extend into extracellular space.
Dr.T.V.Rao MD 42
Cell Wall

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 Summary of the differences between
Gram positive & Gram negative bacteria

Property of bacteria Gram Positive Gram Negative

Thickness of wall 20-80 nm 10 nm

Number of layers in wall 1 2

Peptidoglycan content >50% 10-20%

Teichoic acid in wall + -

Lipid & lipoprotein content 0-3% 58%

Protein content 0% 9%

Lipopolysaccharide 0 13%

Sensitive to penicillin Yes Less sensitive

Digested by lysozyme Yes Weakly

Dr.T.V.Rao MD 44
 Cytoplasmic (Plasma) membrane
Thin layer 5-10 nm, separates cell
wall from cytoplasm
Acts as a semipermeable
membrane: controls the inflow and
outflow of metabolites
Composed of lipoproteins with small
amounts of carbohydrates
Dr.T.V.Rao MD 45
 Other Cytoplasmic Components
Ribosomes protein synthesis
Mesosomes
1. Multilaminated structures formed as
invaginations of plasma membrane
2. Principal sites of respiratory enzymes
3. Coordinate nuclear & cytoplasmic division during
binary fission
4. More prominent in Gram +ve bacteria
Intracytoplasmic inclusions reserve of
energy & phosphate for cell metabolism e.g.
Metachromatic granules in diphtheria bacilli
Dr.T.V.Rao MD 46
 Nucleus
No nucleolus
No nuclear membrane
Genome
single, circular double stranded DNA.
Haploid
Divides by binary fission

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 Additional Organelles
1. Plasmid
Extra nuclear genetic elements consisting
of DNA
Transmitted to daughter cells during
binary fission
May be transferred from one bacterium
to another
Not essential for life of the cell
Confer certain properties e.g. drug
resistance, toxicity
Dr.T.V.Rao MD 48
 Additional Organelles
2. Capsule & Slime layer
Viscous layer secreted around the cell
wall.
Polysaccharide / polypeptide in nature

a) Capsule sharply defined

structure, antigenic in nature
Protects bacteria from lytic
enzymes
Inhibits phagocytosis
Stained by negative staining
using India Ink
Can be demonstrated by
Quellung reaction (capsule
swelling reaction) Dr.T.V.Rao MD 49
 Additional Organelles
3. Flagella
Long (3 to 12 m), filamentous surface
appendages
Organs of locomotion
Chemically, composed of proteins called flagellins
The number and distribution of flagella on the
bacterial surface are characteristic for a given
species - hence are useful in identifying and
classifying bacteria
Flagella may serve as antigenic determinants
(e.g. the H antigens of Gram-negative enteric
bacteria)
Presence shown by motility e.g. hanging drop
preparation Dr.T.V.Rao MD 50
Types of flagellar arrangement
Polar/ Monotrichous single
flagellum at one pole

Lophotrichous tuft of flagella at one

pole

Amphitrichous flagella at both

poles

Peritrichous flagella all over

Amphilophotrichous tuft of flagella

at both ends
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 Cocci do not have flagella

Peritrichous monotrichous
(or amphi, or lophotrichous

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 FLAGELLA

Some bacteria are motile

Locomotory organelles- flagella
Taste environment
Respond to food/poison
chemo taxis
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 Flagella
embedded in cell membrane
project as strand
Flagellin (protein) subunits
move cell by propeller like action

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 Additional Organelles
4. Fimbriae/ Pili
Thin, hairlike appendages on the surface of many Gram-
negative bacteria
10-20 long, acts as organs of adhesion (attachment) - allowing
bacteria to colonize environmental surfaces or cells and resist
flushing

Made up of proteins called pilins.

Pili can be of two types
Common pili short & abundant
Sex pili - small number (one to six), very long pili, helps
in conjugation (process of transfer of DNA)
http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/yespili.html

http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/nopili.html

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 Additional Organelles
5. Spores
Highly resistant resting
stages formed during
adverse environment
(depletion of nutrients)
Formed inside the
parent cell, hence called
Endospores
Very resistant to heat,
radiation and drying and
can remain dormant for
hundreds of years.
Formed by bacteria like
Clostridia, bacillus
Dr.T.V.Rao MD 56
 The cycle of spore formation and germination

At the beginning of spore formation, a septum forms,

separating the nascent spore from the rest of the cell and all of
the genetic material of the cell is copied into the newly-forming
cell. The spore contents are dehydrated and the protective
outer coatings are laid down. Once the spore is matured it is
released
l from the cell. On germination, the spore contents
rehydrate and a new bacterium emerges and multiplies.
Dr.T.V.Rao MD 57
Shape & position of bacterial spore
Oval central

Spherical central Non bulging

Oval sub terminal

Oval sub terminal

Oval terminal Bulging

Spherical terminal

Free spore
Dr.T.V.Rao MD 58
 Spores
Some bacteria can form very tough
spores, which are metabolically
inactive and can survive a long time
under very harsh conditions.
Allegedly, some bacterial
spores that were embedded
in amber or salt deposits for
25 million years have been
revived. These experiments
are viewed skeptically by
many scientists.
Dr.T.V.Rao MD 59
 Spores
Spores can also survive very high or low temperatures and
high UV radiation for extended periods. This makes them
difficult to kill during sterilization.
Anthrax
Spores are produced only by a few genera in the Firmicutes:
Bacillus species including anthracis (anthrax) and cereus
(endotoxin causes ~5% of food poisoning)
Clostridium species including tetani (tetanus), perfringens
(gangrene), and botulinum (botulism: food poisoning from
improperly canned food)

Dr.T.V.Rao MD 60
Pleomorphic & Involution forms
Pleomorphism great variation in shape
& size of individual cells e.g. Proteus
species
Involution forms swollen & aberrant
forms in ageing cultures, especially in the
presence of high salt concentration e.g.
plague bacillus
Cause defective cell wall synthesis
Dr.T.V.Rao MD 61
 Bacterial Taxonomy
Includes three components:
1. Classification : orderly
arrangement
2. Identification of an unknown
unit
3. Nomenclature : naming the
units
Dr.T.V.Rao MD 62
Bacterial Taxonomy: Classification
Orderly arrangement : Kingdom
Division Class Order Family Tribe Genus
Species
Phylogenetic classification represents a branching
tree like arrangement. One characteristic being used
for division at each branch or level
Molecular or Genetic classification based on the
degree of genetic relatedness of different organisms
Intraspecies classification based on biochemical
properties (biotypes), antigenic features (serotypes),
bacteriophage susceptibility (phage types)

Dr.T.V.Rao MD 63
 Bacterial Taxonomy: Nomenclature
Two kinds of name are given to
bacteria
Casual / common name for local use,
varies from country to country
e.g. typhoid bacillus
Scientific / International Name same
all over world, consists of two words (in
Italics)
e.g. Salmonella typhi, Staphylococcus
Dr.T.V.Rao MD 64
 Microscopy helps to Measure
and Observe the Bacteria
Measurement
Microorganisms are very small
Use metric system
Metre (m) : standard unit
Micrometre ( m) = 1 x10-6 m
Nanometre (nm) = 1 x10-9 m
Angstrom () = 1 x10-10 m
Dr.T.V.Rao MD 65
 Why we should be Stain Bacteria
Bacteria have nearly the same refractive
index as water, therefore, when they are
observed under a microscope they are
opaque or nearly invisible to the naked
eye.
Different types of staining methods are
used to make the cells and their internal
structures more visible under the light
microscope. Dr.T.V.Rao MD 66
 What is a Stain
A stain is a substance that adheres to a cell, giving
the cell color.
The presence of color gives the cells significant
contrast so are much more visible.
Different stains have different affinities for
different organisms, or different parts of
organisms
They are used to differentiate different types of
organisms or to view specific parts of organisms
Dr.T.V.Rao MD 67
 Simple staining
Methylene blue, Basic fuchsin
Provide the color contrast but impart the
same color to all the organisms in a smear
Loffler's ethylene blue: Sat. solution of M. blue
in alcohol - 30mlKoH, 0.01% in water -
100mlDissolve the dye in water, filter. For
smear: stain for 3. For section: stain

Dr.T.V.Rao MD 68
 Simple Staining Easier to Perform
But has Limitations

Simple easy to use;

single staining agent
used; using basic and
acid dyes.
Features of dyes: give
coloring of
microorganisms; bind
specifically to various
cell structures

Dr.T.V.Rao MD 69
 Differential Stains
Differential Stains use two or more stains
and allow the cells to be categorized into
various groups or types.
Both techniques allow the observation
of cell morphology, or shape, but
differential staining usually provides
more information about the
characteristics of the cell wall
(Thickness). Dr.T.V.Rao MD 70
 Gram staining
Named after Hans
Christian Gram,
differentiates
between Gram-
positive purple and
Gram-negative pink
stains and is used to
identify certain
pathogens.
Dr.T.V.Rao MD 71
 Gram staining - Principles
Gram staining is used to determine gram status to
classify bacteria broadly. It is based on the composition
of their cell wall. Gram staining uses crystal violet to
stain cell walls, iodine as a mordant, and a fuchsin or
safranin counterstain to mark all bacteria. Gram status
is important in medicine; the presence or absence of a
cell wall will change the bacterium's susceptibility to
some antibiotics.
Gram-positive bacteria stain dark blue or violet. Their
cell wall is typically rich with peptidoglycan and lacks
the secondary membrane and lipopolysaccharide layer
found in Gram-negative bacteria

Dr.T.V.Rao MD 72
 Gram Staining Steps

1. Crystal violet acts as the primary stain. Crystal violet may also
be used as a simple stain because it dyes the cell wall of any
bacteria.
2. Grams iodine acts as a mordant (Helps to fix the primary
dye to the cell wall).
3. Decolorizer is used next to remove the primary stain (crystal
violet) from Gram Negative bacteria (those with LPS imbedded
in their cell walls). Decolorizer is composed of an organic
solvent, such as, acetone or ethanol or a combination of both.)
4. Finally, a counter stain (Safranin), is applied to stain those cells
(Gram Negative) that have lost the primary stain as a result of
decolorization
Dr.T.V.Rao MD 73
Structure and Reactivity to Gram
Staining.

Dr.T.V.Rao MD 74
Dr.T.V.Rao MD 75
Gm+ve cocci & Gm-ve bacilli

Dr.T.V.Rao MD 76
 GRAM-POSITIVE BACTERIA
GRAM-POSITIVE BACTERIA
are characterized by having
as part of their cell wall
structure peptidoglycan as
well as polysaccharides
and/or teichoic acids. The
peptidoglycans which are
sometimes also called
murein are heteropolymers
of glycan strands, which are
cross-linked through short
peptides.

Dr.T.V.Rao MD 77
 What are Gram Negative Bacteria
Gram-negative bacteria are those bacteria that
do not retain crystal violet dye in the Gram
staining protocol. In a Gram stain test, a counter
stain (commonly safranin) is added after the
crystal violet, coloring all Gram-negative bacteria
with a red or pink color. The test itself is useful in
classifying two distinct types of bacteria based on
the structural differences of their cell walls. On
the other hand, Gram-positive bacteria will retain
the crystal violet dye when washed in a
decolorizing solution.

Dr.T.V.Rao MD 78
 Gram negative bacteria
On most Gram-stained
preparations, Gram-
negative organisms will
appear red or pink because
they are counterstained.
Due to presence of higher
lipid content, after alcohol-
treatment, the porosity of
the cell wall increases,
hence the CVI complex
(Crystal violet -Iodine) can
pass through. Thus, the
primary stain is not
retained. Dr.T.V.Rao MD 79
 Gram Negative Bacteria
Also, in contrast to most
Gram-positive bacteria,
Gram-negative bacteria
have only a few layers
of peptidoglycan and a
secondary cell
membrane made
primarily of
lipopolysaccharide

Dr.T.V.Rao MD 80
ACID FAST STAINING

Dr.T.V.Rao MD 81
 Acid-Fast Stain
Acid-fast cells contain a
large amount of lipids and
waxes in their cell walls
primarily mycolic acid
Acid fast bacteria are
usually members of the
genus Mycobacterium or
Nocardia
Therefore, this stain is
important to identify
Mycobacterium or Nocardia

Dr.T.V.Rao MD 82
 Ziehl-Neelsen stain
Ziehl-Neelsen staining is used to stain
species of Mycobacterium tuberculosis
that do not stain with the standard
laboratory staining procedures like Gram
staining.
The stains used are the red colored
Carbol fuchsin that stains the bacteria
and a counter stain like Methylene blue
or Malachite green.
Dr.T.V.Rao MD 83
 Acid-Fast Organisms
Primary stain binds cell wall mycolic acids
Intense decolorization does not release
primary stain from the cell wall of AFB
Color of AFB-based on primary stain
Counterstain provides contrasting
background

Dr.T.V.Rao MD 84
 AFB Staining Methods
Zeihl
Neelsens-hot
stain
Kinyouns-cold
stain
Modifications
Dr.T.V.Rao MD 85
ALBERTS STAINING FOR
C.diptheria

Dr.T.V.Rao MD 86
 Diphtheria is Serious Disease
When you suspect Diphtheria
In all cases of suspected
cases of Diphtheria, stain
one of the smears with
Gram stain
If Gram stained smear
shows morphology
suggestive of C.diptheria,
proceed to do Albert
staining which
demonstrates the presence
or absence of
metachromatic granules.
Dr.T.V.Rao MD 87
 Appearance of C.diptheria
C.diptheria are thin Gram positive bacilli, straight
or slightly curved and often enlarged (clubbing) at
one or both ends and are arranged at acute
angles giving shapes of Chinese letters or V shape
which is characteristic of these organisms (Fig 1).
Present in the body of the bacillus are numerous
metachromatic granules which give the bacillus
beaded or barred appearance. These granules are
best demonstrated by Alberts stain.

Dr.T.V.Rao MD 88
 Albert staining
Albert stain I Albert stain II
Toluidine blue 0.15 gm
Malachite green 0.20
Iodine 2.0 gm
gm Potassium
Glacial acetic acid 1.0 iodide 3.0 gm
ml
Alcohol(95%) 2.0 ml Distilled water
Distilled water 100 ml 300 ml

Dr.T.V.Rao MD 89
 Albert staining Procedure
Cover the heat-fixed smear with Albert
stain I. Let it stand for two minutes.
Wash with water.
Cover the smear with Albert stain II.
Let it stand for two minutes.
Wash with water, blot dry and
examine.

Dr.T.V.Rao MD 90
How the C.diptheria appear
To demonstrate
metachromatic
granules in
C.diptheria. These
granules appear
bluish black whereas
the body of bacilli
appear green or
bluish green.
Dr.T.V.Rao MD 91
 Programme created by Dr.T.V.Rao MD
from several resources in world wide
web, and Thankful for Dr. Ekta
www.medmicrobes from basic
programme on Bacterial cell
Email
doctortvrao@gmail.com

Dr.T.V.Rao MD 92