2. Introduction:
Dr.T
.V.Rao MD 2
• 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
3. Antioni van Leeuwenhoek
s
• 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 magnification
of over 200x with simple
ground lenses
Dr.T
.V.Rao MD 3
4. How a Microscope Works with..
Ocular Lens
(Magnifies Image)
Objective Lens
(Gathers Light,
Magnifies
And Focuses Image
Inside Body Tube)
Body Tube
(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
microscope magnifies it (again). 4
Dr.T
.V.Rao
MD
5. The Light Microscope
• many types
–bright-field microscope
–dark-field microscope
–phase-contrast microscope
–fluorescence microscopes
• compound microscopes
–image formed by action of 2 lenses 5
Dr.T
.V.Rao
MD
6. 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
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
• Binocular: a microscope having two eyepieces.
6
Dr.T
.V.Rao
MD
7. 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
8. The Bright-Field Microscope
• Produces a dark image against a
brighter background
• Has several objective lenses
–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
Dr.T
.V.Rao
MD
9. 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
Dr.T
.V.Rao MD 9
11. Background Information
Prokaryotes
Dr.T
.V.Rao MD 11
• Prokaryotes represent two domains,
bacteria and archaea.
• Archaea live in Earth’s extreme
environments.
• Bacteria are the most abundant and
diversified organisms on Earth.
12. Dr.T
.V.Rao MD 12
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
13. Differences between prokaryotic & eukaryotic cells
Character Prokaryotes Eukaryotes
Nucleus Nuclear
membrane
Absent Present
Nucleolus Absent Present
Chromosome One circular One or more paired
and linear
Cell division Binary fission Mitosis
Cytoplasmic
membrane
Structure and
Composition
fluid phospholipid
bilayer, lacks sterols
fluid phospholipid
bilayer containing
sterols
Function Incapable of
endocytosis
(phagocytosis and
pinocytosis) and
exD
or
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Capable of
endocytosis and
exocytosis
13
14. Differences between prokaryotic & eukaryotic
cells
Character Prokaryotes Eukaryotes
Cytoplasm Mitochondria Absent Present
Lysosomes Absent Present
Golgi apparatus Absent Present
Endoplasmic
reticulum
Absent Present
Vacuoles Absent Present
Ribosomes 70 S
Dr.T.V.Rao MD
80 S
14
16. 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 don’t 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
17. Size of Bacteria
• Unit of measurement in
bacteriology is the micron
(micrometre, µm)
• Bacteria of medical importance
–0.2 – 1.5 µm in diameter
–3 – 5 µm in length
Dr.T
.V.Rao MD 17
18. 18
Eukaryotic cell Prokaryotic cell
Gram +
Gram -
(e.g. animal)
Nucleoid
Rough endoplasmic
reticulum
Nucleus
Cell membrane
Cytoplasm
Mitochondria
Flagellum
Cell wall
Outer membrane
Granule
Cell wall
Pili
Capsule
Cell (inner) membrane
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19. Shapes of Bacteria
• Cocci – spherical/ oval shaped
• Bacilli – rod shaped
• Vibrios – comma shaped
• Spirilla – rigid spiral forms
major groups
• Spirochetes – flexible spiral forms
• Actinomycetes – branching filamentous
bacteria
• Mycoplasmas – lack cell wall
Dr.T
.V.Rao MD 19
20. Bacteria Have One of Three Cellular
Shapes
• Rods (bacilli)
• Coccoid-Shaped
• Spirilla
Dr.T
.V.Rao MD 20
21. Arrangement of bacteria: Cocci
Sarcina – groups of eight
Cocci in chain - Streptococci
Coccus
Cocci in pair – Diplococcus
Tetrad – groups of four
Cocci in cluster - Staphylococci
Dr.T
.V.Rao MD 21
22. 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.
Dr.T
.V.Rao MD 22
27. Structure of Bacteria
• All cells have 3 main components:
– DNA (‘nucleoid”)
• genetic instructions
– surrounding membrane (“cytoplasmic
membrane”)
• limits access to the cell’s 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
28. Typical shapes of bacteria
28
Dr.T
.V.Rao MD
Most bacteria retain a particular shape; a few
are pleiomorphic
34. Difference Between
Gram-Negative
and Gram-Positive Bacteria
Gram-Negative 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.
Dr.T.V.R
Appear blue/purple.
ao MD 34
35. 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
37. 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
38. Dr.T
.V.Rao MD 38
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
39. 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.
Active transport. Dr.T
.V.Rao MD 39
40. Outer Membrane
Dr.T
.V.Rao MD 40
Gram negative bacteria
• major permeability barrier
• space between inner and outer
membrane
–Periplasmic space
store degradative enzymes
• Gram positive bacteria
• no Periplasmic space
41. Gram positive cell wall
The Gram-positive cell wall
Dr.T
.V.Rao MD 41
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
42. 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
44. Dr.T
.V.Rao MD 44
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
45. Dr.T
.V.Rao MD 45
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
46. Other Cytoplasmic Components
as
e
• Ribosomes – protein synthesis
• Mesosomes –
1. Multilaminated structures formed
invaginations of plasma membran
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
47. Nucleus
nded DNA.
• No nucleolus
• No nuclear membrane
• Genome –
–single, circular double stra
–Haploid
–Divides by binary fission
Dr.T
.V.Rao MD 47
48. 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, toxiD
cr.iTt
.Vy
.Rao MD 48
49. 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
50. 50
Dr.T
.V.Rao MD
Additional Organelles
3. Flagella –
– Long (3 to 12 µm), 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
51. 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
51
Dr.T
.V.Rao MD
53. FLAGELLA
• Some bacteria are motile
• Locomotory organelles- flagella
• Taste environment
• Respond to food/poison
– chemo taxis
53
Dr.T
.V.Rao MD
54. • Flagella
– embedded in cell membrane
– project as strand
– Flagellin (protein) subunits
– move cell by propeller like action
54
Dr.T
.V.Rao MD
55. 55
Dr.T
.V.Rao MD
Additional Organelles
4. Fimbriae/ Pili –
– Thin, hairlike appendages on the surface of many Gram-
negative bacteria
– 10-20µ 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
56. 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
56
Dr.T
.V.Rao MD
57. 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
relleased from the cell. On germination, the spore contents
rehydrate and a new bacterium emerges and multiplies.
57
Dr.T
.V.Rao MD
58. Shape & position of bacterial spore
Oval central
Spherical central
Oval sub terminal
Oval sub terminal
Oval terminal
Spherical terminal
Free spore
Non bulging
Bulging
58
Dr.T
.V.Rao MD
59. 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.
59
Dr.T
.V.Rao MD
60. 60
Dr.T
.V.Rao MD
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)
61. 61
Dr.T
.V.Rao MD
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
62. 62
Dr.T
.V.Rao MD
Bacterial Taxonomy
• Includes three components:
1.Classification : orderly
arrangement
2.Identification of an unknown
unit
3.Nomenclature : naming the
units
63. 63
Dr.T
.V.Rao MD
Bacterial Taxonomy: Classification
• Orderly arrangement :
Division – Class – Order – Family –
Species
Kingdom –
Tribe – Genus –
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)
64. 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
65. 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 (Å) = 1 x10-10 m
Dr.T
.V.Rao MD 65
66. 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. 66
Dr.T
.V.Rao MD
67. Dr.T
.V.Rao MD 67
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
68. Dr.T
.V.Rao MD 68
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
69. 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
70. 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). 70
Dr.T
.V.Rao MD
71. 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
72. Dr.T
.V.Rao MD 72
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
73. Gram Staining Steps
Dr.T
.V.Rao MD 73
1. Crystal violet acts as the primary stain. Crystal violet may also
be used as a simple stain because it dyes the cell wall of any
bacteria.
2. Gram’s iodine acts as a mordant (Helps to fix the primary
dye to the cell wall).
3. Decolorizer is used next to remove the primary stain (crystal
violet) from Gram Negative bacteria (those with LPS imbedded
in their cell walls). Decolorizer is composed of an organic
solvent, such as, acetone or ethanol or a combination of both.)
4. Finally, a counter stain (Safranin), is applied to stain those cells
(Gram Negative) that have lost the primary stain as a result of
decolorization
77. 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
78. Dr.T
.V.Rao MD 78
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.
79. 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. 79
Dr.T
.V.Rao MD
80. 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
82. 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
83. Ziehl-Neelsen stain
83
• 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 gree
Dn
r.T
.V
..Rao MD
84. Acid-Fast Organisms
Dr.T
.V.Rao MD 84
• 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
87. 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
88. Dr.T
.V.Rao MD 88
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 Albert’s stain.
89. Dr.T
.V.Rao MD 89
Albert staining
• Albert stain I
• Toluidine blue 0.15 gm
Malachite green 0.20
gm
Glacial acetic acid 1.0
ml
Alcohol(95%) 2.0 ml
Distilled water 100 ml
• Albert stain II
• Iodine 2.0 gm
Potassium
iodide 3.0 gm
Distilled water
300 ml
90. Dr.T
.V.Rao MD 90
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.
•
91. 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
92. Dr.T
.V.Rao MD 92
• 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
