In very easy way learn about bacteria

1. Bacteriology
Basics
Morphology, Classification, Staining
Methods
IMRAN TARIQ
(ASSISTANT PROFESSOR)

2. Introduction:
• The kingdom protista has been classified into
prokaryotes and eukaryotes. Bacteria are
prokaryotes, white fungi, algae, protozoa are
eukaryotes.
• Eukaryotic cell types - Animals, plants, fungi,
protozoans, and algae
– Prokaryotic cell types - bacteria & blue green
algae
2

3. 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
3

4. 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
4

5. 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
5

6. 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
exocytosis
Capable of
endocytosis and
exocytosis
6

7. 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 80 S
7

8. Differences between prokaryotic &
eukaryotic cells
Character Prokaryotes Eukaryotes
Cell Wall Present Animals & Protozoans –
Absent
Plants, Fungi & Algae -
Present
Composition Peptidoglycan –
complex
carbohydrate
Cellulose or chitin
Locomotor
organelles
Flagella Flagella/ Cilia
8

9. 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 9

10. 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
10

11. 11
Eukaryotic cell Prokaryotic cell
Gram +
Gram -
Cell wall
Rough endoplasmic
reticulum
Mitochondria
(e.g. animal)
Nucleoid
Nucleus
Cell membrane
Cytoplasm
Flagellum
Cell (inner) membrane Outer membrane
Ribosomes
Granule
Cell wall
Capsule
Pili

12. Shapes of Bacteria
• When viewed under the microscope, most
clinically significant bacteria appear in three
basic shapes, they are ; Cocci (from Kokkes
meaning berry) are spherical or oral. Bacilli
(from bacillus meaning rod) are rod shaped.
Spiral Other then three basic bacterial shapes
some bacterial are square, filaments and large
number produces buds, lancet (spneumoid
bean – shaped (Neisseria gonnorhea).
12

13. 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
13

14. Bacteria Have One of Three Cellular
Shapes
• Rods (bacilli)
• Coccoid-Shaped
• Spirilla
14

15. •Cocci These spherical bacteria vary in
diameter between 0.5  m - 2  m. They occur
as single cells (micro cocci)
• Pairs of cocci (Diplococci)
• Chains (Streptococci)
• Packets (Staphylococci)
• Cube – shaped cellular packets (Sarcinae )
15

16. Arrangement of bacteria: Cocci
Cocci in pair – Diplococcus
Sarcina – groups of eight
Tetrad – groups of four
Cocci in chain - Streptococci
Cocci in cluster - Staphylococci
Coccus
16

17. Spherical Bacteria

18. Bacilli
• This morphotype consists of cylindrical
cells, which are either large or small rods.
The bacilli are usually 0.2 – 1  m in
diameter, with lengths of approximately
10  m being common. Most rods occur
single, but some form long chains called
as stretptobaccilli.
18

19. Arrangement of bacteria: Bacilli
19

20. Rod-Shaped Bacteria

21. Other shapes of bacteria
• Spiral: In reality, the spirilla are bacilli twisted
into helix. the rigid spirilla are surrounded by a
cell wall and are represented by genera
spirillum and vibrio. Flexible spirilla with more
than one way are typical of the spirochaetes,
represented by the genera treponema,
spirocheta, leptespira, borrelia and cristispira.
Actinomycetes are branching filamentous
bacteria, so called because of fancied
resembled to the radiating rays of sun when
seen in tissue lesion (from actis meaning ray
and mykes meaning fungus).
21

22. Other shapes of bacteria
Comma shaped
Spirochetes
Spirilla
22

23. Spiral-Shaped Bacteria
Spirochete:
Borrelia
burgdorferi

24. Bacterial Morphology
24

25. Anatomy of a Bacterial Cell
25

26. 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
26

27. Typical shapes of bacteria
Most bacteria retain a particular shape; a few
are pleiomorphic
27

28. Typical prokaryotic structures
Working from the outside in…
28

29. 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
29

30. Structure & Function
of Cell Components

31. CELL WALL
• Outermost layer, encloses cytoplasm
1. Confers shape and rigidity
2. 10 - 25 nm thick
3. Composed of complex polysaccharides
(peptidoglycan/ mucopeptide) - formed by N
acetyl glucosamine (NAG) & N acetyl muramic
acid (NAM) alternating in chains,
held by peptide chains.
31

32. 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. Cell wall also serves as primary sieve in the transport
molecules into the cell. It also functions as an anchor for
the capsule, as well as pilli, fimbriae, flagella. It is primary
receptor for the absorption of specific viruses and
bacteriophages
8. Several antibiotics may interfere with cell wall synthesis
e.g. Penicillin, Cephalosporins
32

33. Peptidoglycan structure
• The polymer contains two sugar derivatives ,
N – acetyl glucosamine N – acetyl muramic
acid alternating in chains which are
crosslinked by peptide chains. Gram positive
cell wall Normally thick, homogeneous cell
wall of gram positive bacteria composed
primarily of peptidoglycan. However gram
positive cell walls, usually contain large
amounts of teichoic acids, polymers of
glycerol joined by phosphate group. Lipids and
proteins are usually absent. 33

34. 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
34

35. 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.
35

36. Functions of teichoic acids
• They may contribute to the overall negative change
density of cell surface. This negative surface charge
and lipoteichoic acids are strong adhesions,
especially in the attachment to mammalian tissues,
and adherence to red blood cells, tooth surfaces and
other positively charged surfaces.
36

37. Bacterial
Cell
Walls
37

38. 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
38

39. 39
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

40. Cytoplasmic (Plasma) membrane
• The cytoplasmic membrane is a layer (5-10 nm) lining the
inner surface of the cell wall separating it from the
cytoplasm. It consists of three layers – outer, middle and
inner layer, chemically, both inner and outer layers are
composed of 70%, protein, the middle (hydrophobic)
layer contains 30% lipid in addition to fatty acids,
phospholipids, transmembrane proteins (permeases)
with small amounts of carbohydrates
• In summary, functionally, the cytoplasmic membrane is
one of most important components of the bacterial cell,
which is site of anabolic and catabolic metabolism and
regulates the transport of molecules into without of the
cell. 40

41. 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. 41

42. cytoplasmic membrane
42

43. Other Cytoplasmic Components
• The bacterial cytoplasm consists of central
nuclear region surrounded by ribosome’s
mixed in a sea of enzymes, cofactors, amino
acids, and vitamin, as well as multitude of
other bone and molecules. It differs from
eukaryotic cytoplasm in not exhibiting
internal mobility and the absence of
endoplasmic reticulum and mitochondria.
These serve as building blocks for cellular
biosynthesis and energy production. The
cytoplasm contains ribosome’s, mesosomes,
inclusions and vacuoles. 43

44. Ribosome’s
• The ribosome’s are the most abundant
inclusion in bacterial cytoplasm. They are
called 70S ribosomes. It consists of 40%
protein and 60% RNA. Eukaryotic ribosome’s
are larger than bacterial ribosome’s. Three
types of RNA exist ribosomal RNA (rRNA),
Transfer RNA (tRNA), messenger RNA
(mRNA)).
44

45. Mesosomes (Chondroids):
• These are vesicular, convoluted or
multilaminated structures formed as
invaginations of the plasma membrane into
cytoplasm’s. They are more prominent in gram
positive bacteria. They are principal sites of
respiratory enzymes in bacteria and are
analogous to the mitochondria of eukaryotes.
Mesosomes are often seen in relation to the
nuclear body and site of synthesis of cross wall
septa, suggesting that they coordinate nuclear
and cytoplasmic division during binary fission.
45

46. Nucleus
• Bacterial nuclei can be demonstrated by acid or ribo
nuclease hydrolysis and subsequent staining for
nuclear material. Bacterial nuclei have no nuclear
membrane or nucleolus. The nuclear DNA is not
associated with basic protein. The genome consists
of single molecule of DNA arranged in the form of
circle. The bacterial chromosome is haploid and
replicates by simple fission instead of by mitosis as in
higher cells. Bacteria may posses extranuclear
genetic elements consisting of DNA. These
cytoplasmic carries of genetic information are
termed as plasmid or episomes. These may account
for drug resistance which may constitute a survival
advantage . 46

47. 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 47

48. Bacterial storage granules and
inclusion :
• These are aggregates of various compounds
that are normally involved in storing energy
reserves or building blocks for the cell. During
their growth and metabolism, bacteria
accumulate variety of cytoplasmic storage
products, as a result of the environment and
metabolism by the cell of large number of
carbohydrates, lipids, proteins. Two major
types of inorganic inclusion bodies are seen.
Polyphosphate granules or volutin granules
Magnetosome. 48

49. Inclusions of
Bacteria
granulose

50. Additional Organelles
Capsule & Slime layer –
– Capsule – usually of uniform consistency
and integrity
– Slime layer – are of ill defined and
loosely formed
– Viscous layer secreted around the cell
wall.
– Polysaccharide / polypeptide in nature
Capsule – sharply defined structure,
antigenic in nature
• Protects bacteria from lytic
enzymes
• Inhibits phagocytosis
• Stained by negative staining
using India Ink
50

51. Flagella –
• Flagella and Motility Flagella are organelles of motility / organs
of locomotion. Each flagellum consists of three distinct parts,
• The filament – external to the cell and connected to the hook
at the cell surface.
• Hook – connects the basal body to the distal portion of
filament.
• The basal body – which is embedded in the cytoplasmic
membrane and portions of cell wall. Gram positive - 1 rings.
Gram negative – 2 rings.
• Chemically, flagella are made up of protein flagellin similar to
keratin or myosin. Flagellin structure of each bacterial species is
sufficiently unique to confer immunologic specificity and useful
in microbiological identification of specific microorganisms.
51

52. In Gram-negative Bacteria
5/03/2012 52
Masdiana Padaga

53. In Gram-positive Bacteria
5/03/2012 53
Masdiana Padaga

54. A TYPICAL FLAGELLA
54

55. Types of flagellar arrangement
• 1. Flagella arranged all round the cell
(peritrichous) eg. Typhoid
• 2. Monotrichous – single flagella situated at
one end eg. Vibrio cholera
• 3. Lopotrichous – Tufts of flagella at one end
eg. Spirilla
• 4. Amphitrichous – Flagella at the both poles
55

56. Types of flagellar arrangement
Polar/ Monotrichous – single
flagellum at one pole
Lophotrichous – tuft of flagella at one
pole
Peritrichous – flagella all over
Amphitrichous – flagella at both
poles
Amphilophotrichous – tuft of flagella
at both ends
56

57. Peritrichous monotrichous
(or amphi, or lophotrichous
Cocci do not have flagella
57

58. Fimbriae/ Pili
– Both Pilli and Fimbriae are attached to the outer most
surface of cell. These hair like appendages, 10-20µ
long. They are confined to gram negative bacteria and
to selected gram positive bacteria (i.e, streptococcus,
actinomyces). The pilli are distinguished from flagella
in being approximately one half their diameter as well
as being distinctively straight or stiff in appearance.
– 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)
58

59. Functions of Pilli or Fimbriae
• These structures function primarily in the
transfer of genetic material between the
bacteria, as well as adherence. Pilli that help
in transfer of genetic material are referred as F
or sex Pilli. F Pilli form physical bridges
between the donor and recipient cells known
as conjugation bridges, which permit the
transfer of DNA between them. The Pilli are
also bacterophage receptors, specific
bacteriophage will absorb to the type of the
Pilli, which are then retracted into the cell. 59

60. 60

61. Spore
• Some bacteria have the ability to form highly resistant resting
stages called spores. Each bacterium forms one spore, which
on germination forms a single vegetative cell. An bacterial
spores are formed inside the parent cell, they are called
endospores. Sporulation occurs after a period of vegetative
growth and is presumed to be related to the depletion of
exogenous nutrient. Sporulation is initiated by the appearance
of clear area, usually near one end of the cell, which gradually
becomes more opaque to form forespore. The fully developed
spore has at its core the nuclear body, surrounded by the
spore wall, a delicate membrane from which the cell wall of
the future vegetative bacterium will develop.
61

62. 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.
• They can be destroyed by
autoclaving at 120°C for 15
minutes
62

63. Shape & position of bacterial spore
Oval central
Spherical central
Oval sub terminal
Oval sub terminal
Oval terminal
Spherical terminal
Free spore
Non bulging
Bulging
63

64. 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.
64

65. 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
• 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)
65

66. Bacterial Growth and
Multiplication
• Transverse or Binary cell division
Growth of bacteria can be separated into two
distinct stages 1. An increase in cell mass such as
elongation of a bacillus, or an increase in size or
volume such as in occurs
2. The division of these cells into two daughter cells
The most common mechanism of bacterial cell
division which can be described as Binary or
transverse division.
66

67. 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.
67

68. • Other mechanism of bacteria cell division
Bacteria such as hyphomicrobium,
corynebacterium and mycobacteria divide by
budding. The budding cell originates as an
outgrowth of the original, or mother cell, which
eventually reach a size equal to that of the
mother cell. the bud then separates to undertake
an independent existence several of the
actinomycetes form filamentous cells that
reproduce by the formation of spores from a
chain of cells or by a simple fragmentation of the
original filament into new cells.
68

69. The bacterial growth curve
• When bacterium is seeded into a suitable liquid medium
and incubated its growth shows a definite course. If
bacterial consists are made at intervals after inoculation
and plated in relation to time, a growth curve is obtained.
The curve shows the following phases.
• Lag phase
• This phase of growth occurs when cells are transferred
from one medium to another or one environment to
another It is a phase of adjustment and represents the
period required for the adaptation of the cells to new
environment. The cells in this phase often increase in total
volume almost two or threefold, but they do not divide.
These cells are rapidly synthesizing DNA, new protein and
new enzymes as a prerequisite to division
69

70. Exponential phase
• – or log phase In this phase, cells are dividing
at both a constant geometric rate as well as
maximum rate. Cellular component such as
RNA, protein, dry weight, and cell wall
polymers are also increasing at a constant
rate, they are much smaller in diameter than
cells in the lag phase. The exponential growth
phase usually comes to an end because of
depletion of essential nutrients, depletion of
oxygen in an acrostic culture or the
accumulation of toxic products. 70

71. Stationary phase
• During this phase, there is a rapid decrease in the rate
of cell division. Eventually, the total number of dividing
cells will equal the number of dying cells, a true
stationery cell population occurs. The cells then direct
their resources towards survival energy generation is
harnessed to maintain osmotic barriers, mobility, and
the repair and synthesis of essential macromolecules.
The energy required to maintain cells in stationery
phase is called Maintenance energy and is obtained
from the degradation of cellular storage products.
After all of the storage products are exhausted, the
cells then degrade their cellular components, which
ultimately leads to death phase.
71

72. Death Phase
• In this stage, the cells reproduce more slowly,
and death overtakes them in increasing
numbers. The cells eventually enter the
logarithmic death phase, during which the
decrease in the number of viatie cells occurs
at a regular, unchanging rate, a rate that
approximates that of the exponential growth
phase but is of negative slope.
72

73. 73

74. 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)
74

75. 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 75

76. 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
76

77. 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. 77

78. 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
78

79. 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
79

80. 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
80

81. 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). 81

82. Gram staining
• Named after Hans
Christian Gram,
differentiates
between Gram-
positive purple and
Gram-negative pink
stains and is used to
identify certain
pathogens.
82

83. 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
83

84. 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. 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
84

85. Structure and Reactivity to Gram
Staining.
85

86. 86

87. Gm+ve cocci & Gm-ve bacilli
87

88. 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.
88

89. 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.
89

90. 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. 90

91. 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
91

92. 92
The Cell Envelope
Gram Positive Gram Negative

93. 93
GRAM POSITIVE
GRAM NEGATIVE
Cytoplasm
Cytoplasm
Lipoteichoic acid Peptidoglycan-teichoic acid
Cytoplasmic membrane
Inner (cytoplasmic) membrane
Outer Membrane
Lipopolysaccharide
Porin
Braun lipoprotein

94. Gram-positive and gram-negative bacteria

95. 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. Appear blue/purple.
95

96. ACID FAST STAINING
96

97. 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
97

98. 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. 98

99. 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
99

100. AFB Staining Methods
• Zeihl
Neelsen’s-hot
stain
• Kinyoun’s-cold
stain
• Modifications
100

101. ALBERT’S STAINING FOR
C.diptheria
101

102. 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.
102

103. 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.
103

104. 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
104

105. 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.
•
105

106. 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.
• 106