INTRODUCTION TO

MICROBIOLOGY
1
Mikros - "small",
Bios - "Life"
-lodge - " study, science"
MICROBIOLOGY
 Microbiology: is the study of microscopic
organisms, such as bacteria, viruses, archaea,
fungi and protozoa. This discipline includes
fundamental research on the biochemistry,
physiology, cell biology, ecology, evolution and
clinical aspects of microorganisms, including the
host response to these agents.
 Microorganisms: organisms that are too small to
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be seen with the unaided eye.
 MEDICAL MICROBIOLOGY
 Medical microbiology is a branch of microbiology that
deals with the study of microorganisms including bacteria,
viruses, fungi, and protozoa of medical importance that are
capable of causing diseases in humans.
 It also includes the study of:
 microbial pathogenesis,
 immunology,
 epidemiology of diseases,
 prevention, diagnosis and treatment of infectious diseases
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 HISTORY OF MICROBIOLOGY

First Period: From Antiquity to the discovery of

the microscope (heuristic).
Second Period: 1675 until the mid-nineteenth
century (morphological).
Third Period: Second half of Century XIX
(physiological).
Fourth Period: Beginnings of the twentieth
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century to the present (immunological and
molecular genetic).
 FIRST PERIOD - HEURISTIC
 The period prior was characterized by speculation about the
existence of microorganisms and their functions.
 Microbial diseases have played a major role in historical events,
such as the decline of the Roman Empire.

 The plague had killed one-third of the population (about 25 million

people).

 Apart from the bubonic plague, measles and smallpox too played
their roles as epidemic diseases causing high mortality and
morbidity.

 The first recorded epidemic of smallpox was in the year 1350 BC in

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Egypt.
Hippocrates suggested the existence of
«miasma»;

Ibn Sina (Avicenna) wrote in the Canon

of Medical Science that the cause of
plague, smallpox and other diseases
are the smallest living beings invisible
to the naked eye, transmitted through
air and water.

These ideas were formulated in the

15th century as a hypothesis of the
Italian physician D. Fracastoro (1478-
1553), who suggested that disease
was caused by invisible living creatures
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(contagium vivum)
ARISTOTLE

Spontaneous
Generation:
Mice grow from
straw + darkness

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 He theorized that ‘a vital force’ forms life. He noticed that
mice were commonly found in barns where grain was
stored. He thought that the mice grew from the grain
and hay, and he coined the term “Spontaneous
generation”.

 They pointed out that boiled extracts of hay or meat

would give rise to microorganisms after sometime.

 No one doubted this for more than a thousand years.

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SECOND PERIOD - MORPHOLOGICAL
(SLOW ACCUMULATION OF OBSERVATIONS)
ANTONI VAN
LEEUWENHOEK
(1673 - 1723)
Described first
microbes. He saw
bacteria and
protozoa.
But he did not
evaluate these
organisms as
agents
of disease.
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 MICROSCOPE

Simple microscopes composed of

double convex glass lenses held
between two silver plates.
His microscopes could magnify around
50–300 times.
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THIRD PERIOD - PHYSIOLOGICAL
(THE CULTIVATION OF THE MICROORGANISMS)
LOUIS PASTEUR
He first coined the term
“microbiology” (for the study of
organisms of microscopic size),
“fermentation”,
“pasteurization”, and the
“Germ Theory of Disease”.
Louis Pasteur went on to
discover three vaccines, the 13
first
was by accident.
 Pasteur for the first time demonstrated that he could
kill many microorganisms in wine by heating and
then rapidly cooling the wine, a process now called
pasteurization.

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 THE GOLDEN AGE OF MICROBIOLOGY
He found that the old culture of chicken cholera was
weakened (he called this “attenuated”), and thus he
discovered how to make the first vaccine for chicken cholera.
Later, he found the causative agent for anthrax and invented
the sheep anthrax vaccine (He attenuated the culture of
anthrax bacillus by incubation at high temperature of 42–43°C
and inoculated the attenuated bacilli in the animals).
He also invented the rabies vaccine.
A vaccine is a form of artificial immunity
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PASTEUR: SHEEP VACCINE FOR ANTHRAX

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ROBERT KOCH, (1843-1910)

He developed “Koch’s
Postulates” which
establish the cause
of disease.

He was known for

quoting Pasteur by
saying “Chance favors a
prepared mind”.
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KOCH’S POSTULATES
1. The microorganism must be present in every case of
the disease but absent from healthy host.
2. The suspected microorganism must be isolated and
grown in a pure culture from lesions of the disease.
3. The isolated organism, in pure culture, when
inoculated in suitable laboratory animals should
produce a similar disease.
4. The same microorganism must be isolated again in
pure culture from the lesions produced in
experimental animals.
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KOCH’S POSTULATES

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SOLID MEDIUM FOR CULTURE OF BACTERIA

 Koch pioneered the use of agar as a base for

culture media. He developed the pour plate
method and was the first to use solid culture media
for culture of bacteria. This development made
possible the isolation of pure cultures that
contained only one type of bacterium and directly
stimulated progress in all areas of bacteriology.

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DISCOVERY OF IMPORTANT BACTERIAL AGENTS
CAUSING HUMAN DISEASES

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FOURTH PERIOD -
IMMUNOLOGICAL AND MOLECULAR GENETIC
(THE CLOSE INTERWEAVING OF THE MICROBIOLOGICAL
SCIENCES).

 Given the advances of the nineteenth century, both

theoretical and methodological, microbiology
ceased to be merely speculative, in order to
consolidate itself as a science and to divide its
object of study into specific areas.
 In this sense, research on infections progressed,
both in the techniques of sterilization and post-
operative care, as well as in their possible cures. 22
STUDY OF VIRUSES
 In 1892, Dmitri Ivanovsky, a Russian scientist
working in St. Petersburg, demonstrated that the
sap of leaves infected with tobacco mosaic disease
retains its infectious properties even after filtration
through filter candles.
 An organism smaller than bacteria (a filterable
agent) that is not observable in the light microscope
and is able to reproduce itself only in living cells or
tissues.

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VIRUS

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MECHANISMS OF IMMUNITY

 In 1884 Elie Metchnikoff demonstrated that cells

also contribute to the immune state of an animal.
He observed that certain white blood cells, which
he termed phagocytes, were able to ingest
(phagocytose) microorganisms and other foreign
material.

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METCHNIKOFF (1845 – 1916)

Began field of
immunology

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CHEMOTHERAPEUTIC AGENTS

 Until the 1930s, there had been no chemical

treatment available to fight bacterial infections in
general.

 Paul Ehrlich demonstrated that dyes react

specifically with various components of blood cells
and the cells of other tissues. He began to test the
dyes for therapeutic properties to determine
whether they could kill the pathogenic microbes. 27
 He developed Salvarsan, an arsenical compound in
1909. The compound was capable of destroying T.
pallidum, the causative agent of syphilis. This
treatment proved effective against syphilis. This
work was of epochal importance, stimulating
research that led to the development of sulfa drugs.

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PAUL EHRLICH (1854 –1915)

Chemotherapeutic
Agents For syphilis

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ALEXANDER FLEMING (1881-1955)

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ANTIBIOTIC

 1928: Alexander Fleming

discovered the first
antibiotic.
 He observed that
Penicillium fungus made
an antibiotic, penicillin,
that killed S. aureus.
 1940s: Penicillin was
tested clinically and mass
produced. 31

Figure 1.5
NOTE:

 Antibiotics are chemicals that kill bacteria.

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 SELECTED NOBEL PRIZES IN
PHYSIOLOGY
 1901 Behring diphtheria antitoxin
 1902 Ross malaria transmission
 1905 Koch TB bacterium
 1908 Metchnikoff phagocytosis
 1945 Fleming,
Chain, Florey penicillin
 1969 Delbruck,
Hershey viral replication
 1987 Tohegawa antibody genetics
 1997 Prusiner prions 33
WHY WE HAVE MICROBIAL
DISEASES
 Mutation leads to evolution of bacteria
 Antibiotic use causes resistance and evolution of
bacteria
 Travel exposes us to microbes we are not used to
 Deforestation (removal of trees, especially in the
tropics, Africa, Central America) destroys the
ecosystem and disturbs natural balance of
microbes

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CLASSIFICATION

 Microorganisms are a heterogeneous group of several

distinct living structures of microscopic size, classified
under the kingdom Protista. The kingdom Protista
includes unicellular organisms, such as bacteria, fungi,
protozoa, and algae. Based on the differences in cellular
organization and biochemistry, the kingdom Protista has
been divided into three groups: prokaryotes, eukaryotes,
and the most recently described archaebacteria.
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 1. Prokaryotes: Bacteria and blue green algae are
prokaryotes. Bacteria are unicellular free living
organisms having both DNA and RNA.

 2. Eukaryotes: Fungi, algae other than blue green,

protozoa, and slime moulds are eukaryotes.

 3. Archaebacteria: These are more closely related

to eukaryotes than prokaryotes. They however do
not include any human pathogens.

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SCIENTIFIC NAME:
 The binomial system was published by C. Linnaeus.
The genus and species are significant taxonomic uses
in binomial nomenclature for each organism. The first
name for genus and second name for species. First
letter of genus should be written in capital letter,
whereas first letter of species, must be write in small.
Name of genus and species for any organism must be
write in Italic from or place line under each genus and
species.
 Ex: Staphylococcus aureus.
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NAME OF BACTERIA ARE DERIVED FROM
1. The name of disease that caused by bacteria. Ex:
Vibrio cholerae= causes cholerae.
2. The locality where the bacteria was first isolated. Ex:
Escherichia coli=from colon.
3. The scientists responsible for isolating bacteria.
Listeria= Lister.
4. Properties of bacterial morphology and physiology.
Staphylococcus aureus= cluster.

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 MICROSCOPY
39
 A BRIGHT-FIELD, COMPOUND LIGHT
MICROSCOPE.

 Resolving power of microscope is the ability of the lens

system to distinguish two closely placed objects as distinct 40

and separate entities.

BRIGHT-FIELD MICROSCOPES

 The purpose is to focus light

directly on the preparation for
optimal visualization against a
bright background.
 Bacteria may be stained by a
variety of dyes, including
methylene blue, crystal violet,
carbol-fuchsin (red), and
safranin (red). The two most
important methods, the Gram
and acid-fast techniques, use
staining, that helps to classify
the organism. 41
 The bright-field microscopy has
many uses.
 It may be used to examine wet
films and fixed colored smears.
 It is useful for demonstration of the
structural details.
 It is also useful for measuring
approximate size of the
microorganisms.

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 THE EFFECT OF IMMERSION OIL ON RESOLUTION

The effect of immersion oil on resolution. (a) Without

immersion oil, light is refracted as it moves from the cover
glass into the air. Part of the scattered light misses the
objective lens. (b) With immersion oil. Because light travels
through the oil at the same speed as it does through glass,43no
light is refracted as it leaves the specimen and more light
enters the lens, which increases resolution.
DARK-FIELD MICROSCOPES
 In dark-field illumination, a
black background is created
by blocking the central light.
Peripheral light is focused so
that it will be collected by the
objective only when it is
reflected from the surfaces of
particles (eg, bacteria). The
microscopic field shows bright
halos around some bacteria
and reveals a spirochete too
thin to be seen with bright-
field illumination.
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 The dark-ground microscopy has
following uses:
 It is useful for demonstration of
very thin bacteria (such as,
spirochetes) not visible under
ordinary illumination, since the
reflection of the light makes them
appear larger.
 It is also useful for demonstration
of motility of flagellated bacteria
and protozoa.

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PHASE MICROSCOPES

 Light rays passing

through a specimen
naturally slow down and
are shifted about 1/4
wavelength out of phase.
A special filter called a
phase plate, which is
mounted in a phase
objective lens

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 The phase-contrast microscopy has
following uses:
 It is immensely useful for examination
of living microorganisms particularly
protozoa (e.g., T. vaginalis,
Entamoeba histolytica, etc.)
 It is useful for examining the internal
structures of a living cell by improving
the contrast and differentiating
structures within the cell that differs in
their thickness and refractive index.

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FLUORESCENCE MICROSCOPES
 Fluorescence microscopy is
similar to dark-field microscopy,
except that the light source is
ultraviolet and the organisms
are stained with fluorescent
compounds. The incident light
generates light of a different
wavelength, which is seen as a
halo (colored in this illustration)
around only the organism
tagged with fluorescent
compounds. For the most
common fluorescent 48
compound, the light is green
 Fluorescence microscopy is widely
used in diagnostic microbiology in
the following ways:

 It is used for direct demonstration

of antigen of a pathogen in clinical
specimens by direct fluorescent
antibody test

 It is also used for the estimation of

antibodies in the serum by indirect
fluorescent antibody test
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IMMUNOFLUORESCENCE.

(a) Aftera fluorescent dye is

covalently linked to an antibody,
the dye-antibody combination
binds to the antibody’s target,
making the target visible under
fluorescent microscopy.
(b) Immunofluorescent staining
of Yersinia pestis, the causative
agent of plague. The bacteria
are brightly colored against a
dark background.
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ELECTRON MICROSCOPE

Electron microscopy shows

structures by transmission of an
electron beam and has 10 to
1000 times the resolving power
of light microscopic methods.

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 Electron microscope is widely used for:
 Rapid detection of viruses directly in clinical specimens.
This is especially useful for detection of noncultivable
viruses.
 Ultrastructural study of various microorganisms.

 (a) The path of electrons through a TEM, as compared to

the path of light through a light microscope

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MORPHOLOGY OF
BACTERIA
Cocci: The cocci (kokkos, berry) are oval or spherical cells.
These may be arranged in pairs (e.g., pneumococci,
meningococci, and gonococci), tetrads (micrococci), chains
(e.g., streptococci), and clusters (e.g., staphylococci).
 Bacilli: The bacilli (bacillus, rod) are rod shaped.
These bacilli may show either of the following
arrangement:
(a) Coccobacilli: Length of the bacteria is approximately the
same as its width, e.g., Brucella.
(b) (b) Streptobacilli: These are arranged in chains, e.g.,
Streptobacillus.
(c) (c) Comma shaped: They exhibit curved appearance,
e.g., Vibrio.
(d) (d) Spirilla: They exhibit rigid spiral forms, e.g.,
Spirillum.
Spirochetes: Spirochetes (spira, coil; chaite, hair) are
slender, flexuous spiral forms, e.g., Treponema
Actinomycetes: Actinomycetes (actin, ray; mykes, fungus)
are branching filamentous bacteria resembling fungi. They
possess a rigid cell wall.
MORPHOLOGY OF BACTERIA

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 MORPHOLOGY OF BACTERIA
 EXTERNAL COMPOSITION OF A PROKARYOTIC
CELL
1. The cell envelope primarily consists of two
components:
a cell wall and
cytoplasmic or plasma membrane.
It encloses the protoplasm, which consists of (i)
cytoplasm,
(ii) cytoplasmic inclusions (mesosomes, ribosomes,
inclusion granules, vacuoles), and
(iii) a single circular DNA Cell wall 59
CELL WALL
 More complex in Prokaryotes (bacteria) than in
Eukaryotes (plants, etc).
 Functions:
 protection from environment
 determination of cell shape
 protection from internal turgor pressure
 antigenic structure
 adhesion to substrate

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CELL WALL
 Composed of peptidoglycan, which is a
combination of peptide (protein) and glycan
(sugar).
 Peptidoglycan is only found in bacteria.
 Mycobacterium and Mycoplasma are the only
bacteria without a normal cell wall.

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 PEPTIDOGLYCAN

 Consists of a chain of two types of sugars (NAM and

NAG) linked by proteins.

 N-acetylglucosamine (NAG)

 N-acetylmuramic acid (NAM)

The sugars are arranged in this order: NAG-NAM-NAG.

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63

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GRAM-POSITIVE CELL WALL
 The Gram-positive cell wall is thick (15–80 nm) and
more homogenous than that of the thin (2 nm)
Gram-negative cell wall. The Gram-positive cell wall
contains large amount of peptidoglycan present in
several layers
 Cell wall consists primarily of teichoic and
teichuronic acids

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BACTERIAL CELL WALLS
GRAM+

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GRAM-NEGATIVE CELL WALL
 The Gram-negative cell wall is much more complex than
the Gram-positive cell wall.
 Peptidoglycan content in the Gram-negative cell wall is
significantly less than the Grampositive cell wall. Only 1–
2 layers of peptidoglycan (2–8 nm) are present just
outside the cell membrane.
 The Gram-negative cell wall outside the peptidoglycan
layer contains three main components—
 (a) lipoprotein layer,

 (b) outer membrane (has a variety of proteins:Porins,

Outer membrane proteins (OMPs)
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 (c) lipopolysaccharides
BACTERIAL CELL WALLS
GRAM-

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ATYPICAL FORMS OF BACTERIA
 Atypical forms of bacteria include
 (i) cell wall deficient forms,

 (ii) pleomorphic bacteria,

 (iii) involution forms

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 CELL WALL DEFICIENT FORMS WITHOUT CELL WALLS OR
WITH DEFICIENT CELL WALLS MAY BE OF VARIOUS TYPES
 Protoplasts: These are defective unstable forms of bacteria
with an intact cytoplasmic membrane but without any cell
wall. In hypertonic media, these are produced from
Grampositive cells on treatment with lysozyme.
 Mycoplasma: These are naturally occurring bacteria
without cell wall. They do not possess any definite shape.
They are very minute in size measuring 50–300 nm in
diameter.
 L-forms: The L-forms do not exhibit any regular size and
shape. They may be spherical or disc shaped, about 0.1–
20 mkm in diameter. They are difficult to cultivate. Some
bacterial species produce L-forms spontaneously. L-forms
in the host may produce chronic infections and are relatively
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resistant to antibiotic treatment

 Pleomorphic bacteria (e.g., Yersinia pestis) may
show considerable variation in size and shape
called pleomorphism.
 Involution forms: The involution forms are those
that on ageing of culture show swollen and aberrant
forms, especially in high salt concentration.

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 GRAM NEGATIVE

O Antigen
LPS

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Inner plasma membrane
Peptidoglycan

Lipid A Outer plasma

(endotoxin) membrane
LPS
 COMPARISON
OF GRAM-POSITIVE AND
GRAM-NEGATIVE CELL WALLS

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GRAM POSITIVE GRAM NEGATIVE
Plasma membrane
Cell Wall

Outer plasma
membrane

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DEMONSTRATION OF CELL WALL
 The cell walls can be demonstrated by
 differential staining procedure,

 electron microscopy,

 plasmolysis,

 mechanical rupture of the cell, and

 serological test by exposure to specific antibodies

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 IDENTIFICATION OF CELL WALL
 The dyes in a Gram stain enter the cytoplasm of both
Gram positive and negative cells.
 The iodine forms large crystals with the dye that are too
large to escape through the cell wall.
 Alcohol dissolves the outer membrane of the gram-
negative cells and leaves small holes in the thin
peptidoglycan layer through which the Crystal Violet-
iodine complex leaks out.
 Although gram-positive and gram-negative cells both
absorb fuchsine (safranin), the pink color of fuchsine is
masked by the darker purple dye previously absorbed
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by gram-positive cells.
GRAM POSITIVE GRAM NEGATIVE

Crystal Violet added

Plasma membrane
Peptidoglycan

Outer plasma
membrane

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GRAM POSITIVE GRAM NEGATIVE

Iodine Added

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GRAM POSITIVE GRAM NEGATIVE

Alcohol
Added

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GRAM POSITIVE GRAM NEGATIVE

Fuchsine
(Safranin)
added

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GRAM POSITIVE GRAM NEGATIVE

Final Result

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THE GRAM STAINING PROCEDURE

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PLASMA MEMBRANE
 In Gram negative organisms, the outer plasma membrane
contains lipopolysachharides (LPS), which means it is
made of lipids (fats) and many sugars (polysaccharides).

 The LPS embedded in the plasma membrane of Gram

negative bacteria contains a string called an O antigen.

 Also within the LPS is an endotoxin called Lipid A.

 Alcohol, soaps, and other detergents easily rupture the

plasma membrane 82
THE CELL MEMBRANE HAS FOLLOWING FUNCTIONS:

 It acts as a semipermeable membrane regulating

the inflow and outflow of metabolites to and from
the protoplasm.
 It helps in electron transport and oxidative
phosphorylation.

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CYTOPLASM
 Cytoplasm contains all the biosynthetic components
required by a bacterium for growth and cell division,
together with genetic material.
 The cytoplasm consists of

 ribosomes,

 mesosomes,

 intracytoplasmic inclusions bodies.

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 RIBOSOMES:
 Ribosomes look like small particles at low magnification in
electron micrographs. They are smaller than their eukaryotic
counterpart with sedimentation of 70S, compared with 80S in
eukaryotes. They consist of two subunits of 30S and 50S,
giving a net 70S. Ribosomes are important because:
 They serve as the sites of protein synthesis;

 They are also the sites of actions of several antibiotics, such

as amino glycosides, macrolides, and tetracyclines.

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RIBOSOMES

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 MESOSOMES:
 These are vesicular convoluted or multilaminated
structures formed as invagination of the plasma
membrane into the cytoplasm.
 Mesosomes are of two types—septal and lateral. The
septal mesosome attached to the bacterial DNA is
believed to coordinate nuclear and cytoplasmic divisions
during binary fission. The function of lateral mesosomes
still remains to be known. Mesosomes are analogous to
the mitochondria of eukaryotes and are the principal sites
of respiratory enzymes in bacteria.

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INTRACYTOPLASMIC INCLUSION BODIES
 are present in the protoplasm
of bacteria. Their main
function is believed to be of
storage.
 They may be of two types:

 (i) organic inclusion bodies,

which usually contain either
glycogen or
polyhydroxybutyrate, and
 (ii) inorganic inclusion bodies, which may be of
polyphosphate granules or sulfur granules. Examples of
intracytoplasmic inclusion bodies include metachromatic
granules or volutin granules, starch inclusions, and lipid
inclusions. Volutin granules, typically present in C. 88

diphtheriae, can be demonstrated by Albert’s stain.

 NUCLEUS
 The bacterial nucleus is neither enclosed in a nuclear
membrane nor associated with any nucleolus. It is haploid and
replicates by simple fission. The nucleus of the bacteria
consists of a single circle of double-stranded deoxyribonucleic
acid (DNA)
 The chromosome is located in an irregularly shaped region
called nucleoid
 The nucleoid is visible through the light microscope after
staining
 Many bacteria also possess smaller circles of
extrachromosomal DNA called plasmids. The plasmids are
double-stranded DNA molecules, usually circular, that can
exist and replicate independently. Plasmids are not required for
host growth and reproduction, although they may carry genes
89
that confer the bacterium with properties such as antibiotics
resistance or the capacity to produce toxins or enzymes.
ENDOSPORES

 Specialized resting cells formed by gram-positive rod

shaped bacteria (Bacillus) when essential nutrients are
depleted

 An example is Clostridium, which causes diseases such

as gangrene, tetanus, botulism, and food poisoning.

 Another example is Bacillus, some species of which

cause anthrax and food poisoning.
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ENDOSPORES
 Only bacteria make endospores.
 They are highly durable, dehydrated cells with thick
walls.
 They are formed inside the cell membrane
 When released into the environment, they can survive
extreme heat, lack of water, and exposure to toxic
chemicals and radiation.
 The vegetative (parent) cell forms one endospore
because a key nutrient becomes unavailable.

91
The spore shows following
structures

Core: The core contains a complete nucleus (chromosome), all of the

components of the protein-synthesizing apparatus, and an energy-generating
system based on glycolysis.
Spore wall: This is the innermost layer surrounding the inner spore membrane. It
contains normal peptidoglycan and becomes the cell wall of the germinating
vegetative cell.
Cortex: It is the thickest layer of the spore envelope containing unusual
peptidoglycan. It is extremely sensitive to lysozyme, and its autolysis plays a role
in spore germination.
92
Protein coat: It is composed of a keratin-like protein containing many
intramolecular disulfide bonds; this layer confers relative resistance to
antibacterial chemical agents
GERMINATION
 The endospore returns to its vegetative state.
 This is triggered by a change in the environment.
 Water enters into the endospore, and metabolism
resumes.
 Only one cell comes from one endospore, therefore
sporulation is not reproduction.
 They are resistant to heating, freezing, desiccation
(drying), use of chemicals, and radiation.
 Endospores can survive in boiling water for several
hours or more. 93
TYPES OF ENDOSPORES
 Terminal
endospore

 Sub-terminal
endospore

 Central
endospore

94
STAGES OF BACTERIAL SPORE FORMATION.

95
IDENTIFICATION OF ENDOSPORES

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 IDENTIFICATION OF ENDOSPORES

Spore stain of (a) Bacillus (b) Clostridium

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GLYCOCALYX
 Outside cell wall
 Usually sticky

 Extracellular
polysaccharide allows
cell to attach

GLYCOCALYX

CAPSULE SLIME LAYER

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 CAPSULE
 Non-slimy protein (made of polypeptides) or sugars
(polysaccharides) covering the bacterium.
 It is neatly organized.
 Not every bacterium has a capsule.
 The capsule itself is an antigen, called the K antigen.
 The capsule has various functions:
1. It contributes to invasiveness of bacteria by protecting
the bacteria from phagocytosis.
2. It facilitates adherence of bacteria to surfaces. It plays a
role in the formation of biofilms.
3. The glycocalyx layer of the capsule may also play a role
99

in resistance to desiccation
 CAPSULE
Capsule is demonstrable by a light
microscope.

When this gel-like layer is narrower,

detectable only by indirect
serological methods or by
electron microscope but not by
light
microscope, it is called a
microcapsule

The capsule can be demonstrated by light microscopy in either living

or stained bacteria : Negative staining with India ink:
It is carried out by mixing a suspension of bacteria with an equal
volume of Indian ink on a slide, covering with a cover slip, and
then examining it under microscope. The capsule appears as a
clear zone around the cell. This method is useful for improving 100
visualization of encapsulated bacteria in clinical samples, such
as blood or cerebrospinal fluid.
IDENTIFICATION OF CAPSULE

101
ANTHONY CAPSULE STAIN OF KLEBSIELLA
PNEUMONIAE.

102
SLIME LAYER

 Slimy protein covering the entire bacterium.

 Not neatly organized.

 Not every bacterium has a slime layer.

 Function is to attach to some structure in the

host.

 Example is the bacteria in the mouth.

103
 FLAGELLUM

 Whip-like tail used for motility.

 Can observe motility in live
cells, but can’t see the flagella.
 Requires a special stain, and
that kills the bacterium.
 Made of a protein called
flagellin.
 Structure:
 Filament
 Hook
 Turning disks within a basil
body. 104
FLAGELLUM
 ATP is needed to turn the disk, which turns the
flagella.
 Bacteria flagella contain a protein called an H
antigen (Flagellar antigen).
 There is a particular strain of E. coli called
O157.H7
 The letter “O” followed by a number indicates the
type of cell wall lipopolysaccharide (LPS) and the
H7 indicates the type of flagellar antigen.

105
 FLAGELLA ARRANGEMENT

Monotrichous (single polar flagellum), e.g., Vibrio cholerae.

Lophotrichous (multiple polar flagella), e.g., Spirilla.
Peritrichous (flagella distributed over the entire cell), e.g.,Salmonella
Typhi, E. coli, etc. 106

Amphitrichous (single flagellum at both the ends), e.g., Spirillum minus

AXIAL FILAMENTS

 Special flagella found only in

spirochetes

 Allows the spirochete to move

in a motion like a corkscrew.

 This allows it to penetrate

tissue.

 Example of a spirochete is the

bacterium that causes syphilis.
107
FUNCTION OF FLAGELLA
 They are primarily responsible for motility of bacteria by
chemotaxis.

 They may play a role in bacterial survival and

pathogenesis.

108
DEMONSTRATION OF FLAGELLA
 The flagella can be demonstrated by direct and indirect
methods.

 The direct methods include direct demonstration of

electron microscope.

 Indirect methods of demonstration of flagella include

demonstration of motility of the bacteria by

 (a) dark-ground microscopy,

 (b) hanging drop method,

 (c) observing spreading type growth on semisolid 109

media, such as mannitol motility medium.
 IDENTIFICATION OF MOTILITY

The hanging drop slide

110

dark-ground microscopy
FIMBRAE (PILI)
 The pili are shorter and straighter
than flagella, although the basic
structure is same
 Hair-like structures made of
helics of protein called pilins.
 In Eukaryotes, they are called
cilia.

Demonstration of pili: The pili can be detected:

 Directly by electron microscope and
 By agglutination of RBCs
111
FIMBRAE (PILI)
 Sex pili: A specialized kind of pili called sex pili is responsible for
the attachment of donor and recipient cells in bacterial conjugation.
These pili are longer (10–20 mkm) and vary 1–4 in number. The
sex pili are of two types:
1. F pili: They specifically adsorb male specific RNA and DNA
bacteriophages. They are encoded by sex factor F and fertility
inhibition–positive resistance factors(fi + R factor ).
2. I pili: They adsorb male specific filamentous DNA phages, encoded
by col factor and fi - R factor.

112
FUNCTION OF PILI
 play a major role in the adherence of symbiotic
and pathogenic bacteria to host cells, which is a
necessary step in initiation of infection.
 Transfer of bacterial DNA takes place through
sex pili during the process of conjugation.

113