1.
Dr. KRITHIKAA SEKAR, MD
Assistant Professor, Microbiology,
SLMCH

2.
 DEMONSTRATION
 SIZE
 SHAPE & ARRANGEMENT
 ANATOMICAL STRUCTURES
- CELL WALL
- CELL MEMBRANE
- CYTOPLASM
- SLIME LAYER & CAPSULE
 APPENDAGES- FLAGELLA & FIMBRIAE
 SPORES

3.
 Unstained Preparation- hanging drop
(motility), dark ground microscopy
(Spirochates)
 Simple staining (single stain)- crystal violet,
gentian violet, carbol fuschin, saffranin
 Negative staining- bacterial capsules (India
Ink)
 Impregnation Techniques- flagella (silver
impregnation)
 Differential staining – Grams stain, Acid
fast stain, Albert’s Stain

4.
 COCCI- spherical or oval cells- eg. Staphylococcus
 BACILLI- rod shaped- eg. E.coli, Brucella,
 COCCOBACILLI-length is same as width, eg.Klebsiella
 SPIROCHAETES- slender flexuous spiral forms. Eg.
Treponema
 VIBRIOS- comma shaped
 SPIRILLA- rigid spiral forms
 ACTINOMYCETES- branching filamentous bacteria
resembling fungi
 MYCOPLASMA- round or oval bodies & interlacing
filaments (cell wall deficient)
 RICKETTSIAE and CHLAMYDIAE- small obligate
parasites classified as viruses, but included in bacteria
due to presence of cell wall, bacterial enzymes and
structural similarities with bacteria

5.
 STREPTO- chains
 DIPLO- pairs
 STAPHYLO- clusters
 TETRADS- groups of four eg. Micrococci
 SARCINA- groups of eight
 CUNEIFORM- chinese letter pattern eg.
Corynebacterium

6.
 Tough & rigid structure
 Made of peptidoglycan –
mucopeptide(murein) composed of N-
acetyl muramic acid & N- acetyl
glucosamine alternating in chains cross
linked by peptide subunits

7.
 tightly cross linked peptides- d-alanine & d-
glutamic acid
 Thickness- 18-80 nm & constitutes 40-80% of
the dry weight
 Techoic acid- water soluble polymers of
glycerol phosphate or ribitol phosphate
residues
 two types – wall techoic acid-ribitol
&membrane techoic acid- glycerol
functions- cell wall stability, association of
wall with membrane, adherence, reproduction

8.
 Polysaccharides- mannose, arabinose ,
rhamnose, glucuronic acid & mannuronic
acid
 Thick peptidoglycan layer
 S LAYER- protein or glycoprotein
molecules that self assemble on the outer
surface of the organism.
 Protect from stressful environments,
inhibit phagocytosis, contribute to
virulence

9.
 Thickness- 3-4 nm
 Loosely crosslinked by d- diaminipimelic acid
or lysine
 Outer membrane- bilayered LPS containing
OMPs – porins, OmpC, D, F, PhoE, LamB,Tsx
contains 3 regions- I- polysaccharide
determining O ag specificity
II- core polysaccharide containing 3-deoxy-
D- mannulooctulusonate(KDO) & heptose
III- glucolipid responsible for endotoxicity

10.
 Lipoprotein layer- connects peptidoglycan
to outer membrane & stabilizes the outer
membrane
• Periplasmic space- space between inner&
outer membranes containing the
peptidoglycan layer and gel like solution
of proteins & membrane derived
oligosaccharides
• Thin peptidoglycan layer

11.
 Accounts for the shape of the cell
 Protects the cell against osmotic damage
 Confers rigidity
 Cell division
 Target site for antibiotics, lysozymes,
bacteriophages
 Carries bacterial antigens

12.
 Plasmolysis
 Microdissection
 Reaction with specific antibody
 Electron microscopy
 Indirect methods- Grams staining & Acid fast
staining, fluorescent staining for acid fast
bacteria

13.
 MYCOPLASMA- stable oval or round forms
 L- FORMS- observed in streptobacillus
monoliformis. Induced by penicillin
 PROTOPLASTS- gram positive bacteria when
placed in hypertonic saline
 SPHEROPLASTS- gram negative bacteria
when subjected to penicillin. Some cell wall
material is retained
 PLEIOMORPHIC & INVOLUTION FORMS-
swollen & aberrant forms resulting from
ageing

14.
 5-10nm thick elastic membrane beneath the cell wall
separating it from cytoplasm
 Composed of lipoprotein. Sterols absent except in
mycoplasma
 Permeases- membrane associated carrier proteins
 FUNCTIONS- selective permiability and transport of
solutes
electron transport and oxidative phosphorylation
excretion
bearing the enzymes and carrier molecules for
biosynthesis

15.
 Colloid of organic and inorganic solutes in
viscous watery solution
 RIBOSOMES- centres of protein synmthesis.
Composed of rRNA of size 10 - 20nm with a
sedimentation constant of 70S
 MESOSOMES- vesicular, convoluted
invaginations from plasma membrane.
more prominent in GPB
principal site of respiratory enzyme
site of synthesis of cross wall septa during
binary fission

16.
 VOLUTIN- ( BABES ERNST GRANULES)
highly refractile, strongly basophilic bodies
consisting of polymetaphosphate.
stained by Albert or Neisser stain
present in diphtheria bacilli
reserve of energy
 POLYSACCHARIDE- stained with iodine
 LIPIDS- stained with sudan black
 VACUOLES- fluid filled cavities covered by
a membrane

17.
 Contains the cell’s genome made of a single
molecule of double stranded DNA arranged in
the form of a circle . Measure about 1mm

18.
 Extranuclear genetic material
 Transmitted to daughter cells either by
binary fission or from one bacterium to
another by conjugation or bacteriophage
 Confer properties like toxigenicity and
drug resistance

19.
 - acid or ribonuclease hydrolysis and
subsequent staining of nuclear material
by Feulgen stain specific for DNA
appear as oval or elongated bodies
 Binary fission and conjugation

20.
 Amorphous viscid bacterial secretion
surrounding the cell wall
Loose undemarcated secretion – slime layer
or glycocalyx
Sharply defined structure- capsule

21.
 Homo or hetero Polysaccharides made of
hexose and pentose sugars plus ribitol,
glycerol and other sugar alchohols
synthesised by the cell membrane with
enzymes- glucosyl and fructosyl
transferases producing an insoluble
glucan matrix
 Anthrax bacilli- polypeptide

22.
leuconostoc & klebsiella– slime layer
pneumococcus- capsule
meningococcus- microcapsule
streptococcus salivarious- capsule & slime
layer

23.
enhances virulence
protective covering
increases invasiveness
adhesion
capsular antigen for identification of bacteria

24.
 Negative staining- india ink or nigrosin-
capsule appears as a clear halo around
the cell
 Positive staining- B.anthracis-
polychrome methylene blue- McFadyean
capsule stain

25.
 Manevals method- background- congo
red
stain- Manevals solution
capsule- unstained halo
MACROSCOPY- encapsulated- smooth
colonies
unencapsulated- rough colonies

26.
Serological methods- Quellung test
(Neufeld reaction)- loopful of
pneumococci + antiserum(
antipneumococcal rabit sera)
observe in oil immersion under phase
contrast microscope
capsules become refractile & visible ,
seperated from the coccal bodies by the
width of the capsule

27.
 Long, filamentous appendages arising at the
cytoplasmic membrane, protruding through
the cell wall into the surrounding medium

28.
monotrichous- single polar flagella- Vibrio
amphitrichous- single flagellum at both the
poles- A.fecalis
lophotrichous- tuft of flagella at one or both the
ends- Spirilla
peritrichous- flagella all around the cell- S.typhi

29.
 V.cholerae- darting
 Listeria- Tumbling
 Clostridium- Stately
 Mycoplasma- Gliding

30.
 SIZE- 5-20µm long, width-13-17nm
 PARTS- FILAMENT- made of flagellin
semirigid, forms a left handed helix and exits
the cell
HOOK- Acts as a sleeve from which the
filament emerges
transmits rotatory motion from basal body to
filament
BASAL BODY- consists of M,S,P,L rings
connected by a rod shaped structure
in gram positive bacteria only 2 rings are seen

31.
 PHASE VARIATION- 2 types of flagella
due to expression of genes coding for 2
different flagellin proteins in the same
bacteria
 flagellar antigen- H-antigen
 Endoflagellum- arises from one pole,
wraps around the cell body interior to
the cell. Eg:- vibro, spirochaetes

32.
 flagellar antigen H used for identification
 MOTILITY- impart spinning movement
driven by the flow of protons into the cell
down the gradient produced by the primary
proton pump
 CHEMOTAXIS, AEROTAXIS, PHOTOTAXIS,
ELECTRON ACCEPTOR TAXIS- movement
of the cell towards the source of attracant by
swimming, tumbling and reorienting itself to
the attractant

33.
 DIRECT METHOD- dark field microscopy &
electron microscopy
 INDIRECT METHODS- Swarming growth of
proteus
craiges tube method- spreading of bacteria on
semi solid agar
hanging drop preparation-motilty of the
bacterium examined on a wet film under high
power
mannitol motility medium- fanning
wet mount preparation

34.
 Hair like appendages protruding from the cell
as straight filaments
 Found in many gram positive and some gram
negative bacteria
 SIZE- 0.1-1.0µm length, 10nm thick. Each
cell possesses 100-500 fimbriae

35.
 ARRANGEMENT- peritrichous &
helically arranged
 Possess antigenic property
 Composed of a protein fimbrillin(pilin)
which form hollow tubes in the cell
membrane
 ADHESINS- minor proteins on the tips of pili
responsible for attachment.

36.
 Common pili- six types- I- responsible
for adhesion and are mannose
sensitive
type II- mannose resistant
 Sex or fertility pili- long pili present in
male bacteria of size 18-20 nm & are 1-
4 in number. Helps in forming
conjugation tubes for transferring
genetic material to female cells

37.
 TWITCHING- motility established by
pili. Bacterium moves in the direction of
the adhering tip resulting in surface
motility. Seen because the pili donot
rotate & lack a basal body

38.
adhesion
antigenic property
inhibiting phagocytosis
transfer of genetic material

39.
 Electron microscopy
 Haemagglutination- tile test- drop of dense
bacillary deposit + red cell suspension on a
white tile at 3-5ºC .
develops coarse clumping within a few
seconds
mannose 0.5% inhibits type I fimbrial
haemagglutination
RBC’s of guinea pigs, fowl, horses & pigs
agglutinate strongly, sheep and human blood
weakly and ox blood scarcely

40.
 Spherical or oval structures formed within the
bacterial cell
 Represents the resting or dormant phase
formed under unfavourable conditions related
to depletion of exogenous nutrients
 Also called as endospores
 In sporulation each vegetative cell forms only
one spore and during subsequent germination
each spore gives rise to only one vegetative
bacterium
 Bacillus and clostridia species form spores

41.
 CORE- it’s the spore protoplast. Contains nucleus,
protein synthesizing apparatus, energy generating
system based on glycolysis.
Vegetative cell enzymes are increased in amount
Contains large amounts of calcium dipicolinate
responsible for resistance
 SPORE WALL- innermost layer surrounding the
inner spore membrane. Made of peptidoglycan and
forms cell wall
 CORTEX- Thickest layer made of peptidoglycan
sensitive to lysozyme. Role in spore germination

42.
 COAT- keratin like protein containing
many intermolecular disulphide bonds.
Impermeable and provides resistance to
antibacterial agents
 EXOSPORIUM- composed of lipids,
proteins and carbohydrates. Consists of
paracrystalline basal layer and hair like
outer region

43.
 Process by which spores are formed. Involves
production of many new structures , enzymes
and metabolites along with disappearance of
many vegetative cell components -
differentiation
 Spore composition determining genes are
activated by association of RNA polymerase
core protein with sigma factor
 Sporulation process takes about 7hrs under
laboratory conditions

44.
 ACTIVATION- spore coat gets damaged
 INITIATION- triggered by L-alanine or
adenosine. Autolysin is secreted that degrades
the cortex peptidoglycan. Water is taken up
releasing calcium dipicolinate and degrades
various spore components by hydrolytic
enzymes
 OUTGROWTH- degradation of cortex and
outer layers results in emergence of new
vegetative cell

45.
NON BULGING- diameter of the spore is
same as or less than the width of bacteria
BULGING- diameter is wider than the
bacillary body
POSITION- central
subterminal
terminal

46.
 GRAMS STAIN- spore appears as clear
unstained ares within the cell
 ZIEHL NEELSON METHOD- 0.25%
sulphuric acid is used. Stain red &bacilli
blue
 MALACHITE GREEN STAIN-5%
aqueous solution of malachite green-
1min
saffranin or basic fuschin – 30sec
spores- stain green& bacilli red

47.
 Indicator for sterilization-
G.stearothermophilus is destroyed by
autoclaving, hot air oven
 Ethylene oxide sterilizer- B.atrophaecus

48.
 https://drive.google.com/file/d/1vq9iEoo6o
meomDXatLYrPicFY2cCQwVF/view?usp=sha
ring
 https://drive.google.com/file/d/1SR8FG6Iu
MHuErW5tUffWoI_II-
jMZTBw/view?usp=sharing