Note

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MBBS 10th batch 2nd year

Microbiology Practical
 CA
Bac

CLED
TCBS

MA
Opt
BA
DCA MA

MA NLF
AST
CA
LF
MA
CA
DCA
CA NA
• Selective For E coli
 ZN stain
• Auramine rhodamine stain can also be used
• Discovered by Ehrlich and modified by ziehl –
and neelsen
• Methylene blue or malachit green for counter
stain
• Nocardia , Bacterial spores , Scolex of tenia ,
cryptosporidium , actinomyces , ascospores of
some yeast can be stained
-ve

+ve
TSI
 -ve

+ve

Simmon’s citrate agar

Bio chemical test
• Negative: No or very few bubbles produced.
• Examples: Streptococcus and
Enterococcus spp
 Citrate utilization test
Principle of citrate utilization test:
The basic principle of this test is to detect the
ability of an organism which can utilize citrate
as a sole source of carbon for their
metabolism with resulting alkalinity. The
citrase enzyme hydrolyses the citrate to form
oxaloacetic acid and acetic acid.
 oxidase test
The oxidase test is used to identify bacteria that
produce cytochrome coxidase, an enzyme of
the bacterial electron transport chain.
 Indole Test
Indole Test: Principle, Indole test is used to
determine the ability of an organism to split
amino acid tryptophan to form the
compound indole. Tryptophan is hydrolysed
by tryptophanase to produce three possible
end products – one of which is indole.
 Urease Test
Principle of Urease Test. Urea is the
product of decarboxylation of amino acids.
Hydrolysis of urea produces ammonia and
CO2.
The formation of ammonia alkalinizes
the medium, and the pH shift is detected by
the color change of phenol red from light
orange at pH 6.8 to magenta (pink) at pH
8.1.
 Triple sugar iron
Principle: Triple sugar iron agar test is used to
determine whether gram negative bacilli utilize
glucose and lactose or sucrose fermentatively
and produce hydrogen sulfide (H2S). It
contains 10 parts of lactose: 10 parts of
sucrose: 1 part of glucose and peptone
Proteus
E.coli
E.coli
Microfilaria
Giardia cyst
Acid Fast Bacilli
Leishmania spp.
E.Histolytica Cysts
Malaria Kit
HIV TRI-DOT test
Entamoeba histolytica cyst
• Shape : Spherical/Oval
• Size: 10-15µm
• Nucleus:
o Early cyst contains single
nucleus and mass of
glycogen and 1-4 cigar-
shaped refractile rods
known as chromatid
body.
o As it matures, the
glycogen mass and
chromatid bodies
disappear.
o The nucleus undergoes
two succesive mitotic
divisions to form two and
finally 4 nuclei (mature
cyst: Quadrinucleated)
Enterobius vermicularis
• Shape : Planoconvex
• Size: (50-60x30)µm
• Color: Colorless(non-bile
stained)
• Outer Membrane(egg
shell): Transparent
• Inner Membrane(Ovum):
Fully developed; a coiled
tadpole-like larva
• It floats in the saturated
Solution
Taenia spp.
• Shape : Spherical/Oval
• Size: 30-45µm
• Color: Brown(bile stained)
• Outer Membrane: Thin and
transparent
• Inner Membrane:
Embryophore: It is thick,
brown, radially striated,
encloses embryo(oncosphere)
Embryo contains six hooklets
(hexacanth embryo)

• Doesn’t floats in the

saturated Solution
Ancylostoma duodenale
• Shape : Oval or elliptical
• Size: (60x40)µm
• Color: Colorless(non-bile
stained)
• Outer Membrane(egg
shell): Transparent
hyaline
• Inner
Membrane(Ovum):
Segmented ovum
generally with 4
blastomeres

• It floats in the saturated

Solution of common salt
Trichuris trichuria
• Shape : Barrel shaped with
projecting mucous plug at
both pole
• Size: (50x25)µm
• Color: Brown (bile stained)
• Outer Membrane(egg
shell): Transparent hyaline
• Inner Membrane(Ovum):
Unsegmented ovum

• It floats in the saturated

Solution
Ascaris lumbricoid (fertilized)
• Shape : Round and oval
• Size: (60-70x40-50)µm
• Color: Brown (Bile stained)
• Outer Membrane(egg shell):
Thick, smooth, translucent with
an uneven outer albuminous
coat (rugosites)
• Inner Membrane(Ovum):
Large, unsegmented ovum
Clear, cresentic area between
the ovum and eggs-shell at
each pole
• It floats in the saturated
Solution
Ascaris lumbricoid (Unfertilized)
• Shape : Elliptical
• Size: (80x55)µm
• Color: Brownish (Bile
stained)
• Outer Membrane(egg
shell): Thinner, with an
irregular outer albuminous
coat (rugosites)
• Inner Membrane(Ovum):
Small, Atropied ovum with a
mass if disorganised, highly
refractile granules of various
sizes
• It floats in the saturated
Solution
Hymenolepis nana
• Shape : Spherical/Oval
• Size: 30-45µm
• Color: Colorless(Non-bile stained)
• Outer Membrane: Thin and
transparent
• Inner Membrane: Embryophore
encloses embryo containing six
hooklets
Clear space is present between
two membranes filled with yolk
granules, polar filaments(4-8)
arising from the poles of
embryophores

• It floats in the saturated Solution

 Hydatid cyst
• It represents scolex of future adult worm
and remains invaginated within vesicular
body
• Hydatid cyst in humans is typically
unilocular, subspherical in shape and is
filled with fluid
• The cyst wall secreted by embryo has
two layers:
a. Outer cuticular layer (Ectocyst):
Laminated hyaline membrane, appears as
white of hard-boiled eggs, 1mm thick,
elastic, when ruptures or incised curls on
itself.
b. Inner Germinal layer(Endocyst):
It is cellular, nucleus and thin
It is a vital layer of cyst and gives rise to :
• Blood capsules with scolices
• Specific hydatid fluid
• Outer layer
• A number of fastidious organisms, including
some bacteria, yeasts, and other fungi, grow
well on BHI. It can also be used to
differentiate between enterococci and group
D streptococci.
Kirby bauer disc diffusion method

Mueller- hinton agr or nutrient

agar is used
Optochin (or ethylhydrocupreine
hydrochloride)