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7/31/2019 Spore Staining+ Capsule
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Spore Staining
&Capsule Staining
7/31/2019 Spore Staining+ Capsule
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OBJECT: To identify the organism in a
given sample
REQUIREMENTS:
Sample, glass slide, wire loop, burner, malachitegreen, safranine, distilled water and cedar wood
oil, and a microscope.THEORY:
When conditions for vegetative growth are notfavorable, especially when carbon and nitrogenbecome unavailable, bacteria of the generaBacillus and Clostridium are able to survive byforming resistant endospores.
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Cont:
Spore formation involves a change in enzyme
activity and morphology. Spore may be positioned at the end of the
bacterium, near to one end (sub terminal), orcentrally.
It may be round, oval, or elongate
Endospores being dense and thick-walled, areable to withstand dehydration, heat, cold, and
the action of disinfectants. A spore is unable to multiply but when
conditions for vegetative growth return, it isable to produce a bacterial cell which is capable
of reproducing.
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PROCEDURE:
Take a clean, dry slide, with wire loop make a
thin smear.
Fix it on flame after air drying the slide.
Flood the smear with Malachite green & leave
it for 10 min, wash it & pour safranine for
15sec.
Wash it with water & put cedar wood oil &
examine under microscope.
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Bacillus anthracis
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Clostridium perfringens
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Clostridium tetani
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Spore staining
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CLINICAL SIGNIFICANCE:
They can live for many years
They can resist antibodies
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Sporogenesis
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Capsule Staining
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OBJECT: To identify the organism in a
given sample
REQUIRMENTS:
Sample, glass slide, wire loop, burner,Crystal
violet, Copper sulphate
THEORY:
Capsules are chemically polypeptide, poly
saccharide except Bacillus anthracis where D-
glutamic acid is present
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Example
B.anthracis---------- D-glutamic acid
Entrobacteric aerogens --- polysaccharide
Strept pneumoniae --------- polysaccharide Staph. pyogens --- hyaluronic acid, N-acetyl
glucosamine
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PROCEDURE:
Make a smear on a clean glass slide, then dry it
Fix the dried smear using alcohol
Note: When preparing the smear, mix the organism
suspension with a drop of normal serum. This will
help to show the capsules
Cover the smear with crystal violet stain, and
heat gently until the steam just begins to rise.Leave to stain for 1 minute.
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Cont..
Wash off the stain with tap water then coverthe smear with the copper sulphate solution
Wipe the back of the slide clean, and place in
a draining rack for the smear to air- dry Examine the smear microscopically, first with
the 40x objective to see the distribution of
material and then with the 100x oil immersionobjective to look for capsulated bacteria.
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Capsule staining
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Capsule staining
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FUNCTION:
Bacteria resist phagocytosis because of the
presence of slimy material
They help to prevent the digestion of bacteria
by antibacterial enzymes like lysozyme
Capsule helps bacteria to increase the
virulence(adherence)
Capsule renders the organisms antigenetically
specific
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RESULT
Bacterial cell .Dark purple
Capsule outline .Pale blue