spore staining+ capsule

7/31/2019 Spore Staining+ Capsule

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Spore Staining

&Capsule Staining


2/19

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.


4/19

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.


5/19

Bacillus anthracis


6/19

Clostridium perfringens


7/19

Clostridium tetani


8/19

Spore staining


9/19

CLINICAL SIGNIFICANCE:

They can live for many years

They can resist antibodies


10/19

Sporogenesis


11/19

Capsule Staining


12/19

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


14/19

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.


15/19

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.


16/19

Capsule staining


17/19

Capsule staining


18/19

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


19/19

RESULT

Bacterial cell .Dark purple

Capsule outline .Pale blue

spore staining+ capsule

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