Spore Staining & Capsule Staining

OBJECT: To identify the organism in a given sample

REQUIREMENTS: Sample, glass slide, wire loop, burner, malachite green, safranine, distilled water and cedar wood oil, and a microscope. THEORY: When conditions for vegetative growth are not favorable, especially when carbon and nitrogen become unavailable, bacteria of the genera Bacillus and Clostridium are able to survive by forming resistant endospores.

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), or centrally. It may be round, oval, or elongate Endospores being dense and thick-walled, are able to withstand dehydration, heat, cold, and the action of disinfectants. A spore is unable to multiply but when conditions for vegetative growth return, it is able to produce a bacterial cell which is capable of reproducing.

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.

Bacillus anthracis

Clostridium perfringens

Clostridium tetani

Spore staining

CLINICAL SIGNIFICANCE:
They can live for many years They can resist antibodies

Sporogenesis

Capsule Staining

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 Dglutamic acid is present

Example
B.anthracis---------- D-glutamic acid Entrobacteric aerogens --- polysaccharide Strept pneumoniae --------- polysaccharide Staph. pyogens --- hyaluronic acid, N-acetyl glucosamine

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.

Cont..
Wash off the stain with tap water then cover the 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 immersion objective to look for capsulated bacteria.

Capsule staining

Capsule staining

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

RESULT
Bacterial cell .Dark purple Capsule outline .Pale blue