Technique CAPSULE STAIN India Ink Method
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Principle The capsule or glycocalyx is a gelatinous outer layer that is secreted by the microbe and remains stuck to it. Capsules may be polysaccharide, glycoproteins or polypeptides depedning on the organism

The large particles of the India ink are unable to penetration into the capsule
Cautions Any stray material in the preparation that is not stained is frequently mistakenly identified as a capsule

1: The film should be of the same thickness as the capsulate organisms.
2: If the coverslip is not pressed down sufficiently, the organisms will drift in the ink and may be obscured by overlying ink.
3: If the coverslip is pressed down too much, the capsules may be distorted.
Method 1: Place a large loopful of undiluted India ink on a slide
2: Mix into this a small portion of the bacterial colony
or
Mix a small loopful of the deposit from a centrifuged liquid culture.
3: Place a coverslip on top of the mixture.
4: Press down under a pad of blotting paper.
Results The capsule appears as a clear zone between the refractile cell outline and the dark background.
Positive control Klebsiella pneumoniae (ATCC e13883)
Negative control Alacilgenes denitrificans (ATCC 15173)
Reagents Undiluted India ink Not all India Inks are suitable. Add 0.3% tricresol as a preservative
Reference J.P. Duguid 1951 J. Path Bact 63: 673

 

 

Technique CAPSULE STAIN Antony's Method
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Principle The capsule or glycocalyx is a gelatinous outer layer that is secreted by the microbe and remains stuck to it. Capsules may be polysaccharide, glycoproteins or polypeptides depedning on the organism

Both the cell and the capsule become stained by the crystal violet. However the crystal violet does not remain bound to the nonionic capsule and is easily dislodged with the copper sulfate solution, which also acts as a counterstain
Cautions DO NOT HEAT FIX because there will be shrinkage
Vigorous washing may dislodge the cells.
Method 1: Add a loopful of a culture grown in either skimmed milk or litmus milk to a microscope slide
2: spread the culture over the slide using the edge of a second microscope slide
3: Air dry
4: stain with 1% crystal violet for 2 minute
5: Wash with a solution of 20% copper sulfate
6: Blot dry
7: Examine under oil immersion
Results The cells are stained a deep blue or purple. The capsules are stained a light blue. The background make appear colourless or light violt depending on the degree of destaining
Positive control Klebsiella pneumoniae (ATCC e13883)
Negative control Alacilgenes denitrificans (ATCC 15173)
Reagents Crystal Violet 1gm/ 100ml distilled water
Copper Sulfate (CuSO4.5H20) 20gm/100 ml of distilled water
Reference Anthony,E.E. 1931 A note on capsule staining. Science 73:319

 

Technique CAPSULE STAIN Hiss's Method
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Principle The capsule is a gelatinous outer layer that is secreted by the microbe and reamins stuck to it. Capsules maybe polysaccharide, glycoproteins or polypeptides depending on the organism

Both the cell and the capsule become stained by the crystal violet. However the crystal violet does not remain bound to the nonionic capsule and is easily dislodged with the copper sulfate solution, which also acts as a counterstain
Cautions Vigorous washing may dislodge the cells.

This method uses a dilute 0.1% crystal violet compared to the 1% crystal violet of Anthony's method
Method 1: Add a loopful of a culture grown in either skimmed milk or litmus milk to a microscope slide
2: spread the culture over the slide using the edge of a second microscope slide
3: Air dry
4: Heat FIX
5: stain with 0.1% crystal violet
6: Gently heat until steaiming (about 1 minute)
7: Wash with a solution of 20% copper sulfate
8: Blot Dry
9: Examine under oil immersion
Results The cells are stained a deep violet. The capsules are unstained against a purple background
Positive control Klebsiella pneumoniae (ATCC e13883)
Negative control Alacilgenes denitrificans (ATCC 15173)
Reagents Crystal Violet 0.1gm/ 100ml distilled water
Reference Hiss, P.J. Jr 1905 A contribution the the physiological differentiation of Pneumococcus and Streptococcus and to methods of staining capsules. J. Exptl Med 6: 317-345