Capsule Stainings
REAGENTS
Crystal violet solution: crystal violet saturated alcoholic solution (10 g per 100 ml 95% alcohol)  5ml, water 95 ml.
Copper sulfate 25% solution: copper sulfate crystals CuSO4 * 5H2O  20 g, water 80 ml. Dissolve the copper in water with the aid of
heat and filter.
PROCEDURE
Dry preparation then cover it with formalin. Let stand for few seconds then drain and
blot dry.
Apply 0.5% crystal violet solution  and heat until steaming. Remove the stain by
washing with 25% copper sulfate solution. Blot dry.

RESULTS
The bacterial cells are stained dark purple and the capsules are light blue.

NOTES
Do not fix the slide in a flame.
REFERENCES
1. H. Raducanescu, V. Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. Augustus B. Wadsworth: Standard Methods of the Division of Laboratories and Research of the New York State Department of Health.
1947, The Williams & Wilkins Company.
Hiss's stain (modified by Wadsworth)
Toluidine blue stain
REAGENTS
Toluidine solution: toluidine blue 0.5 g, ethanol .10 ml, fenic acid 3 g, distilled water ad 100 ml.
PROCEDURE
Dry and heat-fix the smear then apply toluidine blue solution. Let stand for 1-2 minutes. Remove the stain, wash and dry the slide.

RESULTS
The bacterial cells are stained blue and the capsules are pink.
Methylene blue stain
Using old, oxidized,  Loffler methylene blue solution (stored over few months)
capsule will be pink-colored and the rest of the cell will be blue.
Giemsa stain (Gustav Giemsa)
REAGENTS
Giemsa solution: glycerin 125 g, methanol 375 ml, methylene azure 0.8 g and eosin 3
g. Better prepare it from commercially available Giemsa powder.
PROCEDURE
Dry the smear then apply Giemsa solution, counting the drops. Add equal quantity (drops) of distilled water on the slide. Let stand for 3-5
minutes. Remove the stain, wash and dry.

RESULTS
The bacterial cells are stained dark violet and the capsules are pink to violet.
NOTES
Do not fix the smear by heat.
(c) Costin Stoica
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