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.
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.
The bacterial cells are stained dark purple and the capsules are light blue.
Do not fix the slide in a flame.
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
Toluidine solution: toluidine blue 0.5 g, ethanol .10 ml, fenic acid 3 g, distilled water ad 100 ml.
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.
The bacterial cells are stained blue and the capsules are pink.
Methilene 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)
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.
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.
The bacterial cells are stained dark violet and the capsules are pink to violet.
Do not fix the smear by heat.
(c) Costin Stoica