Method is used for Leptospira and Treponema staining.
Ruge solution: acetic acid (glacial) 1 ml, formalin (neutral) 2 ml, distilled water 100 ml,
Mordant solution: tannic acid 5 g, phenol 1 g, distilled water 100 ml,
Fontana solution (ammoniacal silver nitrate solution): add NH3 solution 10%, drop by
drop to AgNO3 solution 0.5% until the precipitate is solved.
Dry then fix the smear with Ruge solution. Wait 5 minutes then repeat fixation 2-3
Wash the slide with alcohol then add mordant solution. Heat it to steam (avoid
boiling). Wash in running tap water.
Add Fontana solution then steam the slide for 20-30 second. The slide should get a
metallic-brownish aspect. Staining may be repeated 2-3 times.
Remove solution, wash and dry the slide.
Leptospira and Treponema are brown-black colored and the background is yellow.
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.
Ammoniacal silver nitrate solution preparation: when the silver nitrate is solved, add ammonium hydroxide drop by drop. A brownish
precipitate will be formed . Continue to add ammonium hydroxide slowly until the precipitate disappears and a faint milky appearance
Solution is active only for few hours.
(c) Costin Stoica