MODIFIED WARTHIN-STARRY METHOD (pH 4.

0)
FOR SPIROCHETES AND OTHER MICROORGANISMS

FIXATION: 10% buffered neutral formalin.

SECTIONS: Paraffin, 6 micrometers.

SOLUTIONS
1% CITRIC ACID SOLUTION
Citric acid 1 .0 gm

Distilled water 100.0 ml

ACIDULATED WATER SOLUTION

Sterile water 1 000.0 ml

Add enough 1% citric acid solution to reach a pH 4.0.

1% SILVER IMPREGNATING SOLUTION

Silver nitrate 1 .0 gm

Acidulated water 100.0 ml

2% SILVER NITRATE SOLUTION 4

Silver nitrate 2.0 gm
Acidulated water 100.0 ml
Place flask in a flotation bath set at 54°C.

5% GELATIN SOLUTION

Gelatin 5.0 gm
Acidulated water 1 00.0 ml

Place flask in a flotation bath set at 54°C.

0.15% PYROCATECHOL SOLUTION

Pyrocatechol 0.15 gm
Acidulated water 100.0 ml
Place flask in a flotation bath set at 54°C.

DEVELOPING SOLUTION
Silver nitrate solution, 2% 1 .5 ml

Gelatin solution, 5% 3.75 ml

Pyrocatechol solution, 0.15% 2.0 ml

Just before use combine in a small beaker in the order given. Mix the silver
nitrate solution and the gelatin solution thoroughly before adding the
pyrocatechol solution.

214 AFIP Laboratory Methods in Histotechnology

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Impregnate in 1% silver nitrate solution at 43°C for 30 minutes.
3. Place slides on staining rack using glass rods. Flood with the developing
solution. Allow color to develop to yellow brown.
4. Wash quickly and thoroughly in hot tap water to stop reaction.
5. Dehydrate and clear through 95% ethyl alcohol, absolute ethyl alcohol, and
xylene, 2 changes each, 2 minutes each.
6. Mount with resinous medium.

RESULTS
Background tan to yellow
Spirochetes and other microorganisms black

REFERENCES
Allen TC, Luna LG, Wear DJ. Modified Warthin-Starry technique. AFIP Letter.
1986;144:3.

Kerr DA. Improved Warthin-Starry method of staining spirochetes in tissue sections.

Am JClin Pathol. 1938;8:63.

❖ Bacteria, Fungi, and Other Microorganisms 215