Modification of Warthin-Starry Silver Technique for the Demonstration of Bacillus Piliformis


By: Robert Dionne, Histotech

Veteran’s Affaire Medical Center

White River Junction, VT 05009


NovaUltra Special Stain Kits


This protocol is the modification of Warthin-Starry Silver Technique of Frank Razzaboni’s procedure (Histo-Logic Vol. XXI, NO.1, Jan./Feb. 1991, Page 214). Frank Razzaboni's protocol was easy and simple, with good results but we always ended up with smudges and shiny spots on the slides. We were using ProbeOn Plus Microscope Slides. One day, I prepared the Developer too early and it became black before using it. The next time, I prepared the Developer during the second rinse in Walpole’s Buffer and the result was much better. I also tried this staining procedure on a new slide: S/P Brand Superfrost* Plus Microscope Slides, the results were great without any smudges on the slides. 


Here is the procedure I follow now with great results.


Staining Procedure


1. Deparaffinize and hydrate to deionized water.

2. Rinse in Walpole’s Buffer.

3. Place slides in a plastic coplin jar, cover with 1% Silver Nitrate, and microwave for

   1.5 minutes at a medium setting. Let slides stand for 5 minutes in this solution.

4. Rinse in Walpole’s Buffer. During this time, prepare the Developer.

5. Flood slides with Developer that has been freshly prepared, 3 times for 1 minute each,

    drain the slides between each flood.            

6. Wash in warm tap water.

7. Dehydrate in two changes of 95% ethanol, followed by two changes of absolute ethanol.

8. Clear in xylene.

9. Mounting.




Bacillus Piliformis stains black and the background is light brown to golden brown.




Image 1: Use original method showing smudges Image 2: Use modified method without smudges






Frank Razzaboni (1991). A Warthin-Starry Silver Technique for the Demonstration of Bacillus Piliformis. HistoLogic. Vol. XXI, No. 1, Jan./Feb. 1991 page 214.