Re: [Histonet] Steiner and Steiner method/H. pylori

Warthin Starry Techniques for Spirochetes

Fixation: Formalin

Technique: Sections cut at 4 microns

Control: Tissue containing Spirochetes

Solutions:

Acidulated WaterAcidulate 1 liter distilled water with 0.1 g citric acid until pH of 3.8-4.4 is reached. A pH of 4.0 is ideal for staining spirochetes. For demonstrating Donovan Bodies of granuloma inguinale, a pH of 3.6 is recommended.

2% Silver Nitrate Solution (developer)Silver nitrate C.P. crystals 0.5 gAcidulated water 25.0 ml

1% Silver Nitrate Solution (impregnation)Silver nitrate C.P. crystals 0.5 gAcidulated water 50.0 ml

0.5% Hydroquinone SolutionHydroquinone crystals, photographic quality 0.35 gAcidulated water 25.0 ml

5% Gelatin SolutionGelatin 1.5 gAcidulated water 25.0 ml

Staining Procedure

   1. Attach slides to automatic stainer, deparaffinize, hydrate to water.
   2. Rinse in distilled water, 2 changes.
   3. Place slides in 1% silver nitrate solution for 45 seconds in microwave. Let stand for 1 minute at room temperature. (Note: alternatively, in a laboratory microwave, heat at 80% power, 60 C for 5 minutes, no standing time required).
   4. Preheat for 45 seconds in microwave in separate flasks: 2% silver nitrate, 5% gelatin, 0.15% hydroquinone. Preheat empty flask in microwave with these.
   5. Mix developer solution in order given: Use warm empty flask:

      2% silver nitrate 1.5 ml5% gelatin 3.75 ml0.15% hydroqinone 2.0 ml
   6. When step 5 is completed, remove slides from silver solution. Do not rinse. Place slides horizontally on a slide rack and cover with developer. Allow sections to develop until they are light yellow to golden brown, approximately 1 minute or less. (Note: developing step can also be carried out in a laboratory microwave at 80% power set for 60 C for 20 seconds).
   7. Rinse thoroughly in 50 ml tap water which is preheated to approximately 56 C in the microwave at 450W for 45 seconds.
   8. Rinse in distilled water.
   9. Attach to automatic stainer, dehydrate and clear. Mount in a xylene soluble mounting medium.

Results:

Spirochetes - blackBackground - pale yellow to light brown

Adapted for the Jefferson Regional Medical Center Histo-Path Laboratory