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

From:"Steven E. Slap"

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

There certainly is a Wrathin Starry microwave method to demonstrate spirochetes, and it does not involve anything radioactive.

Here is Joyce Moore's version, as it was taught to me:

 Warthin Starry Techniques for Spirochetes

Fixation: Formalin

Technique: Sections cut at 4 microns

Control: Tissue containing Spirochetes


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.


Spirochetes - blackBackground - pale yellow to light brown

Adapted for the Jefferson Regional Medical Center Histo-Path Laboratory

best regards,
Steven Slap
At 4:30 PM -0500 12/9/03, Derek and/or Lynda Leopold wrote:
Hi list,
Would anyone care to comment on the Steiner and Steiner (microwave) method for spirochetes?  I susupect my lab's pathologists are interested in it for viewing H. pylori, yet in all the "HP" talk on this list I have yet to hear it mentioned.  How does it compare from a tech safety standpoint?  As a brand-new tech, I still do crazy things like read the safety warnings on ingredients, and that whole uranyl nitrate radioactive thing is sort of bothering me.  We use no protective equipment when doing this stain (which the doctors still call "Warthin Starry", incidentally, but hey---they're making the big bucks....)
I'll take anyone's 2 cents...
Lynda Leopold

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