[Histonet] Steiner and Steiner method/H. pylori

Steven E. Slap siksik03 <@t> comcast.net
Wed Dec 10 11:24:52 CST 2003

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
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
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