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Electron Microscopy Sciences

Technical Data Sheets

Modified Steiner Using Chapman's Modification

EMS Catalog #26119

Fixation

10% Buffered Neutral Formalin

Section

4-6 Microns

Staining Procedure

  1. Deparaffinize and re-hydrate tissues to distilled water.
  2. Sensitize sections in Zinc Formalin at 60°C in a water bath for 15 minutes (cover jar loosely). Preheat this solution.
  3. Rinse in distilled water, 3 times for 2 minutes each.
  4. Place in Silver Nitrate, 1%, in a Coplin Jar. Place in microwave (800 watts); start and run for 80 seconds. Remove from microwave and allow to stand in hot silver solution for 20 minutes (cover jar loosely).
  5. Rinse in distilled water 3 times for 2 minutes each.
  6. Dehydrate in 2 changes of 95% ethanol followed by 2 changes of 100% ethanol.
  7. Treat with Gum Mastic, 2.5%, for 5 minutes.
  8. Allow to air dry for 1 minute.
  9. Rinse in distilled water (2 changes). Slides may stand in distilled water while reducing solution is being made, or longer if necessary.
  10. To prepare reducing solution, mix just before use:

    a. Gum Mastic, 2.5% 10.0 ml
    b. Hydroquinone, 2% 25.0 ml
    c. Ethanol, 100% 5.0 ml

    Filter and then add:
    d. Silver Nitrate, 0.04% 2.5 ml

    DO NOT filter this silver!
  11. Reducing solution at 45°C in water bath for 5-30 minutes or until sections have developed satisfactorily with the spirochetes at desired intensity and the background light yellow. Avoid an intensely stained background by checking microscopically. DO NOT PREHEAT THIS SOLUTION.
  12. Rinse in distilled water to stop reaction.
  13. Dehydrate through graded alcohol's clear in, Xylene (not Xylene substitute) and mount with Permount .

Results

Spirochetes, Donovan Bodies, General BacteriaLegionnaries' Disease Bacteria dark brown or black
Background bright yellow to light brown

Note: Acid wash all glassware used with silver solutions!

References

Margeson, L.S. Chapman CM: Use of Zinc Formalin as a sensitizer in Silver Stains for Spirochetes. Journal of Histotechnology , 19:2 p. 135-138, 6-96.

Product Information

Zinc Formalin
Silver Nitrate 1% Aqueous
Gum Mastic, 2.5%
Hydroquinone, 2%
Silver Nitrate, 0.04%
Modified Steiner Using Chapman's Modification