Silver Stain (Modified GMS)

Reagent Preparation

Prepare working Silver Methenamine solution by combining 8 ml Borax solution, 100 mL deionized water and contents of Silver Methenamine reagent vial. Mix until dissolved. Use once, then discard. Note: To increase tests per vial to two, Silver Methenamine may be dissolved in 100 mL of deionized water and stored tightly capped. Stable refrigerated (2-8 °C) for 1 month. When ready to use, take 50 mL of Silver Methenamine solution and add 4 mL of Borax solution. Return unused portion of Silver Methenamine solution to refrigerator. Pre-warm all reagents to room temperature before use.

Procedure

  1. Place working Silver Methenamine solution in 62 °C water bath.
  2. Deparaffinize tissue sections and hydrate to deionized water.
  3. Place in periodic acid solution for 5 minutes for fungi or 11 minutes for basement membrane.
  4. Rinse in 6 changes of deionized water.
  5. Place slides in working Silver Methenamine solution. Examine after 20 minutes for fungi and opportunistic organisms (should be dark brown) or 30 minutes for basement membrane (should be black). Return slides to working Silver Methenamine solution until desired intensity is achieved.
  6. Rinse in room temperature deionized water 6 times.
  7. Tone sections in Gold Chloride solution for 30 seconds.
  8. Rinse in room temperature deionized water 6 times.
  9. Place sections in Sodium Thiosulfate solution for 2 minutes.
  10. Wash well in running tap water.
  11. Counterstain according to personal preference.
  12. Dehydrate in alcohol, clear in xylene and mount.

Results

Fungi – Purplish-brown to black
Pneumocystis – Purplish-brown to black
Basement Membrane – Black
Background – Light green or yellow (depending on counterstain)

Silver Stain (Modified GMS)

Reagents

     
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