Gms staining procedure (Grocott’s Methenamine Silver stain)

The Grocott’s Methenamine Silver (GMS) staining procedure is a widely used histological technique. It is used for detecting fungi. It can also identify certain bacteria and basement membrane structures.

Here is the step-by-step procedure of Gms staining.

Materials Needed

1. Deparaffinized and hydrated tissue sections.
2. Chromic acid solution (5%).
3. Sodium bisulfite solution (1%).
4. Methenamine silver solution:
   • Methenamine (Hexamethylenetetramine).
   • Silver nitrate.
   • Borax.
5. Gold chloride solution (0.2%).
6. Sodium thiosulfate solution (5%).
7. Light green counterstain (optional).
8. Distilled water.
9. Staining jars or dishes.
10. Microscope slides.

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Grocott’s Methenamine Silver Procedure

Step 1: Deparaffinization and Hydration

1. Deparaffinize the paraffin-embedded tissue sections using xylene.
2. Rehydrate through a series of graded alcohols (100%, 95%, 70%) to distilled water.

Step 2: Oxidation

1. Place the slides in 5% chromic acid solution for 10 minutes.
   • Chromic acid oxidizes polysaccharides in the fungal walls to aldehydes.
2. Rinse slides thoroughly in running tap water for 1-2 minutes.

Step 3: Aldehyde Blocking

1. Immerse the slides in 1% sodium bisulfite solution for 1-2 minutes.
   • Sodium bisulfite solution removes excess chromic acid.
2. Wash in running tap water for 1-2 minutes.

Step 4: Methenamine Silver Impregnation

1. Prepare the methenamine silver solution freshly before use:
   • Mix methenamine, silver nitrate, and borax in distilled water.
   • Heat the solution to approximately 50-60°C.
2. Place the slides in the heated methenamine silver solution for 10-20 minutes.
   • Keep the solution at a constant temperature to ensure even staining.

Step 5: Toning

1. Rinse the slides briefly in distilled water.
2. Place the slides in 0.2% gold chloride solution for 1-2 minutes.
   • This step enhances contrast by toning the silver deposits.

Step 6: Fixation

1. Rinse the slides briefly in distilled water.
2. Immerse in 5% sodium thiosulfate solution for 1-2 minutes.
   • This solution removes any unreacted silver and prevents nonspecific staining.

Step 7: Counterstaining

1. Counterstain the slides lightly with light green for 1-2 minutes.
   • NOTE: This provides contrast to the silver-stained elements. This steps is optional.

Step 8: Dehydration and Mounting

1. Rinse the slides in running tap water, then in distilled water.
2. Dehydrate through graded alcohols (70%, 95%, 100%).
3. Clear in xylene and mount with a coverslip using a synthetic resin.

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Grocott’s Methenamine Silver stain Results

• Fungal : Black.
• Background: Green (if counterstain is used) or pale depending on the counterstain used.
• Other elements: Basement membranes may also appear black.