Gomori’s Silver Methenamine Impregnation for Fungi*

*Modified Grocott’s

Purpose

To demonstrate fungi.

Principle

Polysaccharides in the fungal cell wall are oxidized to aldehydes by the chromic acid. The aldehydes are detected by the reduction of the silver complex.

Solutions

5% aqueous Chromic Acid

  • Chromium trioxide 5g
  • Distilled water 100 ml

Store in a brown glass bottle

Safety: Chromic Acid is VERY toxic, use in fume hood

Expiry: 2 months

 

1% aqueous Sodium Metabisulfite

  • Sodium metabisulfite 1g
  • Distilled water  100 mL

Store in a screw cap bottle

Expiry: 2 months

 

5% aqueous Borax

  • Sodium tetraborate (borate) 5g
  • Distilled water 100 ml

Store in brown bottle in fridge

Expiry: 6 months

 

3% aqueous Hexamethenamine (hexamethylenetetramine)

  • Hexamethenamine 3 g
  • Distilled water 100 ml

Store in brown bottle in fridge

Expiry: 1 month

 

5% Silver Nitrate

  • Silver nitrate 5 g
  • Distilled water 100 ml

Use acid cleaned glassware to prepare

Store in brown bottle in fridge

Expiry: 1 month

 

Working Silver Solution

Solution A: Mix together in acid washed cylinder and transfer to an acid washed Erlenmeyer

  • 5% aqueous borax 1.25 mL
  • Distilled water 25 mL

Solution B: Mix together in acid washed cylinder and then add to solution A in the acid washed Erlenmeyer

  • 5% aqueous silver nitrate 1.25 mL
  • 3% aqueous hexamethenamine (hexamethylenetetramine) 25 mL

 

Working Silver Solution

Mix Solution A and B separately; then add them together for the working silver solution and place in an acid washed coplin jar. This solution should be made up fresh and discarded after use.

 

0.2% Gold Chloride (can be saved and re-used)

Prepare by diluting a 1% Gold Chloride solution (sodium tetrachloroaurate):

  • 1% gold chloride 10 ml
  • Distilled water 40 ml

Expiry 1% Gold Chloride: 6 months

 

5% Sodium Thiosulfate (store in fridge)

  • Sodium Thiosulfate 5 g
  • Distilled water 100 ml

Expiry: 6 months

 

Counterstains

Light green, fast green FCF, or nuclear fast red (Kernechtrot) can be used. We will use a very dilute light green.  It is important that the counterstain be light here, so as not to obscure small amounts of organism.

Light Green

Stock Light green Solution (2%)

  • Light green SF (yellowish)  2 g
  •   Distilled water  100 mL
  • Glacial acetic acid  0.2 mL

Working  Light Green Solution (0.2%)

  • Stock solution  5 mL
  • Distilled water  45 mL

 

METHOD

  1. Deparaffinize sections and bring to water.
  2. Oxidize in 5% aqueous chromic acid in the fume hood for 1 hour at room temperature, at 60°C for 10 minutes or microwave for 1 min. If using the microwave, place microwave in the fume hood, do NOT tightly cover, put plastic bag around the coplin jar to retain spills and be cautious when removing in case the coplin jar is warm: Chromic acid is VERY toxic. Make working methenamine silver solution while tissues are oxidizing in chromic acid.
  3. Wash in running tap water for a few seconds.
  4. **Working Methanamine Silver solution should be placed in a 60°C waterbath now to pre-warm
  5. Rinse in 1% aqueous sodium metabisulfite (removes excess chromic acid) for 1 min.
  6. Wash in tap water 5-10 min.
  7. Wash in 3-4 changes of distilled water
  8. Using nonmetallic forceps, place sections in freshly mixed pre-warmed working solution of methenamine-silver nitrate in a 60°C water bath for 17 minutes; then rinse in distilled water and check color microscopically (fungi should be dark brown). If impregnation is not sufficient, return to silver solution and check every 3-5 minutes. (intended color-paper bag brown)
  9. Rinse in 6 changes distilled water
  10. Tone in 0.2% aqueous gold chloride for 2-5 minutes.
  11. Rinse in distilled water
  12. Place in 5% aqueous sodium thiosulfate for 2-5 min.
  13. Wash well in tap water
  14. Counterstain in light green for 2 minutes
  15. Rinse in tap water
  16. Dehydrate quickly, clear and mount

Results

Fungi and basement membrane – black

Mucin – taupe to dark grey

Inner parts of mycelia and hyphae – old rose

Background according to counterstain

Notes

  1. Use paraffin coated or plastic forceps to remove slides from methenamine silver nitrate solution.
  2. Counterstain only lightly.
  3. All glassware should be acid washed.
  4. Store stock solution of silver nitrate, methenamine and borax in fridge in brown bottles
  5. Use charged slides

Control

Any tissue positive for fungi.

References

IWK. (2007). IWK Laboratory Histology Staining Manual, Special Stains.

Carson, F. L., & Cappellano, C. H. (2015). Histotechnology: A Self-Instructional Text (4th ed.). American Society of Clinical Pathologists Press.

License

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Gomori's Silver Methenamine Impregnation for Fungi* Copyright © 2022 by NSCC is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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