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
- Deparaffinize sections and bring to water.
- 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.
- Wash in running tap water for a few seconds.
- **Working Methanamine Silver solution should be placed in a 60°C waterbath now to pre-warm
- Rinse in 1% aqueous sodium metabisulfite (removes excess chromic acid) for 1 min.
- Wash in tap water 5-10 min.
- Wash in 3-4 changes of distilled water
- 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)
- Rinse in 6 changes distilled water
- Tone in 0.2% aqueous gold chloride for 2-5 minutes.
- Rinse in distilled water
- Place in 5% aqueous sodium thiosulfate for 2-5 min.
- Wash well in tap water
- Counterstain in light green for 2 minutes
- Rinse in tap water
- 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
- Use paraffin coated or plastic forceps to remove slides from methenamine silver nitrate solution.
- Counterstain only lightly.
- All glassware should be acid washed.
- Store stock solution of silver nitrate, methenamine and borax in fridge in brown bottles
- 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.
