Fungi - RCPAQAP

Aim of special stain:: Identification of Fungi
Diagnostic applications:: Mycosis
Material supplied for surveys:: Placenta tissue inoculated with Aspergillus cut at 3µm
Recommended control:: Tissue/s containing Fungi

1. Grocott’s Methenamine Silver Method

Safety: Universal safety precautions must be adhered to whilst performing this method, including the appropriate use of personal protective equipment (gowns, gloves, eyewear).

Reagents – commercial source unknown, unless specified:

1. 5% chromic acid
a) Chromic acid	5 g
b) Distilled water	100 mL

2. 1 % sodium metabisulphite
a) Chromic acid	5.0 g
b) Distilled water	100 mL

3. 3% methenamine (hexamine)
a) Methanamine	3.0 g
b) Distilled water	100 mL

4. 5% silver nitrate
a) Silver nitrate	5.0 g
b) Distilled water	100 mL

5. 5% Sodium Tetraborate (borax)
a) Sodium tetraborate	5.0 g
b) Distilled water	100 mL

6. Methenamine silver solution: (Prepare fresh)
a) 3% Methenamine	25mL
b) 5% Silver nitrate	1.25mL (measure with pipette)
Mix well until the white precipitate dissolves then discard 1.25ml of this solution.
c) Add distilled water	25mL
d) 5% borax	2mL
Mix well and filter into a 50ml Coplin jar

7. 0.1% gold chloride (commercially available)

8. 5% sodium thiosulphate
a) Sodium thiosulphate	5.0 g
b) Distilled water	100 mL

8. 5% sodium thiosulphate
a) Sodium thiosulphate	5.0 g
b) Distilled water	100 mL

8. Light green
Stock light green	1% light green in 1% acetic acid
Working light green:	a) Stock light green	5mL
	b) Distilled water	45mL

Method:

Rehydrate sections.
Oxidize in 5% chromic acid for 1 hour.
Rinse in water.
Sodium metabisulphite for 1 minute.
Wash in water for 5 minutes.
Rinse in 3 changes of water.
Place in working silver solution (which has been pre-heated to 60°C) for 8 minutes.
Rinse in distilled water and check microscopically.
If staining is not complete return to silver solution at 60°C, check at 2 to 5 minute intervals.
When staining is complete rinse in distilled water.
Tone in 0.1% gold chloride for 2 minutes.
Rinse in distilled water.
Fix in 5% sodium thiosulphate for 2 minutes.
Rinse in distilled water, wash in water.
Counterstain in working light green for 20-30 seconds.
Rinse in water.
Dehydrate, clear and mount.

Results

Fungi – black
Mucin – grey to black
Background – light green

2. Periodic Acid Schiff Stain with light green for Fungi

*Bring Schiff’s reagent to room temperature before use (low-temperature retards reaction).

Reagents – commercial source unknown, unless specified:

1. 1% Periodic acid

2. Schiff’s reagent

3. Aqueous light green
a) 0.2% light green stock solution:
Light green	0.2 g
Distilled water	100 mL
Glacial acetic acid	0.2 mL
b) Light green working solution:
Stock light green	10.0 mL
Distilled water	50.0 mL

Method:

Dewax and bring to water.
Rinse well in distilled water.
Oxidize in periodic acid for 5 minutes.
Rinse well in distilled water for a few minutes.
Coleman’s Feulgen or Schiff’s for 15 minutes.
Wash in running water for 10 minutes approx.
Light green working solution for 1 minute (adjust if necessary).
- Green counterstain is recommended for demonstrating fungi
Blot dry, briefly dehydrate in alcohol.
Clear and mount.

Results:

Fungi and glycogen – Magenta
Background – Green

Disclaimer:

These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.

References

2019 RCPAQAP Technical General Survey 3.
2024 RCPAQAP Technical General Survey 2.
Jeanine H. Bartlett. Theory and practice of Histological Techniques. John D Bancroft; Marilyn Gamble. 6^th edition. New York, NY: Churchill Livingstone; 2008:320-322.
Luna LG. Manual of histologic staining methods of the armed forces institute of pathology. 3^rd Ed. New York: McGraw-Hill;1968:159-160.