Fungi and pneumocystis will be sharply stained black with the silver (Figure 1).
Figure 1. Budding yeast of Blastomyces dermatitidis in canine lung.
Grocott’s Methenamine (GMS) Silver Staining Procedure
Purpose:
This standard operating procedure (SOP) establishes the standard for staining
fungi and pneumocystis using the GMS staining procedure.
Reagents and Materials:
Reagents
0.5% Periodic Acid
Periodic Acid 2.5gm
Distilled water 500ml
5% Borax
Sodium Borate, tetra 5gm
Distilled water 100ml
Methenamine –Silver, Stock
Sigma Diagnostics Co.
1 vial 110mg
Distilled water 100mlMethenamine - Silver, Working
Methenamine Silver, Stock 25ml
Distilled water 25ml
5% Borax 2ml.25% Gold Chloride
Gold Chloride 1gm
Distilled water 250ml2% Sodium Thiosulfate
Sodium Thiosulfate 2gm
Distilled water 100ml
Light Green
Light Green, SF Yellow 1gm
Distilled water 499ml
Acetic Acid 1ml
Materials
Tissue slides and control slides for special stain requested.
Microwave to bake slides.
Staining rack, 20 slide capacity.
Coverslips and synthetic mounting media.
Hazards
and Precautions:
See Material Safety Data Sheets.
Sample
Preparations:
Paraffin tissue section slides cut at 4 microns.
Calibration
and Standardization:
A tissue section slide containing fungi and pneumocystis is stained with each
procedure.
Procedure:
1. Deparaffinize
and hydrate to distilled water.
2. 0.5% Periodic Acid – 10 minutes.
3. Distilled water rinse.
4. Place in Methenamine Silver Working Solution in a plastic coplin jar with
the lid
loosely applied.
Microwave at 50% power for 90 seconds.
If additional staining is necessary microwave 30 seconds more.
5. Distilled water rinse.
6. Gold Chloride – 30 seconds.
7. Distilled water rinse.
8. 2% Sodium Thiosulfate – 1 minute.
9. Distilled water rinse.
10. Light Green – 2 minutes / Routine H&E Procedure.
Make fresh:
Light green 10ml
Distilled water 40ml
11. Distilled water rinse.
12. Dehydrate, clear and coverslip.
Positive
control: Dog lung with Blastomyces dermatitidis (Figure 1)
Results:
Fungi and pneumocystis will be sharply stained black with the silver (Figure
1).
Figure 1. Budding yeast of Blastomyces dermatitidis in canine
lung.
Effective:
July 1, 2004
Cherie Chapman
James R. Turk
References:
Sheehan DC, Hrapchak BR: Theory and Practice of Histotechnology, 2nd Ed, CV Mosby, St. Louis, 1980, pp.245-246.Armed Forces Institute of Pathology Laboratory Methods in Histotechnology, eds. Prophet EB, Mills B, Arrington JB, Sobin LH, American Registry of pathology, Washington, D.C. , 1992, pp.225-227.