Grocott’s Hexamine- Silver Borate Stain: Introduction, Principle, Procedure, Result Interpretation

Introduction of Grocott’s Hexamine- Silver Borate Stain

Grocott’s  Hexamine-Silver borate stain is the special staining method of choice for a large majority of histopathology laboratories for the demonstration of all fungi.

Principle of Grocott’s Hexamine- Silver Borate Stain

The exposure of formalin-fixed sections or methanol treated sputum is to chromic acid that reacts with fungal cell wall polysaccharide components to form chromic acid-aldehydes. A hexamine-silver solution at an alkaline pH reduces them and this causes fungal structures to be selectively blackened

Requirements for Grocott’s Hexamine- Silver Borate Stain

Equipment and reagents required for staining are as follows-

1. Coplin jars
2. Dropping bottles
3. Coverslips
4.  Dissecting needles, forces, scalpels, etc.
5. Ordinary filter papers
6. Mounting media (DPX or Canada balsam mountant)
7. Slide washing tray
8. Methanol ( in the case of sputum)
9. 5% chromic acid
10. Sodium metabisulfite
11. Distilled water (D/W)
12. hexamine solution ( incubating solution)
13. 0.1 % aqueous gold chloride
14. 3% sodium thiosulphate
15. 0.2% light green in 0.2% acetic acid
16. Microscope

Reagent preparation of Grocott’s Hexamine-Silver borate stain

1. 15% sodium tetraborate ( borax) in D/W
2. Methenamine silver
   5% silver nitrate in D/W- 5ml
   3% Methenamine in D/W- 100ml
   Add Silver nitrate to methenamine solution, gently shaking until precipitate dissolves. The mixture will keep for 1-2 months at 4°C.
3. Incubating solution ( Working solution)

Borax ( solution 1):  5ml
D/W: 25 ml
Methenamine silver (Solution 2)): 25 ml
Ideally, solutions a,b, and D/W should be preheated to 56° C and mixed prior to use. As silver the solution starts degenerates, borax is added.

• % aqueous chromic acid (chromium trioxide)
• 1% aqueous sodium metabisulphite
• 5% sodium thiosulphate
• 1% aqueous gold chloride
• 2% light green in 0.2% acetic acid

The procedure of Grocott’s Hexamine- Silver Borate Stain

1. Take sections to water or if sputum fixes smear of sputum with methanol.
2. Oxidize in 5% chromic acid (Chromium – tetraoxide) for 1 hour.
3. Wash well in tap water.
4. Rinse in 15 sodium meta bisulfate.
5. Wash in tap water for 5 minutes.
6. Rinse in D/W then place preheated (56° C) silver incubating solution in a dark place for 20-30 minutes.   Note: Check control sections for dark brown fungus; if not, return to solution and check at 3-minute intervals until the right color is reached.
7. Rinse well in D/W.
8. Treat in 0.1 % aqueous gold chloride for 4 minutes.
9. Rinse in D/W.
10. Place in 3% sodium thiosulphate for 5 minutes.
11. Counter strain with 0.2% light green in 0.2% acetic acid for 1 minute.
12. Blot dehydrate clear and mount.

Result Interpretation of Grocott’s Hexamine- Silver Borate Stain

Fungal elements: Black

Other oxidizable carbohydrates, including glycogen: Black

Background: Green

Keynotes on Grocott’s Hexamine- Silver Borate Stain

1. Keep the stock hexamine-silver solution at 4°C and out of direct sunlight. It will last 1-2 months in the refrigerator. Give it a thorough shake if a white precipitate forms; it should disperse.
2. Extending the period in chromic acid too long will cause the carbohydrates to oxidize to carboxylic acid, preventing the silver stain from being taken
3. If your sections have a lot of silver precipitates, it’s probably because you used low-grade
4. Other oxidizable carbohydrates, including glycogen, may take this stain black and therefore reporter may have the ability to differentiate fungal structure from other than fungal elements.

Further Reading 

1. https://skinpathonline.wordpress.com/2011/06/22/grocott-hexamine-silver-special-stain-for-fungus-%E2%80%93-method-and-tips
2. https://stainsfile.info/stain/metallic/gomori.htm
3. Gray, Peter. (1954). The Microtomist’s Formulary and Guide. Originally published by: – The Blakiston Co.Republished by:– Robert E. Krieger Publishing Co.
4. Drury, R A, and Wallington, E A,(1967). Carleton’s histological technique., Ed. 5.
   Oxford University Press, London, England.
5. Hayashi, I., Tome, Y. and Shimosato, Y.,(1989) Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner. Stain Technology, v 64, p 185.