Gomori's Methenamine Silver
for Glycogen and Fungi
This method is also known as Grocott’s or Grocott-Gomori’s methenamine silver.
Materials
- Chromium trioxide, 5% aqu.
- Neutral red, 1% aqu. or Light green SFy, 0.2% in 0.2% acetic acid, or Progressive hemalum and eosin
- Sodium bisulfite, 1% aqu.
- Sodium thiosulfate, 2% aqu.
- Yellow gold chloride, 0.1% aqu.
- Stock Methenamine silver
Material Amount Methenamine, 3% aqu. 100 mL Silver nitrate, 5% aqu. 5 mL Shake until the precipitate redissolves. Silvering of the container indicates deterioration.
- Working Methenamine silver
Material Amount Stock Methenamine silver 25 mL Distilled water 25 mL Borax* 0.1 g Make just before use and preheat to 50°C.
*Or 2 mL of a 5% aqueous borax solution (Grocott)
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.
Protocol
- Bring sections to water via xylene and ethanol.
- Oxidise with 5% chromic acid (chromium trioxide) for 60-90 minutes.
- Rinse well with tap water.
- Bleach with sodium bisulphite for 1 minute.
- Rinse well with tap water.
- Rinse with distilled water.
- Treat with methenamine silver solution at 50°C. until impregnated (up to 3 hours)
- Wash with distilled water.
- Tone with 0.1% gold chloride solution for 5 minutes.
- Rinse with distilled water.
- Fix in 2% sodium thiosulphate for 5 minutes.
- Wash well with running tap water.
- Counterstain with light green, neutral red or a light H&E.
- Rinse with tap water.
- Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Expected Results
- Oxidisable carbohydrates, including glycogen and fungi – black
- Background – as counterstained
Notes
- Methenamine is also known as hexamethylenetetramine and hexamine.
- Borax is sodium tetraborate. Grocott’s modification adds the 0.1 g of borax as 2 mL of a 5% aqueous solution.
- Aqueous solutions of chromium trioxide are usually referred to as chromic acid. Ten minutes in a 10% aqueous solution will usually give the same result as 60 minutes in a 5% solution.
- Toning is a variable step. Untoned sections give dark brown material on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black material on a pale grey-brown background. Others tone longer (a few minutes) to produce black material on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.
- This method depends on a similar principle to Bauer’s chromic acid Schiff method, but in which the aldehydes produced by oxidation reduce a silver solution instead of combining with Schiff’s reagent to form a red compound. Consequently, those materials which are red in a Bauer’s stain will be black in Gomori’ stain, i.e. it is not specific for glycogen but will demonstrate any carbohydrates which can be oxidised to aldehydes, including fungi, and itis often used for that purpose.
- In a similar method Hayashi, Tome and Shimosato recommended that thiosemicarbazide should be applied to the section after oxidation. Thiosemicarbazide has the formula H2NNHCSNH2. The hydrazine group (H2NNH-) combines with any aldehydes present. The thiocarbamyl group (-CSNH2) is a more powerful reducing agent than the aldehydes it replaces and reduces the methenamine silver solution more rapidly and with higher contrast.Immediately following step 5:
- Place sections in 1% aqueous thiosemicarbazide for 10 minutes.
- Wash well with tap water, and carry on from step 6.
- It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide. The MSDS says:
- Flash Point: n/a
- Lower Explosive Limit: n/a
- Upper Explosive Limit: n/a
- Unusal Fire and Expl.rds: none identified
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by The Blakiston Co.
Republished by Robert E. Krieger Publishing Co. - Drury, R A, and Wallington, E A, (1967).
Carleton’s histological technique., Ed. 5.
Oxford University Press, London, England. - 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.
