The Grocott Hexamine-Silver special stain is the method of choice for a large majority of histopathology laboratories for the demonstration of all fungi. The formalin-fixed sections are exposed to chromic acid which reacts with fungal cell wall polysaccharide components to form chromic acid-aldehydes. These then reduced by a hexamine-silver solution at an alkaline pH. This causes them to be selectively blackened. It should be noted that this method is not specific for fungi but rarely fails to demonstrate any fungi within the test tissue.
Below is the author’s method of choice.
5% aqueous chromic acid (chromium trioxide)
1% aqueous sodium metabisulphite
Stock hexamine-silver solution = 100ml 3% aqueous hexamine + 5% aqueous silver nitrate.
Working hexamine-silver solution = 2ml 5% aqueous sodium tetraborate (borax) + 25ml distilled water. Mix, then add 25ml stock hexamine-silver solution.
0.1% aqueous gold chloride
5% sodium thiosulphate
0.2% light green in 0.2% acetic acid
1. Take sections to water.
2. Treat sections with chromic acid for 1 hour.
3. Wash thoroughly in tap water.
4. Treat with sodium metabisulphite solution for 1 minute.
5. Wash well in tap water.
6. Wash well in several changes of distilled water.
7. Treat sections with working hexamine solution (preheated in coplin jar at 56 degrees Celsius) at 56 degrees Celsius for 10-20 minutes. Check control sections to see if fungi are a dark brown colour, if not return to solution checking regularly at 3 minute intervals until correct colour achieved.
8. Wash in several changes of distilled water.
9. Treat sections with 0.1% aqueous gold chloride for 3 minutes.
10. Wash well in distilled water.
11. Treat sections with 5% sodium thiosulphate for 5 minutes.
12. Wash well with tap water.
13. Counterstain with 0.2% light green in 0.2% acetic acid for 1 minute.
14. Wash well in tap water.
15. Dehydrate, clear and mount.
– As with all other silver stains wash everything that you are going to use thoroughly with distilled water.
– Store the stock hexamine-silver solution at 4 degrees Celsius away from sunlight. It will keep for 1-2 months. If a white precipitate forms give it a good shake and it should redissolve.
– Do not extend the time in chromic acid too long as this can over oxidize the carbohydrates to carboxylic acid and therefore not take up the silver stain.
– Do not reduce the time in chromic acid as this will lead to under oxidation and therefore no take up of the silver stain.
– The chromic acid can be reused but its efficiency will decrease after each use.
– If the control sections are failing to stain even after extending the staining time in the heated hexamine-silver solution you have more than likely forgotten to add the borax. If so, you can just add it and continue with the stain.
– If you have a large amount of silver precipitate over your sections it is probably due to using low-grade silver.
– If you forget the sodium thiosulphate step you will not realise until after retrieving the slide from storage further in the future, as the remaining silver not removed will react with sunlight turning black.
– Try to keep your counterstain fairly light as dark counterstaining can mask fungal elements.
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