Special Stains - Grocott - Gomori Methenamine Silver Staining Technique

The Grocott Gomori Methenamine Silver Method is used to demonstrate Fungi. The cell walls of the fungi are rich in polysaccharides which, when oxidised by chromic acid, are converted to dialdehydes. These groups directly reduce silver ions from the methenamine silver solution to metallic silver. Sodium thiosulphate is used to fix the metallic silver and also remove any unreduced silver.

Method

1.      Take sections to water.

2.      Oxidise in 5% chromic acid for 1 hour

3.      Wash in tap water. Bleach in 1% Sodium metabisulphite for 5 minutes

4.      Rinse in several changes of distilled water

5.      Place in pre-heated incubating solution (25ml Grocott solution made from (3% hexamine and 10% silver nitrate) + 25ml H20 +5ml borax) at 60°C in the dark for 20 minutes or until fungi are blackened and background is clear

6.      Wash well in several changes of distilled water

7.      Tone in 0.2% gold chloride for 4 minutes

8.      Wash in water

9.      Fix in 5% sodium thiosulphate for 5 minutes

10.Wash in water. Counterstain in light green working solution for 20 seconds

11.Wash in 1% acetic acid

12.Wash, dehydrate, clear and mount

 Results

Fungal Hyphae and Yeast Bodies - Black

Background -    Pale green

 

The results can be seen below, the fungi are shown up clearly in black while the background is stained green to provide better contrast.

Photo – Grocott x10