Histological Staining Techniques
Gomori, 1946; Grocott, 1955.
Gelatine coated slides recommended
but not essential. Use a positive control section.
Dewax sections, rinse in alcohol, rinse in water.
Place sections in 5% chromic acid (chromium trioxide) - 1 hour. Prepare
silver solution and allow to warm up half way through this step.
Wash well in tap water.
Decolourise in 1% sodium metabisulphite - 1 minute.
Wash in tap water then in several changes of distilled water.
Treat in silver solution in a coplin jar preheated in a water bath at 60oC
(solution will be at about 56oC) - 10 minutes.
Check microscopically after rinsing all slides with distilled water. Positive
control should show dark brown fungal hyphae against a lighter or clear
background. If underdone replace the slides for 2 minutes and check again
after rinsing. Continue up to 1 hour maximum. (Solution is probably no
good if it takes this long.) 15 minutes is about right.
Tone in 0.2% gold chloride (sodium tetrachloroaurate) - 2 minutes.
Rinse in distilled water.
Fix in 5% sodium thiosulphate - 5 minutes.
Wash in tap water.
Counterstain in 0.2% light green in 0.2% acetic acid - 1 minute.
Dehydrate, clear and mount.
Fungal hyphae, (also mucins, chitin, melanin, amoebae and glycogen)
Background - green.
Stock solution (hexamine silver)
5mls of 5% aqueous silver nitrate.
100mls of 3% aqueous hexamine.
Store at 4oC keeps for 1-2 months.
To 25mls of the above stock solution add 2mls of 5% aqueous borax,
(sodium tetraborate, kept in 37oC incubator to prevent crystal
formation), and 25mls of distilled water and mix. Put the solution into
a clean coplin jar in a 60oC water bath and allow to reach temperature
Note: If the background becomes too silver-stained it may be partially
removed by going back to the gold toning step and repeating it for 5 minutes.
(Continue the rest of the steps again as normal.)
Return to index page