Laboratory Protocols

• Cryotomy
• Histological Staining Techniques
  • Index
• Histological fixatives
• Peroxidase-anti-peroxidase Technique
• Preparation of tissue section adhesives
• Processing tissues for light microscopy (basics) (PDF 1.1mb)
• Processing tissues for light microscopy (detailed)
• Sample collection guidelines
• Setting up the light microscope
• Specimen preparation for electron microscopy

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 60^oC (solution will be at about 56^oC) - 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.

Results:

Fungal hyphae, (also mucins, chitin, melanin, amoebae and glycogen) - black.
Background - green.

Stock solution (hexamine silver)

5mls of 5% aqueous silver nitrate.
100mls of 3% aqueous hexamine.
Store at 4^oC keeps for 1-2 months.

Working solution

To 25mls of the above stock solution add 2mls of 5% aqueous borax, (sodium tetraborate, kept in 37^oC incubator to prevent crystal formation), and 25mls of distilled water and mix. Put the solution into a clean coplin jar in a 60^oC water bath and allow to reach temperature before use.

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.)

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