Histological Staining Techniques

Gram/Twort for bacteria
Twort, 1924; Ollett, 1947.
Use a positive control section.


Gram +ve organisms - blue/black.
Gram -ve organisms - red/pink.
Nuclei - red.
Collagen, red blood cells, cytoplasm - varying shades of green.

Neutral red/fast green stock solution (keeps for 1 year)
Add 50ml of 0.2% fast green in 95% ethanol to 450mls of 0.2% neutral red in 95% ethanol.
When preparing the fast green and neutral red solutions use covered conical flasks, stir the solutions for several hours and then allow to stand overnight to ensure maximum dissolution of the dyes. Filter before mixing to make the stock solution.

Working solution
Add 15mls of the above stock solution to 45mls of distilled water.
Mix and use immediately in a closed coplin jar to prevent precipitation.

Lillie's crystal violet

Dissolve 10g crystal violet in 100mls 95% alcohol.
Dissolve 4g ammonium oxalate in 400mls distilled water.
Add the two solutions together and stir the mixture for 3 hours. Filter.

Lugol's iodine

Iodine - 1g.
Potassium iodide - 2g
Distilled water - 100ml
Place the iodine and potassium iodide in a small quantity of the water and allow them to dissolve fully before adding the remainder of the water.

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