Laboratory Protocols
Histological Staining Techniques
Gram/Twort for bacteria
Twort, 1924; Ollett, 1947.
Use a positive control section.
- Dewax sections, rinse in alcohol, rinse in water.
- Stain with Lillie's crystal violet - 1 minute. (Filter stain onto slide.)
- Wash briefly in tap water.
- Lugol's iodine - 1 minute.
- Drain the slide and flood with acetone to differentiate for 2-5 seconds.
- Rinse in distilled water.
- Counterstain with neutral red/fast green in a closed coplin jar - 5 minutes.
- Wash in distilled water.
- Dehydrate, clear and mount. Prolong the alcohol step until neutral red stops coming out of the section.
Results:
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.