|From:||David Anderson <firstname.lastname@example.org> (by way of histonet)|
We use a Gram-Twort stain based on the method found in Bancroft's Theory and
Practice of Histological Techniques.
a. 0.5% crystal violet in 25% alcohol.
b. Gram's iodine
c. Twort's stain
0.2% neutral red in ethanol -- 9 ml
0.2% fast green in ethanol ---- 1 ml
Distilled water --- 30 ml
Mix before use.
1. Hydrate slides to water.
2. Crystal violet 5 minutes. Rinse in water
3. Gram's iodine 5 minutes. Rinse in water
4. Decolorize with acetone until no more blue color runs off (reaction is
immediate). Rinse in water.
5. Twort's stain 5 minutes. Rinse in water.
6. Dehydrate, clear and mount.
The original procedure dries the sections after step 3, then uses 56 degree
C. acetic-alcohol (2% acetic acid in absolute alcohol) as the decolorizer in
step 4 for 15-20 minutes. We've found acetone works quite well and is much
Acetic-alcohol can be used after the Twort's stain (between steps 5 and 6)
to remove excess red color from the tissue, which can mask gram negative
organisms. The fast green gives a nice contrasting color in the background.
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