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Star Republic: Guide for Biologists

Gram stain

Gram-positive organisms will be stained blue/black, Gram-negative organisms and nuclei will be stained red.

1. Prepare Crystal violet stain, Weigerts iodine and 0.1% Nuclear fast red:

Crystal Violet
Crystal violet 2 g
95% Alcohol 20 ml
Ammonium oxalate 0.8 g
ddH2O 80 ml

Weigerts Iodine
Potassium iodide 2 g
Iodine crystals 1 g
ddH2O 100 ml

0.1% Nuclear fast red
Nuclear fast red 0.1 g
Aluminum sulphate 2.5 g
ddH2O 100 ml

2. Take sections to water.

3. Stain in filtered crystal violet for 2 minutes.

4. Rinse in tap water briefly.

5. Stain in Weigerts iodine for 2 minutes

6. Rinse in tap water briefly.

7. Differentiate in acetone briefly until the section is colourless.

8. Rinse briefly in tap water.

9. Counterstain in 0.1% nuclear fast red for 3 minutes.

10. Wash for 3 seconds in tap water.

11. Dehydrate, clear. Mount sections in DPX