Gram Staining Protocol

1. Deparaffinize sample and hydrate to distilled water
2. Place slides of staining rack
3. Add 1ml of crystal violet solution to the sample. Alternativley gentian violet can be used
4. Add 250ul of 5% sodium biocarbonate
5. Gently mix solutions for one minute
6. Rinse with water
7. Cover sample with Gram's iodine solution and incubate for 1 minute
8. Rinse with water
9. Blot with filter paper until dry
10. Incubate breifly in acetone. This step should be short as longer incubation will cause some gram positives to become gram negative. The time of incubation is usually determined by the decolorization of the background tissue. Repeat if necessary.
11. Cover sample with basic fuchsin solution for 3 minutes
12. Rinse with water
13. Blot excess water with filter paper
14. Dip slide in acetone
15. Dip slide in 0.1% picric acid in acetone
16. Rinse in acetone
17. Rinse in 50% acetone - 50% histolene
18. Rinse in 100% histolene. Repeat if necessary
19. Mount slide

Gram positive bacteria stain blue and gram negative bacteria stain red.