Gram Stain

Gram staining serves to differentiate bacteria that have a thick peptidoglycan cell wall (Gram positive)
and those who do not (Gram negative)

Procedure:

1. Smear sample on slide (add a drop of water before)

2. Heat fix with lighter
3. Crystal Violet 1% 1 min
4. Wash
5. PVP-iodine 1 min
6. Wash
7. Decolorize in acetone (2 sec) or 95% ethanol (5-10 sec)
8. Wash
9. Add Safranin 1% 30 s
10. Wash
11. Mount in Nail polish and inspect under 100x with oil

Materials used for Gram staining (from left, 1%Crystal Violet, Iodine solution,IPA,1% Safranin)

Results: Gram positive bacteria – blue-purple colour

Gram negative bacteria- pink-red colour

Gram positive bacteria (mouth mucosa)

Gram negative

E.coli (UTI)

Gram +ve and -ve from culture