Principle: Gram staining depends on bacterial cell wall & cytoplasmic membrane. When bacteria are stained with crystal violet & mordant with lugols some bacteria resist decolorization by alcohol & others do not. The bacteria which resist are called gram positive & those which are decolorized are gram negative. Gram positive bacteria stain blue & gram negative bacteria stains red.
- Fixation: The slide is flamed 3-4 times on the opposite side of the smear. Thus the smear is fixed.
- Primary staining: Crystal violet is coated over the smear & kept for 1 minute. It is then washed with tap water.
- Mordanting: lugols is coated over the smear & kept for 1 minute. It is then washed with tape water. It is done to increase the penetration power of lugols .
- Decolorization: It is done by alcohol & is continued till the violet color comes out from the smear. The slide is then washed with tap water.