5Answer: Gram’s staining is the test used to differentiate between gram positive and gram negative bacteria.
Process of gram’s staining:
1.prepare bacterial smear on the clean glass slide
2. pass the slide through the flame for 2-3 times this process is called heat fixing
3. Apply crystal voilet (Primary stain) on smear for 1 min and rinse it with water
4. Apply gram’s iodine (mordant) for 1 min and rinse with water
5. Then wash it with 95% alcohol which acts as decolourizer for 10-20sec and rinse it
6. Apply saffrannin which acts as seconadry stain for 1 min and wash with water
Air dry it and observe under a microscope.
Primary stain– First cells are stained with crystal voilet hence called as primary staining. Primary stain gives purple or blue colour when it binds to the bacterial cell wall.
Mordant- Iodine acts as mordant. It is added after primary staining. it plays an important role in intensifying the primary stain and fixing of cells.
Decolorizer– Ethyl alcohol acts as decolorizer. It dehydrates the peptidoglycan layer of the bacterial cell wall and shrinks it.
Counterstain– Safrannin is used as counterstain. When cells doesnot retain the primary stain. secondary staining is performed using safrannin. The cells which takes up safrannin appears red in colour.
We observe that all the gram +ve bacteria appear purple or blue in color whereas all the gram – ve bacteria appear pink or red.
6. This happens because the gram positive bacteria contains thick peptidoglycan layer in their cell wall and the lipid content is low which helps in reataining the primary stain and appears purple in color. whereas gram negative bacteria contain thin peptidoglycan layer in their cell wall and high lipid content and it does not retain the crystal voilet stain during decolorising and it takes up the secondary stain,