Gram staining is a differential staining technique used to distinguish between gram-positive and gram-negative bacteria based on their cell wall properties. The process involves primary staining with crystal violet, forming a complex with iodine, followed by decolorization with ethanol and counterstaining with methyl red or carbolfuchsin. The color retention indicates the type of bacteria: gram-positive bacteria retain the violet stain while gram-negative bacteria appear red or pink after decolorization.

1. GRAM STAINING
Differential Staining

2. Principle :
The gram staining is differential staining it
involves the differentiation of gram –ve and +ve
bacteria . The primary stain crystal violet forms
complex with iodine leads formation of crystal
violet magnesium ribo nucleate iodine and
crystal violet iodine complex, these are forms
in gram +ve and -ve bacteria respectively. In
gram +ve bacteria the colour of primary stain is
retained and in –ve bacteria colour of primary
stain was not retained after adding
decolourising agent. Gram –ve bacteria counter
stained by methyl red and carbolfuschin forms
red or pink colour at end.

3. Reagents used in
Grams staining
1.Primary stain: crystal
violet
2.Mordant stain: Grams
iodine
3.Decolourising agent:
Ethanol
4.Counter stain: Methyl red
or carbolfuschin

4. 1.Primary stain : Carbolfuschin
The carbolfuschin is the primary stain forms crystal
violet magnesium ribonucleate iodine complex in
grams +ve bacteria, it retained after the adding
decolourising agent . The colour of primary stain was
retained because gram +ve bacteria containing waxy
material on cell wall, these resistance to entry of
decolourising agent.

5. 2. Mordant: Iodine
The iodine forms complex with crystal violet that is
water insoluble compound.
3. Decolourising agent
The decolourising agent decolourises complex
formed by primary stain and mordant iodine
solution.

6. 4. Counter stain: methyl red
The counter stain used differentiate the gram +ve and
gram –ve bacteria. It It stains only the gram –ve
bacteria and cell of gram +ve bacteria was
impermeable to the entry of methyl red through it. It
was added after decolourisation.
Note: Some type of grams staining involves counter
stain as carbolfuschin in place of methyl red

7. Procedure involved in Grams staining
1. Smear making
2. Heat fixing
3. Primary staining
4. Decolourising
5. Counter staining
6. Obeserve under microscope
Report:
1. If the colour of the primary stain violet is left in side the cell wall of
bacteria reported as Gram +ve bacteria
2. If the colour of primary stain does not retains and appears red or pink
colour.