It was originally devised by Christian Gram (1984). The technique involves 4 steps:
1. Primary staining with methyl violet (or crystal violet or < gentian violet).
2. Iodine application
3. Decolourisation with an organic solvent (ethanol, acetone or aniline)
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4. Counterstaining with a contrasting dye (carbon fustian, safranine or neutral red).
Method
1. Apply methyl violet over the bacterial smear in a slide. Stain for one minute. Then wash off the stain with water.
2. Apply Gram’s iodine and allow it to act for one minute. Wash off the slide in water.
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3. Decolorize the slide in 70 per cent alcohol for not more than 5 seconds. Wash the slide immediately with water.
4. Counter stain with dilute carbon fuchsine for one minute. Wash it in water. Blot the slide dry. Examine the slide under oil immersion objective.
Importance of Gram staining
Gram staining differentiates bacteria into two broad groups. 1) Gram positive bacteria 2) Gram negative bacteria.
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Gram positive bacteria resist decolurisation and retain the primary stain. They appear violet in color.
Gram negative bacteria are decolourised by organic solvents. So they take up counter stain and appear red in color.
Gram positive and Gram negative bacteria differ in several characteristics such as growth requirements, antibiotic sensitivity and pathogenicity.