Diseases of Vegetable, Ornamental and Spice Crops

Exercise - 12: TO PERFORM GRAM’S STAINING

The Gram staining is a differential staining method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. This staining method is named after the Danish Bacteriologist Hans Christian Gram (1853 - 1938) who originally devised it in 1882 in City Hospital Berlin, Germany (but published in 1884).

Gram staining consists of four components:

• Primary stain (Crystal violet, Methyl violet or Gentian violet)
• Mordant (Gram's Iodine)
• Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture)
• Counterstain (Dilute carbol fuchsin, safranin or neutral red)

Procedure:

1. Smear bacteria on a glass slide.
2. Stain the slide with crystal violet for 1-2 min.
3. Pour off the stain and wash the slide with water.
4. Treat the smear with few drop of Gram's Iodine and allowed to act for 1-2 minutes.
5. Pour off the stain again wash the slide in water.
6. Decolorize the slide by washing in absolute ethyl alcohol or acetone. The process of decolorization is fairly quick and should not exceed 30 seconds for thin smears.
7. Wash slide thoroughly with water to remove the acetone. Do not delay with this step.
8. The smear is finally treated with few drops of counterstain such as safranin for 2 minutes.
9. The slide is washed in water; excess water is removed using a blotting paper, dried in air and heat over a flame before observing under microscope.

Observations: Those bacteria that hold on to primary dye iodine complex and remain violet are called Gram positive and those which get decolorized and subsequently take up counterstain (pink/red) are called Gram negative.