Gram stain

The ing method is named after the Danish bacteriologist Hans Christian Gram (1853 –1938) who originally devised it in 1882 (but published in 1884), to discriminate between pneumococci and Klebsiella pneumoniae bacteria in lung tissue. It 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 reaction divides the eubacteria into two fundamental groups according to their stainability and is one of the basic foundations on which bacterial identification is built. ing is not used to classify archaea, since these microorganisms give very variable responses.

ing 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)

The original description of staining technique by Christian Gram in a publication titled "The differential staining of Schizomycetes in tissue sections and in dried preparations" in Fortschitte der Medicin; 1884, Vol. 2, pages 185-189 was slightly different from what we use today. The primary stain used was aniline gentian violet, mordant was Lugol's iodine (iodine-potassium iodide in water), decolorizer was absolute alcohol and bismark brown was the counterstain.

Procedure:

The smear on a glass slide is covered with few drops of one of the primary stains. Gentian violet is a mixture of methyl violet and crystal violet. The primary stain renders all the bacteria uniformly violet. After a minute of exposure to the staining solution, the slide is washed in water.

The smear is treated with few drop of Gram's Iodine and allowed to act for a minute. This results in formation of a dye-iodine complex in the cytoplasm. Gram's iodine serves as a mordant.

The slide is again washed in water and then decolorized in absolute ethyl alcohol or acetone. A mixture of ecetone-ethyl alcohol (1:1) can also be used for decolorization. The process of decolorization is fairly quick and should not exceed 30 seconds for thin smears. Acetone is a potent decolorizer and when used alone can decolorize the smear in 2-3 seconds. A mixture of ethanol and acetone acts more slowly than pure acetone. Decolorization is the most crucial part of ing and errors can occur here. Prolonged decolorization can lead to over-decolorized smear and a very short decolorization period may lead to under-decolorized smear.

After the smear is decolorized, it is washed in water without any delay. The smear is finally treated with few drops of counterstain such as dilute carbol fuchsin, neutral red or safranin.

The slide is washed in water; excess water is removed using a blotting paper, dried in air and heat fixed before observing under microscope.