Microbiology 48 = Identification of Bacteria (Part-03)= Gram Staining Techniques and Procedure

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The Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques.

Procedure - (Main steps only)

(1) First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells a purple color.
(2) Next, Gram’s iodine, a mordant, is added. A mordant is a substance used to set or stabilize stains or dyes; in this case, Gram’s iodine acts like a trapping agent that complexes with the crystal violet, making the crystal violet–iodine complex clump and stay contained in thick layers of peptidoglycan in the cell walls.
(3) Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Cells that have thick peptidoglycan layers in their cell walls are much less affected by the decolorizing agent; they generally retain the crystal violet dye and remain purple. However, the decolorizing agent more easily washes the dye out of cells with thinner peptidoglycan layers, making them again colorless.
(4) Finally, a secondary counterstain, usual safranin, is added. This stains the decolorized cells pink and is less noticeable in the cells that still contain the crystal violet dye.

The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative. Gram-positive bacteria constitute a cell wall, which is mainly composed of multiple layers of peptidoglycan that forms a rigid and thick structure. Its cell wall additionally has teichoic acids and phosphate. The teichoic acids present in the gram-positive bacteria are of two types – the lipoteichoic acid and the teichoic wall acid. The cell wall is known as murein. As Gram-positive bacteria lack an outer lipid membrane, when correctly referring to their structure rather than staining properties, are termed monograms. The outer lipid membrane possessed by Gram-negative bacteria means that, when referring to their physical structure, they are termed sidearms.

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