Fundamentals of Microbiology

2.3.3 Principle

The Gram – positive bacteria will retain the crystal violet and appear deep violet in colour. The Gram-negative bacteria lose the crystal violet on decolorization and are counter stained by the safranine and appear red in colour. Iodine solution is used as a mordant that fixes the primary stain in or on a substrate by combining with the dye to form an insoluble compound-mordant, for the first stain.

The reactions are associated with the structure and composition of the cell wall.

The cell walls of Gram-negative bacteria are thinner than that of Gram-positive bacteria and contain a higher percentage of lipid content. During the staining of Gram-negative bacteria, the alcohol treatment extracts the lipid. This results in increased porosity or permeability of the cell wall. The crystal violet-iodine complex, thus can be extracted and the Gram-negative bacteria is decolorized. The cells subsequently take up the colour of the counter stain safranin.

The cell walls of Gram-positive bacteria with lower lipid content become dehydrated during alcohol treatment. The pore size decreased, permeability is reduced and the CV-I complex can - not be extracted. Therefore, the Gram-positive cells remain purple.