Gram Staining Principle

The basic principle behind this technique is that some bacteria have the ability to retain the dye while the other bacteria don’t have the ability to retain the dye after staining with crystal violet dye. This differentiation is possible due to the difference in the cell wall of bacteria.

1. Gram positive bacteria have high peptidoglycan content and two types of teichoic acids, wall teichoic acid and lipoteichoic acid. Wall teichoic acid keeps the cell wall intact and lipoteichoic acid have affinity toward anionic crystal violet dye. When we pour crystal violet (first step), it gets attached to lipoteichoic acid and reaches to the cell cytoplasm where it bind to magnesium ion and forms a CV-Mg-RNA complex, while in gram negative bacteria there is no teichoic acid and have less content of peptidoglycan. So the dye did not penetrate so well and forms a weaker complex.
2. In Second step, iodine is poured which act as a mordant i.e., it intensify the color and form a stronger complex in gram positive bacteria which is known as CV-Mg-RNA-I complex.
3. In third step, ethanol is poured that has a dual property (1) lipid solvent and (2) dehrydation. So lipid gram positive has low lipid content, which get easily dissolved and form small pores which easily get shrink also that prevent washing off of complex in gram positive bacteria and appear purple at this time and in Gram-negative bacteria have a high content of lipid this results in formation of large pores which does not get shrink so easily. This causes to flood off the complex from gram negative bacteria and appear colorless this time.
4. In fourth step, safranin is poured which stains the gram negative bacteria red in color.

Gram stain is a technique to impart color to the bacterial cell to differentiate between gram-positive bacteria and gram-negative bacteria based on cell wall composition. Gram Staining is a laboratory procedure that consists of four reagents crystal violet (primary stain), iodine (mordant), decolorizer (ethyl alcohol), and safranin (counter stain) to stain the bacterial cell. Hans Christian Gram is a Danish bacteriologist who named this stain and developed this method in 1884.

The basic function of this technique is to differentiate between bacteria based on the chemical and physical properties of the cell walls. The difference in the cells can be identified by the cell wall as the gram-negative bacteria has a thin cell wall due to which the violet stain gets washed out with ethanol whereas the cell wall of gram-positive bacteria is thicker because of which violet stain stays out and give pink color to the bacteria.