GRAM STAINING

To gram stain, an investigator smears a sample of bacteria on a slide, soaks it in a violet dye and then treats it with iodine. The slide is then rinsed with alcohol and counterstained with a pink dye called safranin.

The cell walls of gram-negative bacteria have a very low affinity for the violet stain, which is rinsed out by the alcohol. Once counterstained with safranin, the gram-negative bacteria appear bright pink to red.

Gram-positive cell walls have a high affinity for the violet stain, and retain it even through the alcohol rinse. When the process is complete, they appear dark purple to brown.

One difference between the two cell types appears to be in the amount of peptidoglycan in the cell wall. Gram positive cell walls are about five times as rich in peptidoglycan as gram-negative cell walls. In addition, gram-negatives have a second membrane (chemically different from the plasma membrane) external to the cell wall, and may also have a gelatinous sheath external to the second membrane.