Gram Stain – A differential staining technique in which cells either stain pink (gram-negative) or purple (gram-positive) depending upon their structural type.

During a gram stain, a bacterial sample is smeared on a microscope slide and allowed to dry. The slide is then stained with a violet dye, treated with acetone-alcohol (a decoloriser) and finally counter-stained with a red dye.

Gram-positive bacteria retain the first violet dye as they have a thick layer of peptidoglycan in their cell walls creating cells which appear violet.

In gram-negative bacteria, the acetone-alcohol washes out the violet dye and the counter-stain is taken up creating cells which appear red. The cell walls of gram-negative bacteria have an outer layer of lipoprotein overlaying a thin layer of peptidoglycan.

Example process:

  • slide stained with crystal violet (1 min),
  • rinsed with water,
  • stained with iodine (1 min),
  • rinsed with water,
  • destained with acetyl-choline,
  • rinsed with water,
  • counterstained with safranin (1 min)