Gram staining is a method used to differentiate two groups of bacteria based solely on the construction of their cell walls, using dye. Gram was the guy who developed it, and staining means, well, staining. With dye.

Gram staining means to stain bacteria with a dye then look at them under a microscope to see who they really are. One comes up red, while the other comes up violet. This differentiates them.

Gram positive and Gram negative

  1. Gram positive bacteria stain violet because their cell walls have a thick layer of special stuff (peptidoglycan) which holds on to the dye after decolourisation
  2. Gram negative bacteria stain red because their cell wall is thinner, and does not hold the dye as well after decolourisation, and are instead stained by the safranin in the final process.

How is Gram staining carried out?

The staining steps are thus:

  1. Staining with water-soluble dye (crystal violet) is carried out.
  2. Gram’s iodine solution is added and forms a complex between the crystal violet and the iodine.
  3. Decolourisation is performed – alcohol or acetone is added, which shrinks and tightens the peptidoglycan layer, and the large crystal violet-iodine complex cannot penetrate. It is then trapped in the cell in Gram positive bacteria.
  4. If the bacteria is Gram negative, the outer membrane is degraded and the thinner peptidoglycan layer cannot retain the crystal violet-iodine complex, and the colour is lost.
  5. Counterstaining (typically with safranin) is performed, which stains it red. The safranin is lighter than the crystal violet, and does not disrupt the colour in Gram positive cells, but Gram negative cells are stained red.


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