What is Gram staining?

Gram staining is a method used to differentiate two groups of bacteria based solely on the construction of their cell walls, using dye. Hans Gram was the researcher who developed this method of colouring the cell wall of a microbe with a dye called gentian violet – a substance that has in the past been used to treat infections, as it has an antibacterial, antifungal action.

This means gentian violet (also known as crystal violet) can only be used to identify dead bacteria (because it kills them).

Gram staining means to stain bacteria with a dye then look at them under a microscope to see who they really are. One sort of bacteria comes up red, while the other sort of bacteria appears violet. This differentiates the bacteria into gram-positive or gram-negative or gram-variable.

Some bacteria common to vaginal infections are gram-positive, but hard to detect using this method because the bacteria’s external cell layer is too thin, and hard to stain.

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/gentian 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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Jessica Lloyd - Vulvovaginal Specialist Naturopathic Practitioner, BHSc(N)

Jessica is a degree-qualified naturopath (BHSc) specialising in vulvovaginal health and disease, based in Melbourne, Australia.

Jessica is the owner and lead naturopath of My Vagina, and is a member of the:

  • International Society for the Study of Vulvovaginal Disease (ISSVD)
  • International Society for the Study of Women's Sexual Health (ISSWSH)
  • National Vulvodynia Association (NVA) Australia
  • New Zealand Vulvovaginal Society (ANZVS)
  • Australian Traditional Medicine Society (ATMS)