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Gram staining
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Gram staining

Gram staining is a method for staining samples of bacteria that differentiates between the two main types of bacterial cell wall.

It is named after the inventor, the Danish scientist Hans Christian Gram (1853-1928), who developed the technique in 1884 to discriminate between pneumococci and Klebsiella pneumoniae bacteria.

Gram Staining a Step by Step Procedure

  1. First, an inoculum is taken from a culture using an inoculation loop and put on a slide. If the culture is solid, it is diluted by adding a drop of water on the slide and mixing with the loop. It is important here to take a very small inoculum so that the end result is a sparse single layer of bacteria. It is a common mistake for beginners to put way too much inoculum at this step.
  2. Heat-fix the specimen by passing the slide through a bunsen flame a few times, without allowing the slide to become hot to the touch.
  3. Add a basic dye to stain the sample. crystal violet or gentian violet are suitable. Allow to stain for 1 minute. The slide should look violet in colour.
  4. Rinse off with water for a maximum of 5 seconds.
  5. Add iodine solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye.
  6. Rinse with water.
  7. Apply 95% ethanol or a mixture of acetone and alcohol several times until no more colour appears to come from the sample.This leaves Gram-positive organisms stained purple and Gram-negative organisms unstained.
  8. Rinse with water.
  9. Apply a suitable counterstain. Opinions vary as to the best choice but suitable stains include safranin or fuchsin.This stains the gram negative organisms.
  10. Blot gently and allow to dry. Do not smear.

Inspect the slide under a
Gram positive organisms will appear blue-black or purple.
Gram negative organisms will appear red.

Gram-positive bacteria have a thick meshlike cell wall made of peptidoglycan which is capable of retaining the violet dye. Gram negative bacteria have a thin cell wall made of a layer of peptidoglycan. They also have an outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space.

As a rule of thumb (which has exceptions), Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane is often hidden by a capsule or slime layer which hides the antigens of the cell and so acts as "camouflage" (the human body recognises a foreign body by its antigens, if they are hidden it becomes harder for the body to detect that bacterium. Often the presence of a capsule will increase the virulance of a pathogen)The human body does not contain peptidoglycan and in fact produces an enzyme called lysozyme which attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also much more susceptible to penicillin.