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Gram staining

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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. Heat fix the specimen.
  2. 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.
  3. Rinse off with water.
  4. Add iodine solution (1% iodine, 2% potassium iodide in water]]) for 1 minute. This acts as a mordant[?] and fixes the dye.
  5. Rinse with water.
  6. 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.
  7. Rinse with water.
  8. Apply a suitable counterstain. Opinions vary as to the best choice but suitable stains include safranin[?] or fuchsin[?].This stains the gram negative organisms.
  9. Blot gently and allow to dry. Do not smear.

Results:
Inspect the slide under a microscope
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 surrounded by an outer membrane containing lipids.

As a rule of thumb (which has exceptions), Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane acts as "camouflage"; 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.



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