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