This is by far the most important staining method in bacteriology. It is a staining technique which is employed for the diagnostic identification of a wide variety of organisms. The mechanism of the Gram stain is not fully understood beyond the identifiable differences in cell wall characteristics between those organisms classified as ‘Gram +ve’ and those classified as ‘Gram –ve’. The Gram +ve organisms are able to retain basic dyes at a higher concentration than the Gram –ve species. Probably, the most important difference is in the permeability of the cell wall during the staining process.
Following staining with crystal violet and treatment with iodine, a dye-iodine complex is formed within the cell. This is insoluble in water but moderately soluble in acetone (or alcohol) which is used as a decolouriser. Under the influence of the decolouriser the dye-iodine complex (blue/black in colour) is retained by the Gram +ve group of organisms but flows freely from the Gram –ve group. Presumably, this is due to the former having a less permeable cell wall. The Gram –ve group can now assume the colour of the chosen counter-stain to distinguish between the two groups.
Gram +ve cocci (Pneumococcus)
Photo: Melville Matheson
Gram stain may show fungal hyphae
Photo: Melville Matheson
