The best known and most widely used bacteriological staining method, it is almost always the first test performed for the identification of bacteria.
The primary stain of the Gram's method is crystal violet. Crystal violet is sometimes substituted with methylene blue, which is equally effective. Bacteria are first stained with the crystal violet and then treated with Lugol's solution, consisting of 1 part iodine, 2 parts potassium iodide, and 300 parts water.
After being washed with ethyl alcohol, the bacteria will either retain the strong blue colour of gentian violet or be completely decolourised. Sometimes a counterstain such as fuchsine or eosin is applied to give the decolourised bacteria a red colour to make them more visible.
Bacteria that retain the crystal violet stain are known as gram-positive; those that do not are known as gram-negative. Organisms that sometimes retain the crystal violet colour and sometimes do not are known as gram-variable.
The difference between Gram-positive and Gram-negative bacteria is in the permeability of the cell wall to these "purple collared iodine-dye complexes" when treated with the decolourising solvent.
Typical gram-positive bacteria are those staphylococci that produce boils; typical gram-negative bacteria are the bacilli that cause whooping cough; typical gram-variable bacteria are the bacilli that cause tuberculosis.
- H. C. J. Gram:
Über die isolirte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten.
Fortschritte der Medizin, Berlin, 1884, 2: 185-189.