Gram's Method

GRAM'S METHOD, proposed by Gram in 1884, is a widely used method for staining certain species of bacteria. It consists of staining the preparation with a rosaniline dye (Methylviolett, Krystallviolett, Gentian-violett, Viktoriablau), treating it with Lugol's solution, and then differentiating with alcohol. Some bacteria stain a black-blue color, whereas other bacteria, as well as tissue elements (with the exception of the chromatin of dividing nuclei, granules of basophilic cells, and the stratum corneum of the epidermis), are decolorized by alcohol. All decolorized tissue elements and microorganisms are counterstained with a contrasting dye (eosin, fuchsin, Neutralrot, etc.). Bacteria that retain the stain are called Gram-positive, and those that are decolorized are called Gram-negative. The following are required for staining: 1. Ehrlich's aniline-water gentian violet solution (keeps for no more than three days). To 100 cm³ of 4 percent aniline water, add 11 cm³ of a saturated alcoholic solution of gentian violet. 2. Lugol's solution—J 1.0, KJ 2.0, H₂O 300.0. Instead of gentian violet in aniline water, its carbolic solution is now used: 1.0 of gentian violet is dissolved in 10 cm³ of absolute alcohol, 2.0 of carbolic acid is added, and then 100 cm³ of water. - Staining of smears: 1. Stain the fixed smear for 3–5 minutes, strictly using only freshly filtered dye. 2. Thereafter, without washing, treat with Lugol's solution for 1 minute. 3. Pour off the Lugol's solution and transfer the smear into alcohol until clouds of dye cease to come off. 4. Rinse thoroughly with water (a properly decolorized preparation should have a steel-gray color after rinsing). 5. Counterstain for 3–5 minutes with Pfeiffer's carbol fuchsin, a 1.5 percent Neutralrot solution, or a 2 percent aqueous solution of vesuvin. - Staining of sections: 1. Lithium carmine for 30 minutes to 1 hour. 2. Rinse in water. 3. Carbolic or aniline gentian violet for 5–10 minutes. 4. Lugol's solution for 1–2 minutes. 5. Differentiate with alcohol until clouds of dye cease to come off; oil (clove, bergamot), xylene, mounting in balsam. This basic Gram method has many modifications, which reduce to replacing alcohol with other decolorizing fluids and gentian violet with other pararosaniline dyes. (See also Fibrin, staining.) The theory of this staining is not fully elucidated. There are two opinions. Some (Stoelzner, F. Reichert) explain it purely chemically: fatty acids, lipoids, and waxy substances, thanks to iodine, vigorously fix amidophenylmethanes and form alcohol- and acid-resistant compounds with the dye. Others (Hottinger) believe that the varying staining capacity of bacteria depends on the degree of dispersion of nucleoproteins within the cell. The smaller the degree of dispersion, the more positive the reaction (staining), because the optical resolution of the stained particles is directly dependent on the size of these particles.