The following are the general methods of employing these reagents for the purpose of staining organisms in sections. Special methods are required for special organisms, but one or two only can be given.

Weigert’s method.—The sections must be placed in a freshly made one per cent. aqueous solution of methyl violet, gentian violet, fuchsine, &c. The solution may be kept at the temperature of the body in an incubator. The organisms will often stain more readily if the section be passed through a 1 in 2000 solution of corrosive sublimate before putting it into the staining fluid. After staining the section is washed in distilled water and then in methylated spirit until it appears almost decolourised. Some prefer to decolourise the tissues by washing in a half per cent. solution of acetic acid instead of methylated spirit. Practice is required before the correct time for decolourising is accurately estimated. The beginner should float a section rapidly on the slide now and then, put on a cover-glass and examine it under a low power to see if the decoloration has been carried far enough. A contrast stain may then be used, such as picrocarmine, after which the section may be mounted in Farrant’s medium: or a weak solution of another aniline colour may be used as a counter stain, after which the section is clarified in xylol, and mounted in balsam dissolved in xylol.

Gram’s method.—Place some aniline oil in a test tube and add ten times its volume of distilled water. Close the end with the thumb and shake very thoroughly. Filter ninety drops into another clean test tube, and add ten drops of a saturated solution of gentian violet or some similar dye. Filter the mixture into a watch glass. Stain sections in it for from three minutes to half-an-hour according to the temperature,—the shorter time for the incubator at 100°, the longer when the sections are stained at the ordinary temperature of the room. Wash in distilled water, and transfer to Gram’s iodine solution until they become black, usually in a few minutes. They are then decolourised in absolute alcohol. This often takes some time. It may be hastened, as Crookshank suggests, by placing the section in clove oil, returning to alcohol, and so on.

Ehrlich’s modification of Gram’s method. The contrast stain is here used first.

Stain the section (e.g., that of a mitral valve in a case of ulcerative endocarditis), in an alcoholic solution of eosine (1 in 1500). Transfer to a solution of some aniline dye, such as gentian violet, dissolved in aniline oil water, exactly as in Gram’s method. The section floats on the surface and spreads out, owing to the alcohol diffusing out. Stain for about twenty minutes. Wash the section in water, and float out (p. [55]) on a glass slide. Allow the water to drain off and add Gram’s iodine slowly from a pipette so as not to disarrange the section. When the section has become quite black pour off the Gram’s solution. Remove all superfluous fluid from the slide with blotting paper, and dry the section by carefully and firmly pressing on it a folded piece of blotting paper. If this is done with care the section need not be injured in the least. Decoloration is effected on the slide with aniline oil, instead of alcohol as in the preceding method. The slide is rocked about so that the colour may be evenly discharged by the aniline. When no more colour comes away, the aniline oil is poured off, the section clarified in xylol, and mounted in Canada balsam.

As soon as the section is decolourised it may be treated with a contrast stain, the most suitable being alcoholic solutions of eosine or Bismarck brown if a blue stain has been employed, or methylene blue if fuchsine has been the first stain used.

The following will be found the most useful stains and contrast stains:—

Stains. Contrast Stains.
Gentian violet.
Methyl violet.
Methylene blue.		  Picrocarmine.
Eosine.
Bismarck brown.
Safranine.
Magenta.
Fuchsine.		Methylene blue.
And vice versâ.