The formula of a slightly modified Romanowsky Stain which gives excellent results is given below:—

Two stock solutions are required—

Solution A.—Methylene blue, pure medicinal1·0 grm.
Sodium carbonate0·5  "
Water100·0 c.c.

Keep in a warm incubator for two or three days, until the solution is distinctly purple in colour. It improves with age.

Solution B.—Eosin, water soluble, extra B.A.1·0 grm.
Water1,000·0 c.c.

This solution must be kept in the dark, in dark-tinted (amber-coloured) bottles, as unfortunately it is decolorized by light.

Before use each stock solution must be diluted. Thus, make up 5 c.c. of each stock solution to 100 c.c. by adding distilled water. For staining, 1 volume of solution A is added to 2 or 3 volumes of solution B. Mix thoroughly by shaking, pour the mixture over the film, previously fixed in absolute alcohol, and stain for ten to fifteen minutes. Wash carefully in running water, then dry. The cytoplasm of a protozoan parasite will be stained blue, the chromatin purplish-red and vacuoles or very tenuous protoplasm will remain colourless.

The exact proportions of solutions A and B, which must be mixed together, should be determined by experiment. Freshly mixed stain must be used on each occasion.

Leishman’s Stain is the precipitate resulting from the interaction of alkaline methylene blue and eosin. The washed and dried precipitate is collected and dissolved in pure methyl alcohol, which acts as a fixative; 0·015 grm. of Leishman powder may be dissolved in 10 c.c. of methyl alcohol for staining films. The film is covered with the solution for one minute, twice the volume of water is then added and mixed with the stain on the slide. The staining is then continued for five to ten minutes, and the film is finally washed with water.

Giemsa’s Stain.—This should be procured ready made. Azure II is a mixture of methylene azure and methylene blue. (Methylene azure is sometimes known as Giemsa’s Azure I.) The formula given by Giemsa himself in 1912 is:—