To use the stain, a few drops are poured on the blood film and covered to prevent evaporation and precipitation. In three minutes, or a little longer, the stain is rapidly poured off, and afterwards the film is washed in distilled water from five to ten seconds until it assumes a pink colour. It is then dried high over the flame, or in the air, and mounted in xylol balsam.
Leishman's stain.—Two solutions are prepared. A, a 1-per-cent. solution of medicinal methylene blue (Grübler) in distilled water, rendered alkaline by 0·5 per cent. sodium carbonate. Heat this to 65° C. for twelve hours, and allow it to stand at the room temperature for ten days. B, eosin, extra B.A. (Grübler), 1 in 1,000 of distilled water. Mix equal volumes of A and B and stand for six to twelve hours, stirring occasionally. Collect the flocculent precipitate in a filter, and wash with distilled water. Dry and powder the filtrate, which has a green metallic lustre and contains the active ingredient of the Romanowsky stain. (This powder has been placed on the market, and can be procured in condensed tablets called soloids.) Make a 0·15-per-cent. solution of the dye in methylic alcohol, and keep in a stoppered bottle.
To use the stain, drop three to four drops of the solution on the unfixed blood film. After about half a minute to one minute or longer add six to eight or more drops of distilled water and mix them by moving about the slide. A precipitate forms at once in the water in successful slides. After five minutes or longer wash off the stain with distilled water, leaving a few drops of the water on the film for over a minute. Dry without heat, and mount in xylol balsam.
Eosin-azur stain, sold by Burroughs and Wellcome in tabloid form, is an azur stain of somewhat similar composition to that of Leishman. One tabloid is dissolved in 10 c.c. of pure methyl alcohol and is used as above; it gives similar but more brilliant results. It may also be used as Giemsa's stain dissolved in 5 c.c. of a mixture of equal parts of glycerin and pure methyl alcohol.
Giemsa's stain.—Azur II. eosin 3·0 grm., azur II. 0·8 grm., dried, powdered, and dissolved in 250 grm. of glycerin, to which are subsequently added 250 grm. of methyl alcohol; heat to 40° C. Shake the mixture and stand for twenty-four hours, and then filter.
Fix the films in methyl alcohol (three minutes). The staining solution is prepared by adding 1 drop of stain to 1 c.c. of distilled water at 30° to 40°. Immerse the film in this for fifteen minutes, wash in stream of water, dry, and mount in balsam.
Laveran's method for staining and preserving permanent preparations. The following solutions are required:—
| Eosin, 1: 1,000 | . . . . | 4 | c.c. |
| Distilled water | . . . . | 10 | c.c.„ |
| Bleu borrel[1] | . . . . . | 1 | c.c.„ |
- ↑ Bleu borrel is methylene blue and silver oxide. It is prepared as follows: In a 150-c.c. glass measure several crystals of
