Before being stained the blood must be fixed to the slide either by heat or by some chemical.

Heat may be used in all cases except when Wright's stain is employed; it must be used with Ehrlich's triple stain to get good results. The films should be exposed to a dry heat of from 100° to 110° C. for ten to fifteen minutes.

Chemicals

The smears are fixed in absolute alcohol or ether or a mixture of equal parts of the two for five to ten minutes. Then dried and stained.

Stains. Wrighfs

It is best to procure this stain ready made. It is employed as follows, as no previous fixing is necessary:

The unfixed film is covered with the solution and stained for 19 a minute. Distilled water is added drop by drop until a metallic scum appears on the surface of the fluid and is allowed to remain two to three minutes. Then wash the film, which is a deep blue or purplish color, until it becomes yellowish or pink. Dry between blotting-paper and mount in balsam.

The erythrocytes will be stained orange or pink; the nuclei of the leukocytes, blue; neutrophile granules, lilac; eosinophile granules, pink; fine basophile granules, deep blue; coarse mast-cell granules, deep purple. The malarial organism stains blue.

Ehrlich's Triacid

Best bought ready made.

After fixing with heat, stain five to eight minutes; wash in running water, dry, and mount.

Erythrocytes stain orange; nuclei of the leukocytes, greenish blue; neutrophile granules, violet or lavender; eosinophile granules, copper red; basophile granules are unstained.

Polychrome methylene-blue (Goldhorn's) is bought ready made.

After fixation for fifteen to twenty seconds in methyl-alcohol, wash in water and, without drying, stain for one to two minutes. Wash thoroughly in running water, dry with blotting-paper, and mount.

This method shows very well granular degenerations of the erythrocytes, the nuclei of erythroblasts and leukocytes, basophilic granules, and most bacteria. It is a very good stain for the malarial organism. If the film is first stained for ten to fifteen seconds in a 0.1 per cent, aqueous solution of eosin, washed, and then the methylene-blue used, a very good picture of the acid-coloring elements is given.

Eosin And Methylene-Blue

Fix the smear in absolute alcohol alone or mixed with an equal quantity of ether. Stain in a 0.5 per cent, solution of eosin in absolute alcohol, to which an equal quantity of water is added, for about five minutes without heating. Wash and dry, then counterstain in a saturated aqueous solution of methylene-blue for about one minute. Wash again, dry, and mount.

Gives a good picture of the nuclei of the basophilic granules and of the malarial organism: eosinophile granules stain red; the protoplasm of the polymorphonuclear leukocytes colors a slight pink; the granules remaining unstained.