(5) Rinse well, dry, and mount. Films upon slides may be examined with an oil-immersion objective without a cover-glass.
(2) Ehrlich's Triple Stain.—This has been the standard blood-stain for many years, and is still widely used. It is probably the best for neutrophilic granules. It is difficult to make, and should be purchased ready prepared from a reliable dealer. Nuclei are stained blue or greenish-blue; erythrocytes, orange; neutrophilic granules, violet; and eosinophilic granules, copper red. Basophilic granules and blood-plaques are not stained (see [Fig. 85]).
Success in staining depends largely upon proper fixation. The film must be carefully fixed by heat: underheating causes the erythrocytes to stain red; overheating, pale yellow. The staining fluid is applied for five to fifteen minutes, and the preparation is rinsed quickly in water, dried, and mounted. Subsequent application of Löffler's methylene-blue for one-half to one second will bring out the basophilic granules, and improve the nuclear staining, but there is considerable danger of overstaining.
(3) Wright's Stain.—Recently the polychrome methylene-blue-eosin stains, dissolved in methyl-alcohol, have largely displaced other blood-stains for clinical purposes. They combine the fixing with the staining process, and stain differentially every normal and abnormal structure in the blood. Numerous methods of preparing and applying these stains have been devised. Wright's stain is one of the best, and is the most widely used in this country. Directions for preparing it are given in most of the newer large text-books upon clinical diagnosis. The practitioner will find it best to purchase the stain ready prepared. Most microscopic supply houses keep it in stock. The method of application is as follows:
(1) Without previous fixation, cover the blood film with the stain, and let stand one minute.
(2) Add water, drop by drop, until a delicate metallic scum forms upon the surface. Let this mixture remain on the preparation for two or three minutes.
(3) Wash in water until the better spread portions of the film have a pinkish tint.
(4) Dry between filter-papers and mount.
The stain is more conveniently applied upon cover-glasses than upon slides. Films much more than a month old do not stain well. In some localities ordinary tap-water will answer both for diluting the stain and for washing the film; in others, distilled water must be used. Different lots of Wright's fluid vary, and a few preliminary stains should be made with each lot to learn its peculiarities. The principal variation is in the amount of water which must be added to obtain the iridescent scum. Sometimes eight or more drops must be added after the scum appears.
When properly applied, Wright's stain gives the following picture (Plate VI): erythrocytes, yellow or pink; nuclei, various shades of bluish-purple; neutrophilic granules, reddish-lilac; eosinophilic granules, bright red; basophilic granules of leukocytes and degenerated red corpuscles, very dark bluish-purple; blood-plaques, dark lilac; bacteria, blue. The cytoplasm of lymphocytes is generally robin's-egg blue; that of the large mononuclears may have a faint bluish tinge. Malarial parasites stain characteristically: the cytoplasm, sky-blue; the chromatin, reddish-purple.