To each 100 cc. of the filtered mixture add 500 cc. of a 0.1 per cent aqueous solution of yellow water-soluble eosin, and mix thoroughly. Collect on a filter the abundant precipitate which immediately appears. When the precipitate is dry, dissolve it in pure methyl alcohol (Merck’s) in the proportion of 0.1 grm. to 60 cc. of the alcohol. To facilitate solution the precipitate is to be rubbed up in a porcelain dish or mortar with a spatula or pestle. This alcoholic solution is the staining solution. It should be kept in a tightly-stoppered bottle. Should it become concentrated through evaporation methyl alcohol in proper quantity should be added.
1. Cover film with a given quantity of staining fluid by means of a medicine dropper.
2. After 1 minute add to the staining fluid on the film the same quantity of distilled water by means of the medicine dropper, and allow the mixture to remain for 2-3 minutes according to the intensity of the stain desired. A longer period of staining may produce a precipitate. Eosinophile granules show best after short staining. The quantity of diluted stain on the preparation should not be so great that some of it runs off.
3. Wash the preparation in water for 30 seconds or until the thinner portions of the film become yellow or pink in color.
4. Dry, and mount in balsam.
Films more than a few hours old do not stain as well as fresh ones.
The red cells are orange or pink in color. Polychromatophilia and punctate basophilia or granular degeneration are well shown. Nucleated reds have deep-blue nuclei, and their cytoplasm is usually bluish. The lymphocytes have dark purplish-blue nuclei and cytoplasm of a robin’s-egg blue, in which a few dark-blue or purplish granules are sometimes present. The nuclei of the polynuclear neutrophilic leukocytes are dark-blue or dark lilac-colored, the granules reddish-lilac. The eosinophiles have blue or dark lilac nuclei, a blue cytoplasm and eosin-red granules. The large mononuclear leukocytes have a dark lilac or blue nucleus, cytoplasm pale blue or blue with dark-lilac or deep purple granules. Mast-cells have purplish or dark-blue nuclei, bluish protoplasm and coarse dark purple or black granules. Myelocytes have dark blue or lilac nuclei, blue cytoplasm, and dark-lilac or reddish-lilac granules. Blood platelets are blue with small violet or purplish granules in their central portions. Malarial parasites have a blue body and lilac or red chromatin. Spirochæte pallida is pale blue.
Giemsa’s Method.
a, One per cent water solution of azur-blau; b, one per cent watery solution of eosin. For staining take 1 cc. of b, add 10 cc. of water, and then 1 cc. of the azur-blau solution. Stain 10 minutes to 1 hour.
Giemsa’s Old Method.