a. Water-soluble eosin, 1.25 grms.
Distilled water, 100 cc.
b. Medicinal methylene-blue, 1 grm.
Distilled water, 100 cc.
Mix equal parts of a and b in an open basin, stirring with a glass rod. Let stand for 24 hours; filter; dry the residue at 50°C. Wash residue thoroughly with distilled water and again dry thoroughly. Take 0.5 grm. of the dried powder and dissolve in 100 cc. of pure methyl alcohol. Filter. Solution keeps well.
1. Make blood-film. Dry in air. Do not fix.
2. Cover film with stain, keeping under watch-glass to prevent evaporation Stain 2 minutes.
3. Wash in distilled water until the film has a pink color. Dry in air. Mount in xylol-balsam.
Neutrophile granules are red, eosinophile rose red, basophile granules violet, red blood cells and central portion of blood-platelets are terra-cotta, leukocyte nuclei and granules in red blood cells are blue, protoplasm of nuclei and outer portion of platelets light blue.
4. MODIFICATIONS OF THE ROMANOWSKY METHOD.
A large group of stains has resulted from various applications of the Romanowsky idea of uniting equimolecular proportions of methylene-blue and eosin, and the solution of the dyes so obtained in some suitable solvent. These dyes consist of mixtures of methylene violet, methylene azure, eosinate of methylene blue, etc., and can be obtained from Grübler and Co. under various names, such as Azur-blau, Bleu Borrel, Giemsa’s stain, Leishman’s stain, etc. Hastings, Leishman, Wright and others have combined the Romanowsky method with that of Jenner by dissolving the new dyes obtained by their various modifications in pure methyl alcohol, so as to form a solution that will fix and stain at the same time. Hastings’ stain is a modification of Nocht’s stain; Wright’s stain is a modification of the Leishman-Romanowsky method. The revised directions given by Wright for making and using his stain are here given. Wright’s method and the Giemsa stain possess all of the staining advantages afforded by the variations of the Romanowsky method, and are alone given here. The former is recommended for blood-work, the latter for the staining of protozoa.
Wright’s Blood-stain.
To a 0.5 per cent aqueous solution of sodium bicarbonate add methylene-blue (B.X or medicinal) in the proportion of 1 grm. of the dye to each 100 cc. of the solution. Heat the mixture in a steam sterilizer at 100°C. for one full hour, counting the time after the sterilizer has become thoroughly heated. The mixture should be placed in a flask of such size and shape that the fluid will not be more than 6 cm. deep. After heating, allow the mixture to cool, placing the flask in cold water if desired, and then filter it to remove the precipitate. When cold the fluid should have a deep purple-red color when viewed in a thin layer by transmitted yellowish artificial light. It does not show this color while warm.