2316 Journal of Applied Microscopy 



stain coming off in blue clouds, the preparation changing from a violet to an 

 eosin red. Before the last trace of violet is washed out, the slide should be 

 removed to water and thoroughly washed till no more color comes off. It is 

 then dried between filter paper and mounted in balsam. 



Several hematologists have recently separated the active staining ingredients 

 of the Romanowsky neutral stain and have improved and simplified the technique 

 of staining. 



Jcnner (1899) used equal parts of a 1.2 per cent, to 1.25 per cent, solution of 

 Griibler's water-soluble eosin, yellow shade, in distilled water, and of a 1 per cent, 

 solution of Griibler's medicinal methylen blue in distilled water mixed in an open 

 vessel — not in a flask — and thoroughly stirred with a glass rod. The mixture 

 is best left for 24 hours. It is then filtered and the residue dried either in the 

 air or more quickly in an incubator or water bath. When quite dry the residue 

 is powdered, shaken up with distilled water and washed on a filter. It is then 

 dried again and powdered. For use thoroughly shake up 0.5 grams of the pow- 

 der in 100 c. c. of pure methyl alcohol (E. Merck, " for analytical purposes ") 

 and filter. The solution keeps well. 



A few drops of this fixing and staining solution are poured upon the dry 

 blood and covered with a watch-glass to prevent evaporation. After 1 to 3 

 minutes' staining the solution is poured off and the preparation at once rinsed 

 in distilled water until the film has a pink color, which usually takes from 5 to 

 10 seconds. The preparation is then dried and mounted in xylol balsam. 



The red corpuscles are stained a terra-cotta color, the nuclei blue, blood 

 platelets mauve, granules of the polymorphonuclear leucocytes and myelocytes 

 red, granules of the mast cells dark violet, bacteria, filarial and malarial para- 

 sites blue, 



LeisJwian (1901) has modified the methyl alcohol method of Jenner, and has 

 secured a greater differentiation of the blood elements. His staining solution is 

 made as follows : 



Solution A, — Medicinal methylen blue (Griibler's), 1 per cent, solution in 

 distilled water, to which is added 0.5 per cent, of sodium carbonate. Heat the 

 solution to 65°C. in a paraffine oven for 12 hours, then let it stand at room tem- 

 perature for 10 days before use. Solution B, — Eosin extra B. A. (Griibler's), 1 

 to 1000 solution in distilled water. Equal volumes of A and B are mixed in 

 an open vessel and allowed to stand for from 6 to 12 hours, being stirred from 

 time to time with a glass rod. The abundant fioculent precipitate which 

 results is filtered off and washed with distilled water until the washings are color- 

 less or only pale blue, then dried and powdered. This greenish powder with a 

 metallic lustre constitutes, or at least contains, the active staining ingedient in 

 Romanowsky's method. The powder is to be dissolved in methyl alcohol 

 (Merck's " for analysis ") in the proportion of 0.15 per cent. This solution 

 does not deteriorate by keeping. 



Three or four drops of this alcoholic solution are allowed to fall upon the 

 blood film, and are distributed over the whole surface by gently tipping and 

 rotating the cover-glass. No attempt is to be made to check evaporation. 

 After about half a minute, double the quantity of distilled water is added and 



