168 PUBLIC HEALTH BACTERIOLOGY 



alkali is used in its preparation. Equal parts of watery 

 solutions of (a) Gruebler's water-soluble eosin (1-2 per cent), 

 and (b) Gruebler's medicinal methylene-blue (i per cent), 

 are mixed, and the mixture allowed to stand for twenty- 

 four hours. A coarse granular precipitate forms, is filtered 

 off, dried at 55 C., washed with distilled water, filtered, 

 washed again, filtered, and dried. Of the dried powder, 

 0-5 grm. is dissolved in 100 c.c. of Merck's methyl alcohol. 

 In use, a few drops are placed on the dried unfixed film for 

 one to three minutes, then poured off, and the slide is 

 washed with distilled water until pink in colour. Dry with 

 filter-paper, and mount in xylol balsam. 



Leishman's Stain. The methylene-blue is alkalinized 

 with 0-5 per cent of sodium carbonate, weaker eosin oolution 

 is used, and the technique of preparation is varied. The 

 stain will keep for a long period. In use, a few drops are 

 placed on the unfixed preparation for fifteen to thirty 

 seconds, the film being tilted from side to side to prevent 

 drying at any part. In this way the film is both fixed (by 

 the methyl alcohol) and stained by one operation. About 

 twice as much distilled water is now added, and the diluted 

 stain allowed to act for five minutes longer. Now wash 

 in distilled water, mount, and examine. 



Giemsa's Stain and Method. This is a modified 

 Romanowsky, of great value in staining Spirochaeta pallida, 

 Vincent's spirilla, protozoa, and Negri bodies. The stain 

 used is methyl-azure, which Giemsa believes to be the essen- 

 tial constituent of the Romanowsky stain. In use, the film 

 is first fixed with alcohol, dried, covered with the stain, dilu- 

 ted, well washed, drained, dried, and mounted. For ordinary 

 staining, fifteen minutes are enough ; for the spirochaete and 

 Negri bodies, one to twelve hours may be necessary. 



Staining. By Romanowsky, red cells are stained 

 orange to pink ; eosinophile granules, red ; neutrophile 

 granules, yellow to lilac ; nuclei, shades of violet ; blood 

 platelets, purplish ; malarial parasites, blue ; chromatin, 

 red to rose-pink. 



Blood Films. May be made on cover-slips or on slides. 

 With cover-slips, touch one to the exuding blood, drop 

 it on another, and then draw the cover-slips apart. For 

 slides, touch a drop of blood near one end of slide, and smear 



