CHAPTER XXXVI. 535 



cytoplasm of the parasites is stained faintly blue their nuclei is 

 reddish purple. 



1008. Romanowsky Methods. The Romanowsky methods depend 

 essentially upon the formation of Azur and other oxidation products of 

 methylene blue, either alone or in combination with eosin. The most 

 generally used are Leishman's stain, Wright's stain and the well-known 

 Glemsa's stain. 



1009. Leishman's Stain. This stain is an extremely useful one ; 

 it is best purchased, but may be prepared as described in 784, 

 p. 385. Films are prepared in the ordinary manner, but are merely 

 dried, not fixed. Five to 10 drops of the stain are poured on from a 

 pipette and allowed to act for thirty seconds. An equal quantity of 

 distilled water is then added and the diluted stain allowed to act for 

 a further period of from five to ten minutes. The preparation is then 

 well washed in distilled water, and is dried and mounted in balsam. 

 Red cells are stained pale pinkish, the nuclei of the leucocytes red, 

 parasites blue and their nuclei reddish purple. See 784 et seq. 



1010. Wright's Stain. This stain is best prepared by dissolving 

 Grubler's methylene blue, 1 grm. in 0-5 per cent, sodium bicarbonate 

 100 c.c. The mixture is then heated at 70 to 80 C. for an hour. Cool 

 and add 500 c.c. of a 0-1 per cent, solution of eosin (the yellowish shade 

 water soluble variety). The solution of eosin should be added rather 

 slowly, with constant stirring, till the blue colour disappears and the 

 mixture is purple ; at this stage the fluid should have a metallic appear- 

 ance on the surface, when a finely granular blackish precipitate is 

 formed. This is collected and dried at 37 C. and a 0-3 per cent, solution 

 of this dye is then made in pure absolute methyl alcohol. When required 

 for use it is diluted with methyl alcohol (4 stain, 1 methyl alcohol). 

 Stain as described for Leishman's method. 



1011. Wilson's Stain. This stain also depends upon the production 

 of polychrome derivatives of methylene blue. It gives very satisfactory 

 results when carefully used, closely resembling those obtained in good 

 Giemsa preparations. Two grms. of silver nitrate are dissolved in 

 15 c.c. of distilled water, and to the solution so obtained is added 

 250 c.c. of a freshly -prepared lime water. The mixture should be shaken 

 well and the precipitate of silver oxide collected on a filter and well 

 washed with distilled water. The precipitate is then dried in an oven 

 at a temperature not above 70 to 80 C. The moist silver oxide pre- 

 pared from AgNCXj and NaOH solution may be used in place of the dry 

 product. To the oxide so obtained 2 grms. of methylene blue dissolved 

 in 200 c.c. of 0-5 per cent, sodium bicarbonate solution is added. The 

 mixture is then gently boiled in a porcelain dish for twenty to thirty 

 minutes, stirring from time to time. Pour off one-third of the contents 

 into a cylinder and then add to the remaining solution in the dish an 

 amount of distilled water equal to that poured off. Boil the mixture in 

 the dish again for twenty to thirty minutes. Again pour one-third of 

 the contents of the dish into the cylinder, and boil the remainder for a 



