750 



THE ANIMAL PARASITES OF MAN 



the detection of various Protozoa parasitic in the blood. Such 

 stains, however, leave something to be desired in the revealing of finer 

 cytological details. Other stains, more especially the haematoxylins, 

 must be employed for cytological purposes. 



Formulae of some of the principal Romanowsky and haematoxylin 

 stains may now be given. 



The underlying principle of the Romanowsky Stain is the reaction 

 between alkaline methylene blue and eosin, forming the so-called 

 eosinate of methylene blue which stains chromatin purplish-red. 

 A solution of medicinal methylene blue after having been subjected 

 to the action of an alkali, such as sodium carbonate, becomes partly 

 converted into certain derivatives, the chief of which are methylene 

 azure and methylene violet. These substances are also present in 

 matured polychrome methylene blue. 



The formula of a slightlv modified Romanowsky Stain which gives 

 excellent results is given below : 



Two stock solutions are required 



Solution A. Methylene blue, pure medicinal ... rogrm. 



Sodium carbonate ... ... ... ... o'5 ,, 



Water ... ... ... ... ... locro c.c. 



Keep in a warm incubator for two or three days, until the solution is distinctly purple in 

 colour. It improves with age. 



Solution B. Eosin, water soluble, extra B. A. ... ... rogrm. 



Water ... ... ... ... ... 1,000-0 c.c. 



This solution must be kept in the dark, in dark-tinted (amber-coloured) bottles, as unfortu- 

 nately it is decolorized by light. 



Before use each stock solution must be diluted. Thus, make up 

 5 c.c. of each stock solution to 100 c.c. by adding distilled water. 

 For staining, i volume of solution A is added to 2 or 3 volumes of 

 solution B. Mix thoroughly by shaking, pour the mixture over the 

 film, previously fixed in absolute alcohol, and stain for ten to fifteen 

 minutes. Wash carefully in running water, then dry. The cytoplasm 

 of a protozoan parasite will be stained blue, the chromatin purplish- 

 red and vacuoles or very tenuous protoplasm will remain colourless. 



The exact proportions of solutions A and B, which must be 

 mixed together, should be determined by experiment. Freshly mixed 

 stain must be used on each occasion. 



Leishman's Stain is the precipitate resulting from the interaction 

 of alkaline methylene blue and eosin. The washed and dried 

 precipitate is collected and dissolved in pure methyl alcohol, which 

 acts as a fixative ; 0-015 grm. of Leishman powder may be dissolved 

 in 10 c.c. of methyl alcohol for staining films. The film is covered 

 with the solution for one minute, twice the volume of water is then 

 added and mixed with the stain on the slide. The staining is then 



