512 PROTOZOA 



eating fluid of the puncturing apparatus will carry sporozoites into 

 the blood. The stages of development in the mosquito require about 

 seven days. 



Staining Methods. EHRLICH'S TRIACID STAIN. Unsuitable for 

 demonstration of malarial parasites. 



JENNER'S STAIN. Clear pictures of parasites, which, however, 

 show no chromatin; hence unsuitable for study of finer differential 

 points. 



NocHT-RoMANOWSKY METHOD. Very suitable, but requires accu- 

 rate mixture of several fluids just before using, which then have to be 

 thrown away. 



WRIGHT'S STAIN. Practically identical with Goldhorn's one-solu- 

 tion stain (vide infra), but less rapid; powerful chromatin stain and 

 general blood stain. 



POLYCHROME METHYLENE BLUE (GOLDHORN). To prepare the stain 

 dissolve 1 gram lithium carbonate in 200 c.c. clean water and add 1 

 gram methylene blue. Shake and dissolve. Pour into porcelain dish 

 over water-bath, stirring frequently until blue color changes to a rich 

 purple. Run through cotton in funnel; make up to 200 c.c. To 100 

 c.c. add 5 per cent, acetic acid until a faint pink is just visible on litmus 

 paper above level of point discolored by the dye. Now add the remain- 

 ing 100 c.c. of dye and allow to stand in open dish for forty-eight hours. 

 Run once more through cotton into clean bottle. 



I have not found it necessary to use distilled water, and have obtained 

 satisfactory results with all the different forms of methylene blue I 

 have been able to obtain during the past five years. I now prefer 

 B-X Gruebler. 



Fix the smear by immersion in commercial wood alcohol for fifteen 

 to thirty seconds; wash well and stain for about ten to fifteen seconds 

 in polychrome; wash and stain for from fifteen to sixty seconds in -^g- 

 per cent, aqueous eosin. Wash again in water and dry in air without 

 heat. Body of parasites blue; chromatin is red to purple. 



Results may be varied by using polychrome or eosin for different 

 lengths of time. Admirable preparations may be obtained, even when 

 there is precipitation, by just rinsing the smear a little in 50 per cent, 

 ethyl alcohol. This will remove any precipitation. 



The simplest method of staining the parasite is probably the follow- 

 ing, recommended by me for the staining of mast-cells : Saturate wood 

 alcohol with methylene blue. Pour on dry smear for five to ten seconds 

 and wash in water. Parasite blue. 



GOLDHORN'S ONE-SOLUTION STAIN. To Goldhorn's polychrome 

 methylene blue (vide supra) add weak, watery ( to Y V per cent.) eosin 

 until the filtrate is of a pale-blue color; the exact amount of eosin will 

 depend upon the degree of alkalinity of the polychrome and upon the 

 amount of unaltered methylene blue in the polychrome. 



The precipitate is washed with water and dried without heat and 

 protected from dust. When absolutely dry it is dissolved in commer- 

 cial wood alcohol, making a 1 to 2 per cent, solution. 



