STUDIES ON AVIAN H^EMOPROTOZOA. 655 



longer than half an hour; I have always found a longer 

 period to be detrimental to the staining. I found this 

 method of fixation to be the best for giving a correct idea of 

 the size and general appearance and morphology of the 

 parasites, whether trypanosomes or intra-cellular forms ; and, 

 for the sake of uniformity, all my figures ai-e of individuals 

 so fixed, so that one may be compared at once with another, 

 without any ulterior considerations having to be taken into 

 account. 



Staining. — Nearly all my preparations are stained by 

 some variety of the Romanowsky method. I have made use 

 of two stains (or stain mixtures) : one of them is the 

 ordinary Giemsa sohition, the other is a combination which I 

 have found particularly good for cultural forms. The Giemsa 

 solution was always used in the customary proportion of one 

 diop of the stain to 1 c.c. of water. The length of time for 

 which slides were allowed to stain varied in different cases. 

 The period required to give the best results varies con- 

 siderably at times, even when the smears have been fixed, so 

 far as can be told, in exactly the same manner. For one 

 thing, the temperature made considerable difference. I used 

 the stain at the laboratory temperature, and whereas in the 

 winter and spring forms in the blood required to be stained 

 for twelve to eighteen hours to be successful, in the summer 

 they would be excellently stained in three or four hours. 



Cultural forms stain much quicker than the parasites in the 

 blood, and need only about fifteen to twenty minutes in the 

 stain ; but the Giemsa solution was found to be not nearly so 

 suitable for smears of cultural forms as the other method 

 which I adopted; by this latter method the parasites them- 

 selves are more sharply stained, while the coagulated layer, 

 which is often unpleasantly prominent as a reddish ground- 

 substance, after Giesma, hardly stains at all. 



In my particular method three solutions are made use of, 

 as follows : 



(1) A 1 per cent, solution of azure I, in equal parts of 

 glycerine and methyl-alcohol. 



