TRYPANOSOMES, TRYPANOSOMIASIS, AND SLEEPING SICKNESS 45 
T.evansi. — Here, as with the other trypanosomes, numerous cultures from the blood 
of different animals have been made from time to time. Seven tubes at 22° C. showed 
growth up to the nineteenth and twenty-second days, but these were contaminated 
with bacteria. One tube showed a growth on the twenty-ninth, another on the thirty- 
third, still another on the thirty-seventh day. These three latter were all room 
temperature cultures, the efforts to transplant the twenty-ninth and thirty- seventh day 
cultures proved failures, nor did the original tubes show any more growth when placed 
in the 22° C. oven, all the trypanosomes quickly dying off. The thirty-third day tube 
was used for inoculating a rat and a guinea-pig without success ; the few remaining 
drops were used for sub-cultures, but no growth resulted from them. The trypano- 
somes in these three tubes, especially that of the thirty-third day, resemble the 
description which Now, McNeal, and Hare have given of their efforts to cultivate 
the Surra trypanosome of the Philippines. The parasites were few in number, were 
all single and actively motile, the majority showed dark granules at their posterior 
ends, and resembled the first two drawings of Fig. 1 and the lower of Fig. 2 (Now, 
etc.) The flagella seemed longer and thinner than in the normal parasites obtained 
from an infected animal. 
T. gambiense. — The difficulties attending the cultivation of this organism are 
many. The parasite does not as a rule appear in great numbers in the blood. If an 
animal is infected with the parasites of Nagana, Surra, etc., once they appear they will 
augment from day to day until the blood fairly swarms. With T. gambiense, on the 
other hand, rats rarely show very large numbers in their blood, and periodicity being 
a feature of this disease, the parasites present one day will be found to be absent the 
next day. This feature is constant in all the animals inoculated. Monkeys, pups, 
and kittens, however, usually have more parasites in their blood than do the other 
animals. Advantage as far as possible has been taken of this fact. With the ordinary 
blood-agar medium no growth takes place, and the trypanosomes die off at once. 
Veal and chicken-meat infusion has been found to be more suitable than beef infusion. 
One to 1 "5 per cent, peptone with 0-25 to 0-5 per cent, sea-salt is added and agar 
from 2*5 to 3*5 per cent. Various animals have been used for bleeding — horse, goat, 
sheep, dog, and rabbit. Almost as satisfactory results have been obtained from the 
blood of the goat and sheep as from that of the rabbit. The blood has been used in 
various proportions — 2 : 2, 2 : 1, 3 : 1 — the most satisfactory proving 2 : I or 3 : 2. 
The trypanosomes for the first few days appear to tolerate the media, but after 
the fourth to seventh days numerous degenerated and dead ones are seen. In 
cultures kept at 22 0 to 23 0 the tubes will at the fourteenth to sixteenth day either 
contain dead trypanosomes or a few partially motile ones. These motile ones may 
present the normal outline of the parasite, but granules are usually present in the 
anterior portion of the body. The macronucleus of these forms is often of a 
granular appearance, staining unevenly, and sometimes fissure-like breaks may 
