44 THOMPSON YATES AND JOHNSTON LABORATORIES REPORT 
After cultivating T. lewisi, attention was paid to T. brucei, using blood agar in 
the proportion 2 : 2 and 2 : I ; the third attempt proved successful. This culture 
was run through five generations before it died outtrom the effects of contamination. 
Efforts were then directed to T. equiperdum, T. equinum, and T". evansi. The culture 
of T. equiperdum was difficult, as the parasites were not present in large numbers, but 
at the fifth attempt two tubes out of nineteen inoculated with blood and exudate fluid 
from a pup, showed motile trypanosomes on the eleventh day. One tube was 
infected with heart blood, the other with oedema fluid obtained from the subcutaneous 
tissue of the thigh. The heart blood tube showed evidences of growth and the 
formation of reduplication forms, but only one in thirty fields, while at the time of 
inoculation large numbers of these forms were seen. The oedema fluid on the 
seventeenth day showed many divisional forms undergoing division. These forms 
were agglomerated together into clumps, not rosettes, as after the manner of T. lewisi 
in culture ; many single well-preserved forms were seen, but the motility of all was 
much lessened. Many deformed and motionless, granular, degenerated trypanosomes 
were seen. At this time a" pup used for keeping the strain running in the laboratory 
died. Resource had therefore to be made to the cultures. The total contents were 
taken and injected into the peritoneal cavity of a puppy. On the eleventh day after 
the inoculation a slight rise of temperature was noticed, but no parasites could be 
found. Nineteen days later, i.e., thirty days from date of inoculation, no parasites 
having been seen it was killed and the whole of its blood inoculated into another pup. 
This pup after a prolonged incubation became infected. The medium used in this 
instance was chicken-broth, in the proportion of meat one to water two with o'5 per 
cent, peptone and C25 per cent, sea-salt adcied, and rendered faintly alkaline, 
agar 2*5 per cent., and defibrinated rabbit blood 2 : 1. 
T. equinum. — Many failures with this parasite have to be recorded. Cultures 
were made on various strengths of beef-broth agar, with different proportions of 
blood, peptone, etc., with no result. Rats, mice, rabbits, and guinea-pigs were used, 
the animals being killed at all stages of the disease. The trypanosomes quickly died 
off. One effort yielded a positive result on a chicken-broth agar tube of similar 
composition to that used for the T. equiperdum culture. It was one tube out of twelve 
inoculated from a rabbit, which had died about three-and-a-half hours before the 
inoculation ot the cultures. The trypanosomes were in the average about one to 
eighteen fields. The tubes examined showed no growth, after the twelfth day only dead 
parasites were found. These tubes were left in the incubator at 22° C, and were not 
examined till the twenty-ninth day, when the majority were found to be overgrown 
with mould. One was not and it contained a few trypanosomes, many of them being 
paired, looking apparently healthy with fair motility. A few transplants were made 
into fresh blood agar tubes, and the rest of the culture was used to inoculate a rat, 
which became infected after an incubation of eight-and-a-quarter days. The trans- 
plants never grew. Further efforts failed to produce cultures. 
