MEDIUM FOR CULTIVATING TRYPANOSOMA BRUCEI. 175 



flask was very rich in trvpauosoines, apparently an active nmltiplication 

 taking place. Several transplants were then made. None of these de- 

 veloped, and on subsequent examinations it was found that the original 

 culture had died out also. It seems probable that it would have lived 

 twenty days and more, had it not been disturbed so early. This seems 

 especially probable, in view of the fact that positive cultures, were 

 obtained in the trials immediately preceding and following, in both of 

 which this same medium was employed. 



In trial ninety-two, only one flask was inoculated, with the blood of a 

 rat dead of nagaua. Examinations on the fifteenth and on the twenty-fifth 

 day were negative, but on the twenty-eighth day active trypanosomes 

 were found. The sub-culture, however, failed. 



Trial ninety-six was the next positive cuture. In this case nine flasks 

 of blood agar were inoculated, each with about one-half drop of blood 

 draw^n from the living heart of an anaesthetized nagana rat. Each flask 

 contained a mixture of blood and agar in the ratio of 2 : 1, the agar 

 being slightly different in each in respect to the amount of meat ex- 

 tractives and common salt present. 4.50 g. of chopped beef were ex- 

 tracted with 4.50 c.c. of water in the usual way. Agar was made directly 

 from this by adding pepton, agar and 10 c.c. N Na.,CO.., per liter, and this 

 is designated as 1:1 meat agar. Other agars were made from this meat 

 extract after it Avas diluted with water, thus a 1 : 2 and 1 : 10 meat- water 

 agar were prepared, and finally one with distilled water in place of the 

 meat extract. Each variety was further subdivided by the addition of 

 salt to one portion, while the other was made without salt. Five of the 

 flasks gave positive growth, four were negative; and the line between 

 the positive and the negative gave the first certain information in regard 

 to the essential constituents of the medium. The agars with 1 : 10 meat- 

 water (with one per cent, salt and with none) both gave positive results. 

 The agars with 1 : 2 meat-water (with i/o per cent, salt and without salt) 

 both gave positive results ; whereas the 1 : 2 meat-water with 1 per cent, 

 salt and the two 1:1 meat-water agar flasks remained negative; indi- 

 cating that an excess of meat extractives is injurious in some way and 

 that 1 per cent, salt is unfavorable. The 1 : 10 meat-water agar was bet- 

 ter than distilled water agar, giving a richer and more lasting growth, 

 as is seen in the interesting fact that one of these 1 : 10 flasks was suc- 

 cessfully transplanted after fifty-three days. 



Of the two agars without meat (with 1 per cent, salt and without salt) 

 one gave a positive result and the other dried out. From the fact that 

 the medium used in the latter case, two days later, gave a growth, it is 

 likely that a positive result would have been obtained in this set, had 

 not desiccation taken place. 



This experiment was repeated (trial ninety-nine), using a mouse as the 

 source of the inoculation. Here again, five of the nine flasks gave good 

 cultures, exactly conflrming the previous set. The 1:10 meat-water 

 agar gave the best cultures of the lot. 



In trial 101 two flasks were inoculated with the blood from a rat dead 

 of the disease. The medium in these flasks was a blood-agar 2 : 1, the 

 agar in one case containing 10 g., in the other 5 g. NaCl per liter. The 

 latter gave a good culture, while the former was negative. This was 

 another demonstration of the harmful effect of too much salt. 



Of the next thirteen trials, six gave positive results, and of these 



