MEDIUM FOR CULTIVATING TRYPANOSOMA BRUCEI. 177 



The observations given thus far relate more particiilai*iy to the isola- 

 tion of the trypauosorae from its natural habitat, the living animal; 

 that is to say, the isolation of the first generation. When we come to 

 the consideration of sub-cultures and the serial transplantation, a slightly 

 different medium is more serviceable. The change from the animal body 

 to the artificial medium is difficult and most trying, but after the 

 trypanosome has once accommodated itself to artificial conditions, it is 

 much easier to keep it growing. A 1 : 2 meat- water agar is then most 

 useful, the larger amount of extractives serving to stimulate more rapid 

 growth. When 1:8 or 1:10 meat- water agar is employed, the growth 

 is slower and not so rich, but on the other hand, the vitality and virulence 

 are retained longer, as is seen in the fact that such cultures will kill 

 animals even after two days' exposure to a temperature of 34°, which 

 renders the ordinary cultures harmless. 



The question naturally occurs, why is the agar necessary? If the 

 trypanosome grows in blood in the living body, why not in pure blood 

 outside ? It is a fact that blood which is extremely rich in trypanosomes, 

 when drawn from the body, becomes at once unfavorable to them. The 

 parasites begin to die almost at once and may all be dead in an hour or 

 so, especially if, as is the case under a cover-glass, air is excluded. When 

 kept in a test tube, the majority of the trypanosomes die rapidly, but a 

 few hardy individuals persist for from two to six days. If, however, 

 the blood is drawn from an animal when the parasites are relatively 

 few, these few are not rapidly killed but gradually die out, as did the 

 few survivors in the rich blood. When the richly infected blood is largely 

 diluted at once after removal from the infected animal with sterile 

 defibrinated blood, the life of all the trypanosomes is prolonged. 



These observations would seem to indicate that the trypanosomes pro- 

 duce substances which are extremely poisonous to themselves, and which 

 would appear to be rapidly destroyed or eliminated from the blood by 

 the organs of the living animal, thus permitting the enormous concen- 

 tration of the protozoa in the blood. May we not suppose the agar to 

 act in the same way, removing by diffusion into itself the unfavorable 

 substances from the thin fluid layer above, while at the same time fur- 

 nishing an abundance of new food material to the latter, and thus favor- 

 ing a rich growth in this fluid? This view is supported by the apparent 

 exhaustion of the medium in a* flask. A small flask containing about 

 30 c.c. of blood agar may have on its surface about i/o c.c. of fluid, or 

 •l/()Oth of its volume. When this flask is inoculated with a large num- 

 ber of trypanosomes from a previous culture, growth comes on rapidly, 

 reaches its maximum in three to four days, degenerates and dies out in 

 ten to twenty days. In a similar flask inoculated with a very small 

 number of trypansomes, the growth may show up first after ten or 

 twelve days, reaches its maximum in sixteen to twenty days, and dies 

 out in thirty to forty days. The growth stops and degeneration begins 

 when the trypanosomes have exhausted the absorption capacity of the 

 underlying agar. When w^e consider that it requires a volume of 30 c.c. 

 of nutrient medium to give a good appreciable growth of Tr. hriicei 

 where all the organisms are developed in a volume of 1/2 c.c, it is at 

 once apparent that a pure liquid medium must be immensely better 

 adapted and at the same time more exactly prepared in order to show 

 any growth at all. In fact, to get a good culture in a simply liquid 

 23 



