174 THE MICHIGAN ACADEMY OP SCIENCE, 



before the nineteenth day, and is more certain if the twentieth to thirtieth 

 day is awaited. So for purposes of classification I have arbitrarily as- 

 sumed that first generations showing well formed, active trypanosomes 

 on or after nineteen days are positive, others negative. In most cases 

 conclusive sub-culture tests were carried out, but sometimes, on account 

 of lack of proper medium, this was not done. As stated above, the growth 

 was positive in two of the six tubes, these two being the 5 and 10 alkali, 

 the intermediate TI/2 alkali, curiously enough, remaining negative. 



The second culture, trial 30, was obtained from a white mouse after 

 death from the disease. Two tubes of the same medium were used, a 

 1% jjer cent, agar with 15 c.c. N NaOH per liter, otherwise resembling 

 the preceding. One of these tubes gave a good culture, which was proven 

 by obtaining a successful second generation from it. 



For the third successful trial (No. 38) a 2:-l blood agar was used, 

 the agar portion containing 3 per cent, pepton, 1 per cent. N NaOH and 

 3 per cent. agar. Two tubes were inoculated from a mouse dead of the 

 disease. One became contaminated, the other gave a good culture of 

 trypanosomes, which was proven by a second and third generation. This 

 first generation was richer than the preceding, apparentl}- because of the 

 greater amount of rabbit blood in the medium. This, and similar ex- 

 periments, has led to the regular use of a 2 : 1 blood agar for the growth 

 of this organism. 



In the fourth successful trial (No. 39) thirteen tubes of various kinds 

 of blood agar were employed. Tw^o gave positive gi'owth. The agar basis 

 for these two was the same, a 3 per cent, agar neutralized according to 

 Thahuann, that is by the addition of f the alkali necessary to render 

 the medium neutral to phenolthalein, in this case IG c.c. N NaOH per 

 liter. The blood added to one tube had been laked by means of distilled 

 water; in the other, ordinary defibrinated blood w^as used. Each showed 

 a good growth, but transplants failed to give a second generation. The 

 trypanosomes, in one of the tubes, remained alive for fifty-five days. 



The next fift}- attempts were all failures, partly because the oncoming 

 winter did not afford a favorable room temperature for the growth, 

 partly also because the cultures could not receive the necessary atten- 

 tion as other lines of work became more important. Later, when the 

 small Erleumeyer flask had replaced the test tube, we began to get cul- 

 tures again. This list of fifty failures served to impress upon us the 

 exacting character of the organism and the necessity of more definite 

 knowledge concerning its requirements for isolation. 



Certain experiments, with various solutions, served to show that an 

 excess of NaOH is more unfavorable to trypanosomes than a similar ex- 

 cess of Na.COg, and hence we came to use the latter altogether in making 

 up the agar constituent. 



The ninetieth trial was the next success, A single flask of 2 : 1 blood 

 agar Avas inoculated with a drop of rat blood, rich in trypanosomes, on 

 December Id, 1903. A positive culture resulted and it was carried 

 through four generations. The agar basis employed for the first genera- 

 tion contained 3 per cent, agar, 2 per cent, pepton, 1 per cent. N 

 Na^CO.,, together with the extractives of 450 g. meat in one liter. 



The ninety-first trial is worth mentioning, although it is not one of 

 the fifteen positive cultures. In this trial a single flask, containing the 

 same medium as in the preceding, was used; on the twelfth day the 



