176 THE MICHIGAN ACADEMY OF SCIENCE. 



it will be sufficient to consider only the more important ones. In trial 

 108 the agar used contained various amounts of alkali, ranging from 5 

 to 20 c. c. of N NaoCOo per liter; a 2 per cent, glucose agar was also tried. 

 The 10, 15 and 20 alkali flasks were positive, the 5 alkali and the 2 per 

 cent, glucose agar failed. The agar was made with 1 : 2 meat extract in 

 each case. 



Trial 111 gave the richest and best appearing first generation we 

 have ever had. In this experiment a 1 : 8 meat-water agar was used. Of 

 the two flasks inoculated, one had 10 c.c, the other 15 c.c. NaoCO, per 

 liter. Both gave most excellent cultures, the more alkaline appearing 

 a little better. 



In the 114th trial, five different kinds of agar were used. A rabbit, 

 just bled to death, was dressed, and its flesh, as well as crushed bones, 

 were extracted by boiling with an equal amount of water. The filtered 

 extract differed from the ordinary meat extract in that it contained a 

 considerable amount of gelatin, sufficient to cause it to solidify at a 

 low temperature. Some of this rabbit flesh extract (1:1), was con- 

 verted directly into agar, one portion without and the other with 2 

 per cent, pepton. In each case 15 c.c. N NaoCO., and 5 g. NaCl per liter 

 were added. Another portion of the extract was diluted to eight times 

 its volume with distilled water and this 1 : 8 rabbit flesh extract was 

 made into agar, with and without pepton, as indicated above, 10 c.c. 

 N NaaCO. and 5 g. salt per liter bein'g added. In this experiment (114) 

 four flasks, containing each one of these four varieties, and one flask of 

 the regular 1 : 8 meat agar which served as a control, were inoculated 

 with blood taken from the heart of an anajsthetized rat. The control 

 and the rabbit agar 1 : 8 with pepton, showed positive growth on the 

 twenty-sixth day ; the others remained negative. 



From these few experiments it is possible to construct a working 

 hypothesis somewhat as follows : We can say that Tr. hrucei requires 

 for its isolation a blood agar, best in the ratio of 2:1, and that the 

 nutrient agar employed must be of a certain composition permitting only 

 a slight variation. Meat extractives may be present up to 1 : 2, i. e., the 

 extractives of 100 g. beef in 200 c:c. of agar. First generations can be 

 obtained on blood-agar which contains no meat extractives, but the 

 growth in that case is slow and never very rich. It may be said that a 

 1 : 8 meat- water is about the optimum. About 2 per cent, of Witte's 

 Pepton is distinctly favorable, as has already been worked out for Tr. 

 leivisi. Common salt, not more than 5 g. per liter, is not harmful, and 

 seems to produce better shaped organisms. The reaction is important 

 and should be about 0.8 to 1 per cent. N acid to phenolphthalein, al- 

 though a variation of 0.5 per cent in either direction is not necessarily 

 fatal. The presence of gelatin is not desirable, as it tends to prevent 

 the formation of the condensation fluid in which the trypanosome grows. 

 Full 2 per cent, agar should be used so that the mixture with two vol- 

 umes of blood, will solidify firmly. Erlenmeyer flasks having a capacity 

 of about 50 c.c. are better than tubes, on account of the thinner layer 

 of fluid over the agar surface and the consequent better aeration and 

 perhaps better diffusion of waste products into the underlying solid 

 medium. The flasks are best sealed with good rubber caps, and should 

 be kept at a constant temperature of about 25° C. With the proper 

 medium a growth is obtainable even when but very few trypanosomes 

 are present in the original material. 



