MEDIUM FOR CULTIVATING TRYPANOSOMA BRUCEI. 173 



AN IMPROVED MEDIUM FOR CULTIVATING TRYPANOSOMA 



BRUCEI. 



WARD J. MACNEAL. 



The successful cultivation of Trypanosoma hrucei was announced some 

 months ago by Professor Novy and myself in "The Journal of the Ameri- 

 can Medical Association"* and later in ''The -Journal of Infectious Dis- 

 eases."t In neither of these publications was a detailed description of 

 the culture medium given, largely because we did not know what con- 

 stituents were essential. In the first set comprising Mtj animals (mice, 

 rats and guinea-pigs) from which cultures were attempted, we were able 

 to report positive results in only four, namely trials 26, 3G, 38 and 39. 

 As an indication of the uncertainty in regard to the essential conditions 

 for success, it is significant that the next successful attempt was trial 

 90; that is to say, that after the announcement of this discovery it was 

 necessary for the authors to make fifty attempts before they could dupli- 

 cate the last positive result. 



At present we have completed 114 culture trials; that is to say, in- 

 oculations have been made from 114 animals. In each case from one to 

 ten tubes or flasks were inoculated with trypanosome blood. Of these, 

 fifteen have been successful (about 13 per cent.). Of the lapt fourteen 

 trials, seven have been successful (50 per cent.) ; so it is apparent that 

 we are getting nearer to the proper medium. 



The first successful trial (No. 20) was made August 27, 1903. A 

 nutrient agar containing the extractives of 450 g, of beef, 5 g. salt, 20 g. 

 pepton, and 20 g. agar in one liter, was given difl'erent degrees of alkalin- 

 ity by adding 121/2, 10, 71/2) 5> 2%, and cubic centimeters of N 

 NaOH to each 1000 c.c. of agar. After tubing and sterilizing, this agar 

 was kept for some time, a month or so, before use. It was liquefied in 

 boiling water, cooled somewhat, and the contents of a tube of each lot 

 mixed with f its volume of defibrinated rabbit's blood, then inclined 

 and allowed to solidify. We prepared thus tubes of blood agar (2:3) of 

 different reactions, from the natural acidity to an alkalinity imparted 

 by 121/2 c.c. N NaOH per liter. 



A white mouse, in the last stages of nagana, was etherized, after 

 which its blood was drawn from the heart by means of a 

 sterile pipette. Each of the six tubes was inoculated with one 

 drop of this blood, then sealed with a rubber cap and kept in a black box 

 at room temperature, from 20° to 30° C. The trypanosomes gradually 

 died out until on the twentieth day an increase in numbers was observed 

 in two of the tubes, which continued for a few days and then the protozoa 

 gradually died out. Transplants made on the twentieth day and kept 

 at 30° gave positive growths, and this strain has been kept up ever since, 

 being now in its twenty-third generation. 



Transplantation, in the case of a first generation, is rarely successful 



*Jour. Am. Med. Assn., Nov. 21, 1903. 

 tJour. Infectious Diseases, I, 1904, p. 1. 



