THE CULTIVATION OF TRYPANOSOMA BRUCEI. 267 



pipette (fig. Ill) and diluted to tlie original blood volume with 0.5 per cent 

 sodium chloride solution. This clear diluted serum was then mixed with the 

 dialyzed agar medium, in a ratio of two to one. The remaining blood cells 

 were likewise diluted with salt solution to the original blood volume and the 

 resulting suspension was added to the dialyzed agar in the same ratio of two 

 to one. 



Serum inactivated for one-half hour at 56°C. was also mixed with the 

 dialyzed agar. As in the previous experiments these media were inoculated 

 with trypanosomal rat blood and incubated at 25°C. 



The results of the examination of these media at the end of 14 or 21 days 

 showed that the diluted and inactivated serum gave practically 100 per cent 

 positive growths, whereas, the diluted blood cell medium gave but 38 per 



Figure III — Pasteur Bulb pipette. Full Size. 



cent successful cultures (chart I). Therefore, it would seem advisable in 

 attempting isolation of Trypanosoma Brueei to employ dilute or inactivated 

 serum rather than defibriuated whole rabbit blood. Successive transplants 

 of the cultural forms on serum agar gave at all times a very rich growth, in 

 fact the growth in some cases became so extremely rich that a white film 

 resulted which was easily detected by the eye. The organisms of nagana, 

 therefore, maintain themselves without the presence of hemoglobin. This 

 fact was also noted in regard to Trypanosoma Lewisi. 



Isolations were attempted using ascitic fluid instead of serum and in no 

 instance were cultures obtained. The culture medium described by Rh. 

 Erdmann* was also used and proved to be a total failure and as a matter of 

 fact the easily cultivated rat trypanosome of Lewis could not be grown 

 successfuly by this method. 



The appearance of Trypanosomes of nagana in vitro is very characteristic. 

 They occur either singly or in groups ranging from a few to many hundreds 



4. Soc. Exp. Biol, and Med. 1914, XII, pp. .57-58. 



