TRYPANOSOMIM] 59 



A small quantity of agar-agar, as used for bacteria cultures, is 

 melted in test-tubes in a hot- water bath at a temperature of 50 C. 

 To this is added an equal quantity of fresh defibrinated rabbit's blood. 

 The mixture should be well shaken, and any bubbles which may have 

 formed are punctured with a hot platinum needle, as, otherwise, they 

 might hinder the condensation of water. The tubes are cooled 

 in an oblique position, closed with a rubber cap, and placed for 

 twenty-four hours in a thermostat at a temperature of 37 C. At the 

 end of that time water will be found to have condensed freely, and 

 tubes which have escaped complete sterilization may be recognized 

 and removed. Blood containing Trypanosomes should be taken with 

 a Pravaz's syringe, under antiseptic precautions, from an artery of the 

 infected animal and mixed with a small quantity of normal saline. 

 The skin at the site of operation should be carefully prepared. The 

 animal may be chloroformed without endangering the success of the 

 experiment. Three " looplets " of the infected blood should be 

 transferred to the condensation water in one of the test-tubes, or 

 three droplets may be conveyed by means of a sterile pipette. 



These cultures are very successful in the case of the Trypanosomes 

 of fishes and of rats, all those varieties, in fact, which do best in the 

 refrigerator. At the end of a few days numerous Trypanosomes are 

 seen, which agglomerate into rosettes and multiply very rapidly. 

 Animals may be inoculated from these cultures or secondary cultures 

 may be made. Each culture is, as a rule, infective for about one 

 month. The culture Trypanosomes differ from those living normally 

 in the blood-stream in the greater delicacy of certain portions of their 

 structure (position of the flagellar nucleus, texture of the protoplasm) . 

 These modifications may be attributed, at least in part, to degenerative 

 changes due to the abnormal medium. The tendency to agglomerate 

 into rosettes is also a sign of degeneration. 



The tsetse Trypanosome, T. brucei, is considerably more difficult 

 of cultivation than the Trypanosome of the rat. Novy and McNeal 

 recommend the following medium, though they did not find it 

 invariably successful : 



The extract from 125 gr. beef in 1,000 c.cm. distilled water. 



Agar-agar 20 gr. 1 



Peptone . . ... . . . . . . . . . . 20 gr. 



Common salt . . . . . . . . . . . . 5 gr. 



Normal (5 '3 per cent.) soda solution . . . . 10 c.cm. 



Of this mixture 1 part by volume is added to 2 parts defibrinated 

 rabbit's blood at a temperature of 55 to 60 C. 



1 Nocht and Mayer found that the medium set better with 25 gr. agar-agar. 



