472 RESEARCH IN PROTOZOOLOGY 



1925Z?), Das Gupta (1925), Howitt (1925), and Thomson and 

 Robertson (1925). 



Intestinal flagellates. The cultivation of intestinal flagellates 

 and various media employed have been discussed at some length in 

 Chapters VH and XVL 



HemoHagellates. All of the LeisJimania organisms and some 

 species of Trypanosoma have been grown on the blood-agar me- 

 dium devised by Novy and MacNeal (1903) modified by Nicolle 

 (1908) and known to protozoologists as N. N. N. medium. 



The agar base has the following ingredients: 



Agar 14 gms. 



Sodium chloride 6 gms. 



Distilled water 900 c.c. 



This mixture is tubed while hot in five cubic centimeter quantities and is 

 autoclaved. Before use, from two to five cubic centimeters of fresh rabbit 

 blood (most easily obtained by cardiac puncture using aseptic technique) are 

 added to each tube which has been heated to about 50° C. The tube is rapidly 

 rolled between the two hands to mix the warm agar and blood. Vibration of 

 the tubes during the rotating procedure must be avoided so that the mixture 

 will be free from bubbles. The tubes are tilted sufficiently so that the 

 contents will harden into slants. They are then incubated at 3"/° C. for 

 twenty-four hours to test for sterility. In using the medium, a small quan- 

 tity of blood or tissue is introduced with a pipette or platinum loop under 

 aseptic conditions into the condensation liquid at the bottom of the tube. It 

 is well to prevent evaporation either by capping the tubes with rubber caps 

 or sheets of tin-foil, or by dipping the cotton phig into boiling paraffin before 

 inserting it into the culture tube. 



This method has been modified for various purposes (see 

 Noller, 1917, and Hoare, 1923). 



The Leptospira medium devised by Noguchi (1924) has been 

 used for the cultivation of various species of trypanosomid^ 

 (see Chapter XXVI 11 for details). 



For culturing the pathogenic trypanosomes Ponselle (1923), has 

 found the following satisfactory: 



Sodium chloride 0.3 to 0.8 gm." 



Peptone (Witte's) 2.0 gms. 



Gelatin 2.0 gms. 



Normal solution of sodium carbonate... i.o c.c. 

 Distilled water loo.o c.c. 



Ingredients must be chemically pure. They are dissolved by heat and the 

 solution is autoclaved. When it has cooled to laboratory temperature, it is 



" Varies according to species of trypanosome : 0.3 gm. for T. brucei, 0.6 

 gm. for T. pecaudi, and 0.8 gm. for T. rhodcsicnsc and T. dinwrphon. 



