478 University of California Publications in Zoology [Vol. 20 



MATERIAL AND TECHNIQUE 



Cultures of Leishmania tropica and Leishmania infantum were 

 grown on N.N.N, media made up in the proportions of 1.4 per cent 

 agar and 0.5 per cent sodium chloride in distilled water. This was 

 adjusted to pH 7.8, was tubed, autoclaved, and stored until needed. 

 One day before being used 30 per cent of defibrinated rabbit blood 

 was added and the tubes incubated overnight, to test them for 

 sterilitj' of the culture media. The tubes were then inoculated and 

 the cotton plugs covered with rubber caps to prevent evaporation. 

 After 10 to 12 days' incubation at room temperature, the surface of 

 the media was found to be covered with minute, dewdrop colonies 

 of the flagellates. This growth was rvibbed off with a platinum loop 

 into 1 c.c. of normal saline and decanted into a centrifuge tube. The 

 washings from 15 to 20 cultures of each species were centrifuged 

 separately for half an hour at 1800 revolutions a minute and the 

 supernatant fluid decanted off. The organisms in the bottoms of 

 the tubes were then diluted with normal saline to which 0.25 per cent 

 phenol had been added until each cubic centimeter contained 

 1,500,000 organisms by direct count in a blood-counting chamber. 



With the suspension thus prepared, two rabbits were immunized 

 to each of the two species of Leishmania by intravenous injections 

 of 2 c.c. each at three day intervals. Seven days after the last injec- 

 tion the rabbits were bled from the ear and macroscopic tests made 

 for the. presence of agglutinins. All four rabbits showed complete 

 agglutination of the specific flagellate at a 1 to 980 dilution of the 

 serum. These results were considerably higher than those of Bandi 

 (1913), who found that immunized rabbits would agglutinate the 

 specific strains of Leishmania infantum at a 1 to 160 dilution of the 

 serum. 



A leishmaniosin was prepared, following the technique used b.v 

 Force and Stevens (1917) in the preparation of typhoidin. This 

 consisted in washing off the growth from about 50 cultures of each 

 species with 1 c.c. of saline, precipitating the protein with 95 per cent 

 alcohol, washing with ether, and drj'ing the residue over concentrated 

 sulphuric acid in vacuum. As a small amount of haemoglobin from 

 the culture media was dissolved in the saline, a control was prepared 

 from washings from sterile culture media. About 0.1 gm. of dry 



