3oo 



Society Proceedings (122). 



Knowles 1 in attempting to repeat the work using the methods of 

 Row and of Cornwall and LaFrenais only obtained two positive 

 cultures out of 129 tubes from thirty-four cases. Meyer and 

 Werner, Cornwall and LaFrenais, and Knowles (54 cultures from 

 12 cases), simply added small amounts of blood to tubes of Nicolle- 

 Novy-MacNeal medium ("N.N.N."). Row and Knowles (34 

 cultures from one case) added \ to 2 c.c. of blood to 20 c.c. of 

 "titrated saline" and then distributed the sediment into N.N.N, 

 medium after incubating the tubes for 24 hours at 22 0 C. 



The low percentage of successful culture by Knowles shows 

 that neither method can be relied upon to give constant results. 

 We have confirmed the findings of Cornwall and LaFrenais 2 that 

 the blood of man is unfavorable to the growth of Leishmania and 

 have found that this is true not only for whole blood but for 

 washed red cells and for serum whether fresh or heated for \ hour 

 at 56 0 C. There was only an occasional feeble growth with blood 

 and red cells, none with serum alone. 



In order to free the blood from red cells and serum the following 

 method was used: 10 c.c. of blood was drawn from a vein at the 

 elbow into one or two cubic centimeters of citrated Locke's solution 

 and immediately expelled into a flask containing 50 c.c. of the 

 same fluid. This diluted blood was centrifuged at a low speed to 

 throw down the red cells only. The supernatant fluid was trans- 

 ferred to another sterile 50 c.c. tube and centrifuged at a high 

 speed. The sediment from this was distributed into tubes of 

 buffered N.N.N, medium adjusted to varying hydrogen-ion con- 

 centrations and incubated at 22 0 C. 



Culture Medium. — This was the Nicolle-Novy-MacNeal 

 medium 3, 4 with the addition of 0.2 per cent, dipotassium phosphate 

 (K2HPO4) as a buffer. The salt-phosphate agar was adjusted to 

 hydrogen-ion concentrations varying from P H 6.8 to P H 8.2. 

 Defibrinated rabbit blood was added in the proportion of one part 

 of blood to three of agar. Leishman-Donovan bodies in spleen 

 pulp and peripheral blood develop into flagellates throughout the 

 range tested with little detectable difference. Cultures from 



1 Knowles, R., Indian Jour. Med. Res., 1920, viii, 140. 



2 Cornwall and LaFrenais, loc. cit., p. 299. 



1 Novy and MacNeal, Jour. Inf. Diseases, 1904, i, 1. 



* Nicolle and Comte, Bull, de la Sociitt de Path. Exotique, 1908, i, 299. 



