180 The Philippine Journal of Science 1922 
These were heated in a flask until all of the agar was dissolved. 
One cubic centimeter of the mixture was placed in each of the 
small test tubes, which were autoclaved. The medium was then 
cooled to about 45° C.; different amounts of defibrinated rabbit’s 
blood were then added and allowed to cool as slants. The or- 
ganisms were streaked on the surface of the agar by means of 
a platinum loop. 
This formula is essentially that of Nicolle,(2) which is a mod- 
ification of Novy-McNeal’s medium for trypanosomes. As modi- 
fied by Nicolle, the medium consists only of agar, sodium chloride, 
water, and defibrinatei rabbit’s blood. The meat extract and 
peptone employed in the original formula are not used. This is 
best known as the triple-N or N. N. N. (Nicolle-Novy-McNeal) 
medium. Nicolle designed it for the cultivation of the organisms 
from a case of infantile splenic anemia which he studied at 
Tunis. As the flagellates he recovered from the spleen showed 
similarity to Leishmania donovani he placed them in the same 
genus, but assigned to them a new specific name—Leishmania 
infantum. . 
It is worthy of note that Cristina and Cannata(1) also suc- 
ceeded in growing Leishmania infantum by using media to which 
various other components were added, such as ascitic fluid, gly- 
cerine, bouillon, rabbit’s serum, etc. 
The results of the cultivation experiments are given in Tables 
1 to 4. 
TABLE 1.—Leishmania infantum grown in 0.5 per cent agar plus different 
percentages of defibrinated rabbit’s blood. Cultures inoculated Decem- 
ber 1, 1920. 
5 per cent blood. 10 per cent blood. 25 per cent blood. |Date of examination.) 
Only one active organism was | Negative____._____- Negative __..._._._. December 2, 1920. 
seen in several microscopic 
fields. 
Very Seare@ste sc ccs O06 ieee eeu MO eae Sa December 3, 1920. 
Numerous organisms ___.._.__.. Good growth ______- Rather poorer | December 6, 1920. 
growth. 
Many active organisms present | Moderate growth; | Slight growth _____. December 11, 1920. 
(fewer than in 2 per cent active ones also 
agar-25 per cent blood exam- in water of con- 
ined on the same day. densation. 
Abundant growth -_-__..._...._| Moderate growth___ do .. December 17, 1920. 
Good gtowth=.. 2.5 os | Good growth _-_....| Poor growth___._.-- December 29, 1920. 
INTERPRETATION OF RESULTS 
The observations of the cultures from time to time as recorded 
in Tables 1 to 4 furnish indications with respect to certain re- 
