THE LEISHMANIA OP ITALIAN KALA-AZAE. 
357 
immersion objective of Zeiss (2'5 mm.), with which it is pos- 
sible to control step by step the most minute structui’al details. 
The cultures of Leishmania develop rapidly, and flourish 
in the Novy-McNeal-]S[icolle medium prepared according to 
the formula of Nicolle, which, I think, it worth while to 
record briefly here : 
Agar-agar, not washed . . . .14 grm. 
Sodium cliloride . . . . . 6 „ 
Distilled water ..... 900 c.c. 
The mixture is dissolved and sterilised in the autoclave, 
taking care to add afterwards some sterile water, as much as 
has been lost during the sterilisation. The agar, not alkali- 
nised nor neutralised, is placed in the sterile tubes in the 
desired quantity (I make use of test-tubes 12 cm. in length 
and about 2 cm. in diameter, and place in each 4 c.c. of agar), 
and then sterilised afresh for ten minutes. The tubes can be 
kept a long time, provided that all desiccation of the agar is 
avoided by closing them with a rubber cap and keeping them 
in the ice-chest, sheltered from light. 
At the moment of preparing the substratum the agar is re- 
dissolved by boiling water and allowed to cool to 45°-55° C., 
at which temperature is added to it, with proper pi-ecantions 
to ensure sterility, one third of its volume of dehbrinated 
rabbit’s blood, introducing it with a sterile pipette holding 
10 c.c., which I change two or three times during the 
operation. The agar and the blood are mixed by shaking the 
test-tube well, but care should be taken that excessive froth 
is not formed. The tubes are then placed on an inclined 
plane in order that the blood-agar may solidify in a form like 
the mouthpiece of a flute, and left thus about twelve hours. 
Closed again with rubber caps they are placed in the incu- 
bator at 37° C., in order that the water of condensation may 
collect in good quantity. There is no advantage in leaving 
them in the thermostat two or three days,as Nicolle prescribes, 
since the water of condensation always diminishes a little. 
Tubes prepared in this manner can be kept in the cold 
chamber at a temperature of about 0°C. for some months ready 
