Cultivation of Leishmania Donovani in Media of Embryonic Chick Tissues

Abstract

For the cultivation of Leishmania donovani a number of media generally containing blood have been advocated. In such media only flagellate forms are found. It is of interest to record the growth of Leishmania flagellates in media of various embryonic chick tissues which have not hitherto been used for the purpose.

The chick embryos employed were fertilized hen's eggs incubated at 37°C. for a varying period of 4 to 20 days. The embryonic chick brain, heart, liver and intestines were removed separately and chipped into fine pieces with sterile scissors in different dishes. They were then suspended in sterile Tyrode's solution having a pH 8.0 in approximate concentration of 0.5 gm. of tissue to 10 cc. of fluid. In some experiments the whole chick embryo from 4 to 7 days old minced finely and suspended in Tyrode's solution was used. With a sterile capillary pipette one drop (about 0.15 cc.) of the embryonic tissue suspension was placed in the center of sterile cover-glass. To this was added a similar drop of Tyrode's solution containing Leishmania donovani from the spleen of an infected hamster. A sterile hollow glass slide measuring 7.5 cm. long, 2.5 cm. wide, and 0.7 cm. thick, with a central pit or well, 1.8 cm. in diameter and 0.4 cm. deep, was smeared with sterile vaseline around the mouth of its well. The hollow glass slide was then inverted over the cover-glass in such a way that the inoculated media fluid on the cover-glass faced the center of the well of the hollow glass. The whole slide together with the cover-glass was then turned over by quick motion so that the hanging drop did not spread to the periphery but remained in the center of the under-surface of the cover-glass. Pressure was gently applied to the latter to ensure airtight sealing of the hanging drop inside the hollow glass. Many such hanging drop preparations were made and incubated at 37°C. and at 20°C. in separate lots. The advantage of such hanging drop cultures is obvious. It permits direct microscopic examinatiion at frequent intervals without contamination.