A simple method for cloning leishmanial promastigotes
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A method of cloning leishmanial promastigotes is described in which mid-exponential phase cultures are diluted to contain approximately 3×103 promastigotes per ml. Hanging-drop preparations made from 0.2–0.4 μl volumes of the diluted culture seen to contain a single promastigote are picked-up in glass capillary tubes. Additional culture medium is taken into the capillaries which are then heat-sealed and incubated at 22° C. Growth of leishmanial promastigotes inside the sealed capillary tubes is followed by direct microscopic observation through the walls of the tubes. When active promastigotes are seen the contents of the tubes are inoculated into small volumes of culture medium. The method is extremely easy to use, requires no specialised apparatus, and has been successfully used with different strains and species ofLeishmania, with up to 100 percent of the cloned organisms growing.
KeywordsSmall Volume Microscopic Observation Capillary Tube Glass Capillary Tube Phase Culture
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