High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.
This report describes a method for growing both bloodstream- and procyclic-form Trypanosoma brucei as colonies on agarose plates. Procyclic colonies, which took 2 weeks to develop, grew with approximately 17% plating efficiency on SDM-79/0.65% agarose supplemented with 20% (vol/vol) conditioned medium. Bloodstream forms were adapted to in vitro growth in liquid HMI-9 medium and then spread on HMI-9/0.65% agarose plates, where they grew to visible colonies in 3-5 days. Plating efficiencies were from 3 to 80%, depending upon the trypanosome variant and experiment. Colonies were proven to be the result of growth from a single cell and contained approximately 10(6) cells at maturity. Colonies were transferred to filters and probed for multicopy and single-copy genes. Potential uses of this method in conjunction with classical and reverse genetic approaches to studying trypanosomes are discussed.
Carruthers
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Laboratory of Molecular Parasitology, Rockefeller University, New York, NY 10021.
Cross
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D000818:Animals; D002455:Cell Division; D003470:Culture Media; D066298:In Vitro Techniques; D012685:Sepharose; D014346:Trypanosoma brucei brucei / Q000254:growth & development*
D003470:Culture Media; D012685:Sepharose