The conditions for luxuriant axenic cultivation of the trypanosomid flagellate in chemically-defined media were determined. Improvements over the hitherto published media include higher concentrations of amino acids, folic acid (where this is the sole source of pteridine) and purines and the inclusion of threonine and Tween 80. Increased oxygenation was obtained by incubating liquid cultures in tubes in a sloped position. Under these conditions consistently excellent growth (up to 2 × 10 organisms/ml.) was obtained and the useful incubation period could be decreased to 4 days. Using the improved medium, qualitative and quantitative studies on the nutritional requirements of the organism were carried out. Growth failed when any one of the following amino acids was omitted from the basal medium: histidine, phenylalanine, isoleucine, leucine, valine, lysine, arginine, tyrosine, methionine, tryptophan. The omission of threonine caused drastically decreased growth rates and lower yields. It was found that phenylpyruvic acid could replace phenylalanine; cysteine, cystathionine or homocysteine could replace methionine; and citrulline, but not ornithine, could replace arginine for . In addition to the amino acid requirements a nutritional need for the following was demonstrated: a carbohydrate, haemin, a purine, thiamine, riboflavin, pantothenic acid, nicotinic acid or its amide, pyridoxal or pyridoxamine, biotin, folic acid and an unconjugated pteridine (biopterin). The natural purines, their nucleosides and nucleotides are nearly equivalent, on a molar basis, in growth promotion. Numerous substituted purines were also tested for their ability to supply the purine requirement. No exogenous source of pyrimidine for nucleic acid synthesis is necessary provided folic acid is present. Folic acid can be omitted from the growth medium provided thymine and methionine are present. The absolute requirement for biotin could not be demonstrated in the present medium without the use of avidin.


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