Cultivation of free-living stages of Trichostrongylus colubriformis in media without bacteria, animal tissue extract, or serum

Trichostrongylus colubriformis was cultured from hatched first-stage to third-stage larvae in bacteria-free media in the absence of animal tissue extract or serum. This was achieved for the first time with a nematode, parasitic in vertebrates, whose rhabditiform larvae are food-dependent. The best media contained enzymatic hydrolysed casein (amino nitrogen:total nitrogen ratio 0.39), yeast extract, phosphatidylcholine from soybean, and a number of chemically defined ingredients, which include a salt solution, a sterol, and an iron porphyrin. The yield of third-stage larvae obtained was up to 17% of all the living larval stages present after incubation. When casein hydrolysate with AN:TN ratio of 0.39 was replaced by casein hydrolysate with AN:TN ratio of 0.53, little or no development to third-stage larvae occurred. Development to infective larvae was shown to be possible in media with soy peptone instead of casein hydrolysate, although to a very limited extent. It is proposed that the free-living stages of the parasite require peptides, whose molecular weights all lie within a narrow range.