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Simplified Methods for Obtaining Purified Oocysts from Mice and for Growing Cryptosporidium parvum In vitro
Bruno P. Meloni and R. C. Andrew Thompson
The Journal of Parasitology
Vol. 82, No. 5 (Oct., 1996), pp. 757-762
Stable URL: http://www.jstor.org/stable/3283888
Page Count: 6
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Seven- to 8-day-old Arc/Swiss mice were infected with 100,000-120,000 Cryptosporidium parvum oocysts. At 8 days postinfection (PI) the jejunum, ileum, cecum, colon, and rectum were removed. Using a simple extraction procedure and purification by Ficoll gradient centrifugation, we rountinely obtained between 3-6 million and up to 15 million purified oocysts per mouse. For in vitro cultivation, purified oocysts were pretreated in a low pH (2.5-3) 0.5% trypsin solution for 20 min, resuspended in supplemented RPMI-1640 containing glucose 0.1 g (5.55 mM), sodium bicarbonate 0.3 g, bovine bile 0.02 g, folic acid 25 µg, 4-aminobenzoic acid 100 µg, calcium pantothenate 50 µg, ascorbic acid 875 µg, penicillin G 10,000 U and streptomycin 0.01 g per 100 ml, and 1% fetal bovine serum (pH 7.4 before filtration), and used to inoculate confluent monolayers of the human adenocarcinoma cell line HCT-8. Incubation was in a candle jar at 37 C. We tested numerous supplements to RPMI-1640, different pHs, and atmospheric conditions and found the parameters described above produced the greatest parasite numbers in vitro. We obtained significantly superior growth of C. parvum grown in HCT-8 cells using the conditions described above than in culture conditions described previously.
The Journal of Parasitology © 1996 The American Society of Parasitologists