Continuous axenic cultivation of Pneumocystis carinii

Continuous axenic culture of Pneumocystis carinii has been achieved. A cult ure vessel is used that allows for frequent medium exchange without disturb ance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle's salt su pplemented with S-adenosyl-L-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, L-cysteine and L-glu tamine, and horse serum. Incubation is in room air at 31 degrees C. The pH of the medium begins at 8.8 and rises to approximate to 9 as the cells grow . Doubling times calculated from growth curves obtained from cultures inocu lated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultu red organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured materi al. Cultured organisms are infective for immunosuppressed rats and can be s tored frozen and used to reinitiate culture.