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Continuous axenic cultivation of Pneumocystis carinii
S Merali1, U Frevert, J H Williams
1Department of Medical and Molecular Parasitology, New York University School of Medicine, 341 East 25th Street, New York, New York, 10010, USA.
Abstract:
Continuous axenic culture of Pneumocystis carinii has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle's salt supplemented with S-adenosyl-L-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, L-cysteine and L-glutamine, and horse serum. Incubation is in room air at 31 degrees C. The pH of the medium begins at 8.8 and rises to approximately 9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated 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 cultured organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture.
Insights
Continuous axenic culture of Pneumocystis carinii was achieved using a novel culture vessel and specialized medium. This breakthrough enables reliable P. carinii cultivation for research and infectivity studies.
Area of Science:
- Microbiology
- Cell Biology
- Parasitology
Background:
- Pneumocystis carinii is an opportunistic pathogen that causes pneumonia in immunocompromised individuals.
- Establishing axenic (pure) cultures of P. carinii has been a significant challenge, hindering research into its biology and pathogenesis.
Purpose of the Study:
- To develop a method for continuous axenic culture of Pneumocystis carinii.
- To characterize the growth conditions and morphology of cultured P. carinii.
- To assess the infectivity and storage potential of cultured P. carinii.
Main Methods:
- Utilized a specialized culture vessel with a collagen-coated porous membrane for organism attachment and frequent medium exchange.
- Employed a modified Minimal Essential Medium supplemented with specific nutrients, horse serum, and incubated at 31°C in room air.
- Assessed organism morphology via stained smears and transmission electron microscopy, and confirmed identity using P. carinii-specific monoclonal antibodies (mAbs).
Main Results:
- Achieved continuous axenic culture of P. carinii, with doubling times ranging from 19 to 65 hours depending on inoculum density.
- Cultured organisms maintained morphology consistent with P. carinii and were labeled by specific mAbs.
- Cultured P. carinii were infective in immunosuppressed rats and could be cryopreserved for reinitiation of cultures.
Conclusions:
- Continuous axenic culture of P. carinii is feasible using the described method.
- The developed culture system supports P. carinii growth, maintains its characteristic morphology, and yields infective organisms.
- This advancement provides a valuable tool for studying P. carinii biology, developing diagnostics, and evaluating therapeutics.