In vitro differentiation of human-derived Pneumocystis carinii

Two major impediments to the development of an in vitro cultivation system for Pneumocystis carinii are lack of an accurate means of quantitation and difficulty in determination of viability over time. Human-derived P. carinii exists as aggregates of cysts and trophozoites in bronchoalveolar lavage fluid. These aggregates persisted over time in an in vitro system consisting of a monolayer of radiated or nonradiated A549 cells, RPMI 1640, and 10% fetal calf serum. Parallel measurements at specified times after introduction into the in vitro system of the number of aggregates, total aggregate area, and total number of cysts varied and appeared to be a function of the number of aggregates initially added into the system. However, cyst density, the number of cysts per unit aggregate area, was independent of the total number of aggregates added into the system. Cyst density was determined by staining an aggregate with a cyst-specific stain, such as toluidine blue, and counterstaining with Diff-Quik, allowing simultaneous visualization of cysts and aggregate area. Preliminary experiments suggested an increase in cyst density over time. Cyst density may be a means of accurate in vitro quantitation of P. carinii.