Measurement of cellular microbicidal activity against Pneumocystis carinii in vitro

As Pneumocystis carinii cysts cannot be cultivated for enumeration of colony forming cells, two alternative approaches to measuring killing of P. carinii by mouse peritoneal cells were investigated. The cells tested were either normal resident peritoneal cells, or cells which were elicited by intraperitoneal injection of the bacterium Listeria monocytogenes. The latter population showed enhanced antibacterial activity against Listeria organisms and enhanced production of H2O2 in the presence of P. carinii, cysts from immunosuppressed rats. To assess killing of P. carinii, cysts were mixed with peritoneal cells at a ratio of 10:1, and after intervals of incubation the peritoneal cells were lysed by saponin treatment. The viability of the cysts was assessed by staining with vital dyes or by uptake of tritiated uridine over 7 h incubation. Viability of cysts was unaffected by saponin treatment, and there was agreement between the two techniques that the elicited peritoneal cells killed approximately twice the number of cysts over a 20 h incubation period compared to normal resident peritoneal cells.