[Isolation and purification of Pneumocystis carinii cyst]

Twenty Wistar rats were injected with cortisone acetate twice a week subcutaneously for 6-12 wks. By 7 weeks Pneumocystis carinii pneumonia was induced in rats successfully. The cysts of Pneumocystis carinii from infected lungs of cortisonized rats were identified by phase-contrast microscopy, Giemsa's stain, Chalvardjian's stain and Gomori's methenamine silver nitrate stain. The process of isolation and purification of Pneumocystis carinii from infected rat lungs included the following three test steps. First, the tissue was cut into small pieces and homogenized and filtered through #60, #100, #200-gauge wire mesh respectively. Secondly, the homogenate was digested with collagenase, the optimal concentration of collagenase being 0.2%. Thirdly, the discontinuous percoll density gradient centrifugation was used to separate P. carinii cysts. The majority of P carinii cysts were present in a density zone of approximately 1.033g/ml and were essentially free from host lung debris, the latter being removed due to their higher density, 1.040g/ml.