Abstract

In situ hybridization was performed to detect rat Pneumocystis carinii in the lung sections. Rats were immunosuppressed by weekly subcutaneous injection of 10 mg/kg methylprednisolone. On the 6th, 8th and 9th week of immunosuppression, the lungs were removed and fixed in 10% neutral formalin. A 22 base oligonucleotide probe complementary to P. carinii 5S ribosomal RNA was commercially synthesized and its 3' terminal was labeled with biotin. In situ hybridization was performed utilizing manual capillary action technology on the Microprobe system. P. carinii were detected along the luminal surface of alveolar pneumocytes, in exudate of alveolar cavities, and also in secretory material of bronchioles. In the 6th week group, positive reaction was observed focally in the peripheral region of the lung sections, but the reaction was observed diffusely in the 8th or 9th week groups. In comparison with Grocott's methenamine silver stain, in situ hybridization technique can detect the organism rapidly, and can detect trophic forms very well. Furthermore, no nonspecific reaction with other pathogenic fungi and protozoa was recognized. Therefore, in situ hybridization can be a good technique to detect P. carinii in the lungs of infected rats.