Localization of silver deposits on Pneumocystis carinii treated with Gomori's methenamine silver nitrate stain

The question which portion of the pellicle of Pneumocystis carinii is actually stained by the Gomori's methenamine silver nitrate (GMS) method was solved with the aid of electron microscopy. Silver particles were specifically deposited on the electron-lucent middle layer of the precyst, cyst and ruptured cyst, whereas such deposition was not found on the pellicle of the trophozoite, probably due to the absence of the electron-lucent middle layer in the trophozoite. These deposits increased in proportion to the duration of staining and to the thickness of the lucent middle layer. Therefore, the lighter colored organisms as observed under the light microscope are considered to be the precysts because they have thinner GMS-positive middle layers than those of cysts. So-called parentheses-like structures or rings are one of the most characteristic light microscopic features of P. carinii cysts when stained with GMS. The present study indicates that these structures must be of cyst wall origin rather than of cytoplasm origin, because the thickened part of the cyst wall showed a dense deposition of silver particles, probably corresponding to the parentheses-like structure. On the other hand we also observed, in the endogeny form of trophozoite, a thin GMS-positive layer in the parent pellicle though this pellicle showed no differences from those of daughter and common trophozoites when observed by means of ultrathin section microscopy.