Freeze-etch studies on the gametocytes of Plasmodium falciparum

Abstract

The Erythrocytic stages of the Honduras strain of the malarial parasite Plasmodium falciparum were maintained in tissue culture medium RPMI 1640. Red blood cells containing gametocytes and other erythrocytic stages were fixed in 2% glutaraldehyde, and cryoprotected in 30% glycerol in H2O. This preparation was freeze-etched for one minute at -98°C in a modified Denton DFE-2 freeze-etch module. Stereo pairs with specimen tilt of 10 between electron micrographs were obtained with a JEM-100B transmission electron microscope equipped with a 60° top entry goniometer stage.The fine structure of the gametocytes of malarial parasites has been studied by conventional electron microscopic methods [stained ultrathin sections (2 and 3)], but this stage of the life cycle has not been demonstated previously with freeze-fracture studies.Since the red blood cells in our preparation also contained other stages of the life cycle, the following combination of characteristics were used to differentiate the gametocytes: presence of microtubules; food vacuoles; and large size.Microtubules are not found in trophozoites, and merozoites are relatively small and do not contain food vacuoles.