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Gametocytes differentiated from ring-stage parasites in microcultures of human blood infected with Plasmodium falciparum. Immature gametocytes could be distinguished morphologically from late asexual trophozoites after approximately 40 h of culture. Differentiation into crescentic forms took several days and the incorporation of [3H]-isoleucine by developing gametocytes was demonstrated. About 1% of red cells contained gametocytes at the maxiumum densities attained. Differentiation of gametocytes occurred either directly from rings placed in culture or from the progeny of subsequent cycles of schizogony and invasiton in vitro. The latter occurrence was confirmed by the development of gametocytes in marker fetal red cells added to cultures, although fetal red cells provide a less favorable environment than those with HbA for growth of the parasites.

Natural isolates of Plasmodium falciparum represent a genetically heterogeneous population of parasites. To obtain stable strains of the parasites for long term experiments, a rapid and definitive method of cloning was developed using micropipets and a micromanipulator. Homogeneous parasite clones prepared by this technique were characterized and compared with the parent isolates during 4 years of continuous culture. The process of phenotypic dominance and selection of drug resistance which occur in nature was also simulated in vitro by evaluating population dynamics of two cocultured isolates of P. falciparum.

Plasmodium falciparum, the human malarial parasite, was synchronized for asexual growth and pulse labeled to determine when RNA, protein, and DNA synthesis occurred. RNA was synthesized during two periods in the 48-h developmental cycle, protein synthesis occurred throughout the cycle, and most DNA was synthesized just before nuclear division (schizogony).

The over-all synthesis of DNA, RNA and protein was studied during the asexual cell cycle of Plasmodium falciparum. A method for stringent synchronization of the culture was developed. Rates of synthesis of the three macromolecules were determined every four hours by labelling the parasites with radioactive precursors during 30 min pulses. There was a peak of synthesis of DNA at 44 hours. Between 33 and 45 hours the rate of synthesis of DNA was exponential. Maximum RNA synthesis was reached at 36 hours. For proteins there were two peaks, one at 24 and the other at 40 hours. Partial degradation of the RNA synthesized by schizonts was detected.

The growth of Plasmodium falciparum in cultures of human red blood cells was studied using acridine orange to stain RNA and DNA, followed by flow cytometric analysis. The cycle of the parasite is characterized by a period of growth, prior to initiation of DNA synthesis, in which a significant increase in red fluorescence is observed, with only a small change in green fluorescence. Following this phase, which is formally similar to the G1 period in mammalian cells, initiation of DNA synthesis is characterized by increases in green fluorescence. Sorting of cells from several regions of the two-dimensional display shows that the distribution of morphological stages correlates with differences in red and green fluorescence. The effect of aphidicolin on the growth cycle of the parasite was also studied.

Employing a simple method of growing ookinetes of Plasmodium falciparum in culture, 40% of mature gametocytes convert to macrogametes, 4% reach the retort-form ookinete stage and 0.45% become mature ookinetes. A single-step gradient centrifugation method on 12.5% Nycodenz enriches both gametocytes and ookinetes.

Human antibodies affinity purified on an adsorbent prepared from a cDNA clone (Ag44) expressing a portion of a rhoptry antigen were used to characterize the synthesis and fate of the antigen in the asexual blood stages of Plasmodium falciparum. The rhoptry antigen is synthesized in the mature trophozoite-stage parasites as a 103 kDa polypeptide, is present in the schizonts and merozoites as a 105 kDa polypeptide, is discharged from the rhoptries and found in the newly invaded red cells as a 110 kDa polypeptide. Anti-Ag44 antibodies immunoprecipitate the antigen and two additional polypeptides of 135 and 150 kDa from lysates of infected cells and from culture supernatants. The three polypeptides are associated in a non-covalent complex that persists in the newly invaded red cells. All the components of the high molecular weight rhoptry complex are antigenic and can be precipitated with immune human serum. The 135 kDa polypeptide is identical to a 140 kDa rhoptry antigen previously identified by a monoclonal antibody.

Errecart (1945) proposed a modified technique for making thick films for the diagnosis of Chaga's disease. We applied his technique to the study of malaria in mammals; the morphology of the blood parasites is very well preserved and allows the identification of species which are frequently not identifiable in ordinary thick films.

Electron micrographs of ultra-thin sections of erythrocytes taken from two Liberian children ill with Plasmodium falciparum malaria show that the appliqué forms of this parasite are clearly within the host cell. The general fine structure of the parasites resembles that of other mammalian malaria parasites, as does the mode of ingestion of host cell material by pinocytosis. Granules of haemozoin were usually found in small vesicles pinched off from the large food vacuole. Scattered through the cytoplasm of infected red cells were narrow clear clefts bounded on each side by two unit membranes. These probably represent the Maurer's clefts seen in light microscopy. The surface of infected erythrocytes was notably distorted, a phenomenon which may have a bearing on the stickiness of the infected red cells in human falciparum malaria and the segregation of these cells in the capillaries. Many uninfected erythrocytes showed a multiple alveolar, blister-like abnormality of a portion of the cell membrane; this was not seen in otherwise comparable blood from a case of P. ovale infection.