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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 cyde of the parasite is characterized by a period of growth, prior to initiation ofDNA 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 Gi period in mammalian

Intra-erythrocytic Plasmodium species can be stained with the DNA binding dye, Hoechst 33342, and the distribution of DNA content determined for parasite populations by flow cytometric measurement of fluorescence. Analysis of this distribution will determine the parasitaemia (percentage of erythrocytes infected), and the percentages of trophozoite infected red blood cells, polyparasitized (trophozoite) red blood cells, and schizont/segmenter infected red blood cells. This analysis is based on the hypothesis that the asexual parasites cycle with single G1 period, and effectively, a single S phase with no significant G2/M period except at schizogony when the genome DNA content is equivalent to 8 N or higher, dependent on the species. Data are presented to support this model.

The fluorescent dye 3,3'-dimethyloxacarbocyanine (DiOC1[3]) is taken up by all cells in mammalian blood which then fluoresce as follows: mature erythrocytes < immature erythrocytes = platelets < leukocytes. A continuous fluorescence distribution can be generated for the red blood cells by flow cytometry and deconvolved into two arbitrary populations, mature and immature erythrocytes (mRBC and imRBC). This analysis mimics the established method of counting imRBC stained with the supravital dyes, newWe have described the use of the cyanine dye 3,3'-dimethyloxacarbocyanine (DiOCJ31) for flow cytometric analysis of blood cells from mice infected with the rodent malaria parasites P vinckei (8) and P chabaudi (9). It was shown that using measurements of low angle forward light scatter (LALS) correlated with those of fluorescence intensity @I), the parasitized (pRBC), immature (imRBC), and mature (mRBC) components of the red blood cell (RBC) population could be quantified. However, because of the high degree of fluorescence overlap between the populations, the analysis was imprecise.In order to improve the analysis of parasitized blood, a protocol using a combination of DiOC1[3] and the DNA binding dye Hoechst 33342 (Ho) has been devised. In this manner, the DNA containing pRBC can be quantified precisely by measurement of Ho fluorescence. The uninfected RBC population, which is composed of RBC in various stages of maturation, can then be analyzed with respect to DiOC1[3] fluorescence after gating on Ho fluorescence. The quantification of the immature RBC population then becomes the same problem as encountered in any normal or anemic blood sample from an uninfected host. This paper describes the flow cytometric analysis of RBC maturity using DiOC1 [3]. Although we have primarily developed the assay in order to pursue goals in malaria research, the procedure will work well for studies in which quantification of an immature subpopulation of mouse RBC is necessary or desirable. Further,

A previous study (Hare JD, Bahler DW: J Histochem Cytochem 34:215, 1986) has shown that the flow cytometric analysis of acridine-orange-stained Plasmodium falciparum growing in vitro generates a complex two-color display, regions of which correlate with the major morphological stages. In this report, four cell cycle compartments (A-D) are defined by characteristic ratios of red and green fluorescence of cells distributed throughout the erythrocytic cycle as well as by the differential effects of several metabolic inhibitors. The primary characteristic of cells in compartment A is the significant increase in red fluorescence. Inhibition of DNA synthesis by either aphidicolin or hydroxyurea causes the accumulation of cells at the interface between compartments A and B, whereas n-butyrate prevents cells in compartment A from reaching the A-B interface. Cells in compartment A display a small increase in green fluorescence which is independent of DNA synthesis but is enhanced by n-butyrate treatment. Cells in compartment B display a continued increase in red fluorescence coupled with a significant increase in green fluorescence, reflecting the onset of DNA synthesis in compartment B. The transition to compartment C is more abrupt and is associated with a marked increase in green fluorescence and little increase in red fluorescence. Compartment D is characterized by an increase in red fluorescence and a continued rise in green fluorescence. It is postulated that these discontinuities in the two-color display reflect not only changes in the rates of RNA and DNA synthesis but also decondensation of parasite chromatin in compartment A as the organism prepares for DNA synthesis, and re-condensation in compartment D as the newly replicated chromatin prepares for segregation into merozoites. The method described promises to provide a sensitive and rapid technique to study the effects of various factors on the growth cycle of the parasite.