Plasmodium species: flow cytometry and microfluorometry assessments of DNA content and synthesis

Fluorescence intensities were established by flow cytometry of different erythrocytic stages of Plasmodium berghei after staining of their DNA with Hoechst-33258 or Hoechst-33342. Parasites were obtained from highly synchronized infections or in vitro cultures. Most fluorescence measurements were performed using a low cost, clinical flow cytometer, equipped with a mercury arc lamp. Cells infected with P. berghei could be readily distinguished from uninfected cells on the basis of Hoechst-DNA fluorescence and single, double, and triple ring infected cells were separated clearly. The relative fluorescence intensities of different developmental stages (merozoites, ringforms, trophozoites, schizonts, and gametocytes) corresponded closely to the relative DNA contents of these stages as measured by microfluorometry. Flow cytometry appeared to be a sensitive and rapid method to measure DNA synthesis during asexual development; a C50 value of 5 microM of aphidicolin, a specific inhibitor of DNA synthesis, was established. Vital staining of parasites in culture was possible with both Hoechst dyes. After removal of Hoechst-33258, normal in vitro development of the stained parasites was observed. After Hoechst staining, the haploid ringforms of P. vivax showed slightly less fluorescence (15%) than ringforms of P. berghei and P. falciparum. No differences in fluorescence intensity were observed, however, by direct microfluorometry after Feulgen-pararosaniline staining, indicating that all three species have the same DNA content.