Aspect of in vitro culture of P. berghei in mouse erythrocytes. A/Follow up of the cultures. Schizonts burst and reinvasion is followed up on Giemsa-stained thin smears. Late stage (arrow head), early stage (arrow), merozoites (empty arrow head), gametocytes (G) A1: aspects of culture at day 3 after seeding showing early and late stages and gametocytes; A2: aspects of culture at day 4 with early and late stages and release of merozoites; A3: aspects of culture at day 5 showing early and late stages and release of merozoites; A4: aspects of culture at day 7 showing trophozoites. B/Double fluorescent labelling of erythrocytes and parasites To confirm reinvasion of red blood cells (RBC) by merozoites, RBC are labeled with a green fluorescent dye (PKH-67) prior to addition to the culture flask. They are added to the parasite culture at a ratio of 1:4. After two days of culture, parasites are labeled using hydroethidine (red fluorescent dye). Double-labeled IRBC are observed with a confocal microscope (Olympus FV1000, ×60). B1-3: aspect of IRBC after co-culture with green labelled RBC (bright field, red and green fluorescence merged). B4: green labelled RBC before adjunction to culture; B5-6: hydroethidine labelled parasites in culture (before adjunction of green RBC).