QUANTIFICATION AND CHARACTERIZATION OF PHAGOCYTOSIS IN THE SOIL AMEBAACANTHAMOEBA-CASTELLANII BY FLOW-CYTOMETRY

Phagocytosis in the common grazing soil amoeba Acanthamoeba castellani i was characterized by flow cytometry. Uptake of fluorescently labelle d latex microbeads by cells was quantified by appropriate setting of t hresholds on light scatter channels and, subsequently, on fluorescence histograms. Confocal laser scanning microscopy was used to verify the effectiveness of sodium azide as a control for distinguishing between cell surface binding and internalization of beads. It was found that binding of beads at the cell surface was complete within 5 min and 80% or cells had bead associated with them after 10 min. However, the tot al number of phagocytosed beads continued to rise up to 2 h. The prolo nged increase in numbers of beads phagocytosed was dye to cell populat ions containing increasing numbers of beads peaking at increasing time intervals from the onset of phagocytosis. Fine adjustment of threshol ds on light scatter channels was used to fractionate cells according t o cell volume (cell cycle stage). Phagocytotic activity was approximat ely threefold higher in the largest (oldest) than in the smallest (new ly divided) cells of A. castellanii and showed some evidence of period icity. At no stage in the cell cycle did phagocytosis cease. Binding a nd phagocytosis of beads were also markedly influenced by culture age and rate of rotary agitation of cell suspensions. Saturation of phagoc ytosis (per cell) at increasing bead or decreasing cell concentrations occurred at bead/cell ratios exceeding 10:1. This was probably a resu lt of a limitation of the vacuolar uptake system of A. castellanii, as no saturation of bead binding was evident. The advantages of flow cyt ometry for characterization of phagocytosis at the single-cell level i n heterogeneous protozoal populations and the significance of the pres ent results are discussed.