FLOW CYTOMETRIC DETERMINATION OF ENDOCYTOSIS OF VIABLE LABELED LEGIONELLA-PNEUMOPHILA BY ACANTHAMOEBA-PALESTINENSIS

Endocytosis of fluorescently labelled cells of Legionella pneumophila (L. pneumophila) by free-living Acanthamoeba palestinensis (A. palesti nensis) has been studied using flow cytometry. L. pneumophila cells we re labelled with CM-DiI, a Lipophilic fluorescent probe under conditio ns that did not modify viability, Coculturing the bacteria with amoeba e was accompanied by rapid endocytosis; after 5 min, 90% of the amoeba e had internalized bacteria. This percentage remained unchanged during further coculture, but the number of bacteria ingested per amoeba inc reased, Moreover, the number of ingested bacteria was found to be depe ndent on the size of the amoeba. The validity of the internalization a nalyzed by flow cytometry was confirmed by observation using epifluore scence and phase contrast microscopy, CM-DiI labelling associated with flow cytometry provides a very valuable technique for the determinati on of bacteria endocytosis by free-living amoeba. (C) 1997 Wiley-Liss, Inc.