An improved fluorochrome microassay for the detection of living and non-living intracellular bacteria in human neutrophils.

Acridine orange fluorescence may be used to distinguish living from non-living intracellular bacteria in individual glass-adherent neutrophil granulocytes (PMN). An improvement of the original assay (Smith and Rommel, 1977; Pantazis and Kniker, 1979) is described which allows differentiation between ingested and cell-adherent bacteria. It is shown that this differentiation is impossible with the original method using wet-mounted preparations. With the improved method, however, using dry-mounted preparations, cell-adherent as well as extracellular bacteria lose their fluorescence. Moreover, the fluorescence of cell nuclei and granula is reduced to a minimum. Phagocytosis kinetics and selective inhibition of the myeloperoxidase of PMN show that living intracellular bacteria fluoresce green and non-living bacteria red in such dry-mounted preparations. The preparations can be stored and interpreted for at least 2 months. Application of this method requires 0.1 ml blood or cell-rich body fluid per preparation and is fast and inexpensive.