Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria.

One of the challenges of phagocytosis research is to differentiate bacteria adherent to a host cell from bacteria which the cell has internalized. To address this question, various techniques such as fluorescence microscopy, electron microscopy, and flow cytometry have been used. We have adapted a flow cytometric method (Fattorossi et al., 1989) to use fluorescence microscopy for studying phagocytosis of fluorescein-labeled Listeria by inflammatory mouse peritoneal macrophages. In this assay, ethidium bromide is used as a quenching agent and is added to cells after they have phagocytosed labeled bacteria. Ethidium bromide causes extracellular FITC-labeled Listeria to fluoresce red-orange, whereas intracellular bacteria are not exposed to the dye and remain green. This process allows distinction between intracellular and extracellular bacteria by simultaneous visualization of both populations.