A one-plate assay for macrophage bactericidal activity.

Human peripheral blood monocytes purified by counterflow centrifugal elutriation (CCE) showed an enhanced capacity to kill Listeria Monocytogenes and Staphylococcus aureus after activation with interferon-gamma (IFN gamma). Bactericidal activity was evaluated by a rapid colorimetric microassay based on the reduction of a tetrazolium dye, MTT. The amount of dye reduced was directly proportional to the number of viable bacteria present in each microwell. Comparison with a bacterial titration plate containing serial dilutions from stock of a known titer allowed calculation of bacterial numbers before and after exposure to macrophages. Results were read in a multiscan plate reader and compared closely with those obtained by serial dilution on agar plates (i.e., colony counts). The entire assay, i.e., culturing the monocytes, treatment with IFN gamma, addition of the bacteria, and quantitation of surviving viable bacteria, was performed in a single microtiter plate.