ENUMERATION OF PHAGOCYTOSED STAPHYLOCOCCUS-AUREUS INSIDE CELLS OF THEMOUSE MACROPHAGE CELL-LINE J774 BY BIOLUMINESCENCE, FLUORESCENCE AND VIABLE COUNTING TECHNIQUES

In order to quantify intracellular Staphylococcus aureus within a macr ophage-like cell line by a bioluminescence technique, the mouse cell l ine J774 and opsonized Staphylococcus aureus were incubated together t o allow phagocytosis to occur. Experiments using UV microscopy and flu orescent stained S. aureus were performed to determine an estimate of the mean intracellular bacterial numbers. For enumeration of intracell ular bacteria by a bioluminescence technique, extracellular bacteria w ere removed by washing. the macrophages lysed mechanically and osmotic ally and treated with apyrase to remove somatic ATP. Bacterial cells w ere washed and the intracellular ATP measured by firefly luciferase bi oluminescence in a luminometer. This new method of enumerating intrace llular bacteria was compared to the conventional method of viable coun ts and found to correlate (r = 0.78). The bioluminescence assay develo ped was found to be a relatively rapid alternative method to the techn iques currently used to enumerate intracellular bacteria and could pro ve advantageous in studies of intracellular killing and effects of ant imicrobial agents on intracellular pathogens.