EVALUATION OF A METHOD FOR MEASUREMENT OF INTRACELLULAR KILLING OF STAPHYLOCOCCUS-AUREUS IN HUMAN NEUTROPHIL GRANULOCYTES

A modified method for measurement of intracellular killing of Staphylo coccus aureus in human neutrophil granulocytes is described. After pha gocytosis of S. aureus the extracellular bacteria were sufficiently re moved by repeated centrifugations and washings of the granulocytes. Th e optimal conditions for incubation of granulocytes for measurement of intracellular killing were found to be 37 degrees C in the presence o f 5% CO2. Under these conditions, stable pH, the viability and the cap acity of the granulocytes for superoxide anion generation were preserv ed. The number of intracellular viable bacteria was determined after l ysis of the granulocytes, which should be done in H2O at pH 11 to ensu re sufficient cell lysis. The kinetics and individual variation of the intracellular killing are described. The intra- or extracellular loca tion of surviving bacteria was studied. After approximately 8 h incuba tion we observed intracellular growth of S. aureus followed by lysis o f granulocytes and extracellular growth of bacteria. Consequently, the incubation period should not be extended beyond 5 to 8 h when the ass ay is used to study the effects of antibiotics on intracellular killin g.