Neutrophil killing of single microorganisms as measured by a new method

A new method of measuring polymorphonuclear leukocyte (PMN) microbicidal activity was developed in which single colony forming units of Staphylococcus aureus were isolated in microtiter wells with or without PMN, centrifuged, and incubated overnight. The percentage of bacteria killed was determined by comparing the number of PMN-containing wells showing bacterial growth to PMN-free controls. This percentage was positively correlated with the number of PMN in each well and was reduced if plates were not centrifuged or if cytochalasin B was added. Killing of either S. aureus or Escherichia coli was enhanced using fresh serum activated by prior exposure to large numbers of E. coli. There was no enhancement using heated serum that had been exposed to E. coli, nor was there enhancement when S. aureus was preopsonized by exposure to fresh serum. This assay circumvents problems in assessing microbicidal activity where aggregation of organisms might otherwise be an issue.