Common methodology is inadequate for studies on the microbicidal activity of neutrophils

Microbicidal activity of neutrophils is usually measured by colony-counting techniques after cell lysis in distilled water. While studying the effect of the reduced nicotinamide adenine dinucleotide phosphate-oxidase inhibitor diphenyleneiodonium (DPI) on the staphylocidal activity of neutrophils, we obtained inconsistent results: various degrees of inhibition in some experiments and no effect in others. The lysis step, i.e., dilution of neutrophils in distilled water, was the source of error. Cell-associated microorganisms were not dispersed effectively by this treatment. We overcame this problem by using water at pH 11 for cell lysis. Under these conditions, killing was inhibited completely and reproducibly by DPI. Here, we show that cell lysis in distilled water is incomplete and leads to an overestimate of microbial killing. This hinders identification of partial defects and makes complete defects appear as partial. We found that DPI-treated neutrophils and chronic granulomatous disease neutrophils were completely defective in killing of Staphylococcus aureus and Candida albicans and partially defective in killing of Escherichia coli after lysis with water pH 11, whereas after lysis in distilled water, killing of S. aureus and C. albicans was approximately 60% and approximately 70% of control killing, respectively, and killing of E. coli was normal. Likewise, killing of S. aureus by myeloperoxidase-deficient neutrophils was severely impaired after lysis in water pH 11 but appeared normal after lysis in distilled water. As most studies about neutrophil microbicidal activity have been performed using distilled water, our findings indicate that previous data about killing defects and the effects of agents that modulate microbicidal activity of neutrophils should be re-evaluated.