A method to detect rabbit neutrophil phagocytosis of Pasteurella multocida

A method was developed to detect neutrophil phagocytosis of bacteria by determining whether neutrophil-associated bacteria were intra- or extracellular. Neutrophils were treated with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride to inhibit degranulation and, consequently, killing of bacteria. Treated neutrophils and opsonized Pasteurella multocida were combined. Following phagocytosis, the suspensions were centrifuged and the pellets were washed to remove non-cell-associated bacteria. The pellets were resuspended and heparin was added to prevent further phagocytosis. Samples were removed, and the number of viable bacteria was determined by a dilution and plate count technique. Streptomycin, an antibiotic that is poorly taken up by neutrophils, was added to kill extracellular bacteria, and the suspensions were incubated for 20 minutes at 37 C, and samples were removed again and bacterial numbers were determined. Percentage killing of bacteria by streptomycin was calculated. Phagocytosed bacteria were protected from the bactericidal action of streptomycin.