Bovine leukocyte phagocytosis and bacteria killing monitored by intracellular acridine orange fluorescence and extracellular fluorescence quenching

The time course of phagocytosis and intracellular killing of serum-opsonized Escherichia coli K12 and Staphylococcus aureus SG511 by glass-adherent bovine peripheral blood polymorphonuclear leukocytes (PMNLs) and cultured monocytes (macrophages) was monitored by fluorescence microscopy of single cells using the acridine orange (AO)/crystal violet (CV) technique. After interaction of glass-adherent leukocytes (20, 40, 60 min, 37 degrees C) with opsonized bacteria, cells were stained with the fluorescent dye AO. Living bacteria stained green, dead bacteria stained orange. The addition of CV to AO-stained bacteria quenched the fluorescence of extracellular bacteria only. CV does not penetrate living bovine PMNLs which allows the discrimination of ingested (fluorescent) and extracellular (nonfluorescent) bacteria during attachment and phagocytosis of bacteria by adherent PMNLs. We investigated quantitatively phagocytosis and intracellular killing of serum-opsonized bacteria by bovine PMNLs from 22 bulls of 4 different Swiss dairy breeds. Within 60 min maximum uptake (approximately 12 bacteria/PMNL) and killing (approximately 80%) of serum-opsonized Escherichia coli K12 and Staphylococcus aureus SG511 were achieved. The AO/CV technique was also used to quantify the uptake and intracellular killing of serum-opsonized Escherichia coli K12 by cultured monocytes (macrophages). Within 60 min maximum uptake of bacteria (approximately 16/MO) was achieved; approximately 83% of bacteria were killed.