Fluorescent staining of intracellular and extracellular bacteria in blood

The fluorescent dye ethidium bromide stains Escherichia coli and Staphylococcus aureus in whole blood. The staining is rapid, relatively specific, and does not require fixation of the sample. Furthermore, stained bacteria can be seen microscopically without the need for a final wash to remove unbound dye. By using lysostaphin, an S. aureus-specific lytic enzyme, we have demonstrated that S. aureus can be stained with ethidium bromide even after phagocytosis. After short periods of incubation with the dye (less than 5 min), bacteria, both intracellular and extracellular, were the predominant fluorescent particles. With increasing time of incubation, blood cell components, notably leukocyte nuclei, began to fluoresce.