Quenching of fluorescence by crystal violet and its use to differentiate between surface-bound and internalized bacteria

Abstract

Phagocytosis is a complex process involving attachment, ingestion and intracellular processing of bacteria by phagocytes. A great difficulty in the evaluation of this process is to differentiate between attachment of the particles to the cell surface and internalization of the particles by the cells. Various techniques have been used to differentiate internalized and surface-attached bacteria in cultured cells, but only a few permit differentiations between surface-bound and internalized bacteria. In this study the quenching of fluorescence by crystal violet on acridine orange stained bacterial biofilm and planktonic bacterial cells is used to differentiate between surface-bound and internalized bacteria within macrophages. Method: One week old Enterococcus faecalis biofilm was grown on perspex and glass substrates in All-Culture medium (nutrient-rich condition) and phosphate buffered saline (nutrient-deprived condition). As model systems, human monocytic (THP-1) and histiocytic (U937) cell lines were used. These cell lines were incubated with the biofilm bacteria for 4 hrs in CO2 incubator at 37°C. The cells and bacteria were stained with acridine orange and quenched with crystal violet to distinguish between surface-bound and internalized bacteria. Results: The presence of green-fluorescing internalized bacteria was detected within the macrophages under the planktonic, nutrient-rich and nutrient-deprived biofilm conditions. All infecting bacteria take up acridine orange and fluoresced green, crystal violet quenched the fluorescence of extra-cellular adhering bacteria so that only fluorescent intracellular bacteria would be visible under fluorescent light microscopy.