Double immunofluorescence microscopic technique for accurate differentiation of extracellularly and intracellularly located bacteria in cell culture

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Abstract
Membranes of viable mammalian cells are impermeable to antibodies but are rendered permeable by treatment with fixatives. Consequently, extracellular bacteria can be stained by specific rhodamine-labeled antibodies before fixation, and intracellular bacteria can be visualized by treatment with specific-fluorescein-labeled antibodies after fixation. The accuracy and simplicity of this method was demonstrated with HEp-2 cell culture monolayers as target cells and an isogenic pair of Yersinia enterocolitica, one of which is phagocytosis-resistant and the other of which is phagocytosis-sensitive. This staining technique ia alsao applicable for studying the interaction of bacteria with macrophages and fibroblasts.