Direct infection of antigen-presenting cells (APCs) with living intracellular bacteria may influence the early innate immune response as well as the following T-cell response. Revealing the identity of primarily targeted cells during infection is therefore an important task, which requires sensitive methods for the intracellular detection of bacteria before their extensive replication. Determination of colony-forming units (cfu) combines – in contrast to microscopy-based methods – a high sensitivity with the specific recognition of viable multiplying organisms. We recently explored an FACS-based ex vivo cell isolation protocol followed by cfu quantification of lysate-derived bacteria in order to make even very few organisms visible. With this approach, the evaluation of cell-subsets during the earliest phase of bacterial infection became feasible. In general, the assay should be transferable to the ex vivo detection of other intracellular (viral or parasitic) pathogens.