FIGURE 2.

Assessment of the intracellular and outer membrane-associated F. tularensis bacteria. Vero cells infected with F. tularensis LVS bacteria were analyzed using a confocal microscope. All cell cultures were treated for 1 h with 20 μg/ml gentamicin following 3xPBS washes. Cell cultures of 5 and 24 h were grown in medium containing 20 μg/ml gentamicin. Double-labeled immunofluorescent images of F. tularensis LVS infections of Vero cells after 0, 5, and 24 h are presented in (A). Bacteria bound to the outer membrane appear green (AI,V,IX) or yellow in merged panels (AIV,VIII,XII), while intracellular bacteria appear exclusively red (AII,VI,X). Nuclei stained with DAPI are presented in images (AIII,VII,XI). The frame size in (AI–XII) is 512 × 512, the dimensions are 70.71 × 70.71 μm, the magnification is x120, and the scale bar = 20 μm. A representative optical slice of the 3D analysis of a nucleus surrounded by bacteria in infected cells is presented in (B). The images represent fluorescent (BI) and surface projections (BII) of the same cell 5 h post infection. Frame size: 512 × 512; dimensions: 73.15 × 73.15 × 23 μm; magnification: x120; scale bar = 20 μm. The ratio between the fluorescent intensity of intracellular versus outer membrane-associated bacteria labeling is presented in (C). All experiments were performed in duplicate and repeated three times. Bacterial growth curves in the cellular fraction of Vero cells were monitored by cfu counts in the presence (red line) or absence of gentamicin (blue line). The experiment was performed in duplicate (D).