FIG 4.

Experimental demonstration of phage sequestration within clusters of phage-resistant bacteria (blue) in a mixed-strain biofilm with phage-susceptible bacteria (red). Purified phages stained with Alexa Fluor 633 (shown in yellow) were added to biofilms grown for 48 h in which resistant cells were inoculated as 95% of the founding population. The central image is a top-down view of a 3D rendering measuring 50 μm × 50 μm × 15 μm (length × width × depth). The inset image is a 2D projection of a vertical slice through a 3D volume at the indicated location. The white arrow in the inset points to an immobilized phage on a cluster of resistant cells. Note that phages are much smaller than the minimum resolvable volume of a confocal fluorescence microscope like the one used here; as a result of this optical effect, the phages appear larger than their true size.