FIG. 5.

LIVE/DEAD viability staining of material recovered from the chinchilla middle ear following GAS infection. Material was recovered 7 dpi with MGAS5005 (A to C) or MGAS5005 Δsrv (D to F) and stained for a two-color fluorescence assay of bacterial viability (LIVE/DEAD BacLight; Molecular Probes). Nonviable cells with damaged membranes are depicted in red (A and D), and cells with intact membranes stained green (B and E). Overlap of both signals appears as yellow staining (C and F). A horizontal Z-slice image (center) and vertically stacked images (margins) show living MGAS5005 bacteria in a concentrated community suggestive of a biofilm (C); alternatively, MGAS5005 Δsrv was found dispersed throughout the macroscopic structure depicted in Fig. 2 (F). Zeiss LSM Image Browser software three-dimensional distance analysis predicted the thickness of the MGAS5005 community to be ∼680 μm (C) and that the MGAS5005 Δsrv community to be ∼696 (F) μm, as indicated by the lines in the respective panels.