FIG 3.
Development of nfxB mutants in a 72-h-old PAO1 flow cell biofilm during treatment with low-dose CIP. The biofilms of PAO1-mCherry-PCD-gfp+ were grown in three independent channels of flow cell chambers with a continuous flow of minimal medium for 72 h and then treated with 0.2 μg/ml CIP for a total of 96 h. Imaging by CLSM was done every 24 h. At least 3 images were taken per channel at every time point. Red represents wild-type cells due to the constitutive expression of mCherry, and green shows nfxB mutants due to the expression of GFP+ via the PCD-gfp+ reporter. z-stacks were generated by using a Zeiss LSM 710 microscope and processed with Imaris 8.2 software (Bitplane). The images show orthogonal 3D biofilm views (left) or a perspective view (right) with an overlay of red and green channel fluorescence.