PAO1 and MS2507 were prepared in the same way as those described in the quantification assay of the biofilm. Freshly prepared bacterial solutions were inoculated into 2 mL of LB for PAO1 and TSB for MS2507 supplemented with various concentrations of cyclic-di-GMP or its analogs in glass-based dishes (coverslip diameter 27 mm, Iwaki) and were incubated statically at 30 °C for 15 h for PAO1 and 9 h for MS2507. The culture supernatant was discarded and the dishes were carefully rinsed three times with 0.85% NaCl and viability staining of unfixed biofilms was performed using BacLight Live/Dead kit (L-7012, Molecular Probes) according to the manufacturer's recommendations to visualize biofilms. The biofilms attached on the polyethyleneimine-coated glass dishes (Iwaki) were observed at × 400 magnification using a confocal laser scanning microscope (LSM5 Pascal, Carl Zeiss Co. Ltd). Images were acquired at a 1-μm interval through the biofilms and five image stacks, each representing a different field of view, and were compiled from at least two independent experiments. The image stacks were analyzed using the comstat program (Heydorn et al., 2000 (link)) and the following parameters were obtained: total biomass; mean thickness, the mean height of the biofilm; maximum thickness; roughness coefficient, a measure of how much the thickness of the biofilm varies; and surface-to-volume ratio, an estimate of the portion of the biofilm exposed to nutrients.
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