CLSM images of single- and two-species biofilms. Biofilms were grown on glass surfaces at 20°C for 48 h under a flow of VNSS medium. Single-species biofilms of Pseudoalteromonas sp. 3J6(pCJS10) (A and B), Paracoccus sp. 4M6 (C), Vibrio sp. D01 (E), and Bacillus sp. 4J6 (G) are shown. The Pseudoalteromonas sp. 3J6(pCJS10) biofilm was visualized by detecting only the GFP fluorescence (A) and by staining the cells with Syto 61 red and overlaying the GFP and the Syto 61 red fluorescences (B). The other single-species biofilms were stained with Syto 61 red (C, E, and G). Two-species biofilms resulted from coinoculations of Pseudoalteromonas sp. 3J6(pCJS10) and Paracoccus sp. 4M6 (D), Vibrio sp. D01 (F), or Bacillus sp. 4J6 (H). Overlays of GFP fluorescence from Pseudoalteromonas sp. 3J6(pCJS10) and of Syto 61 red fluorescence from all cells of the biofilms are displayed (D, F, and H). Maximal projections in the xy plane (top of each panel) and three-dimensional (3D) shadow projections (bottom of each panel) are presented. Bars, 30 μm.