*2.5. Scanning Electron Microscopy (SEM)*

For SEM analysis, the 72 h biofilms were carefully fixed with 2.5% glutaraldehyde solution for 12 h at 4 ◦C, then dehydrated in a series of ethanol (30, 50, 70, 80, 85, 90, 95, and 100% ethanol) and sputter-coated with gold. Specimens were examined at 10,000× magnification [25]. Three specimens were tested for each group and each sample was taken with three images.

#### *2.6. Confocal Laser Scanning Microscopy (CLSM)*

The bacteria and extracellular polysaccharides (EPS) of 72 h biofilms were labeled with SYTO 9 (Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA) and Alexa Fluor 647 labeled dextran conjugate (Molecular Probes), respectively, as previously described [26]. Biofilm images were captured using a confocal laser scanning microscope (Olympus FV1000, Tokyo, Japan). The image collection gates were set to 495–515 nm for SYTO 9 and 655–690 nm for Alexa Fluor 647. Each biofilm was scanned at five selected positions, and then the confocal image series was generated by optical sectioning of each of these positions. Three-dimensional reconstruction of the biofilms and the quantification of EPS/bacteria biomass were performed with IMARIS 7.0.0 (Bitplane, Zurich, Switzerland). The EPS/bacteria ratio was calculated with ImageJ software [26,27]. Three specimens were tested for each group.
