Biofilm Spatial Structure on PDMS and GNP/PDMS

The spatial structure of single- and dual-species biofilms of P. aeruginosa and S. aureus on PDMS and GNP/PDMS surfaces was assessed by CSLM as described by Lima et al. [7 (link)]. S. aureus and P. aeruginosa + S. aureus biofilms were first counterstained with 6 µM SYTO^®9 (Invitrogen Life Technologies, Alfagene, Portugal). All biofilm samples were then observed using a 40× water immersion objective lens (Leica Microsystems, Wetzlar, Germany) in an inverted microscope Leica DMI6000-CS with 488 nm argon and 633 nm helium-neon lasers. Image stacking was acquired with a 1 μm thickness for each sample at a minimum of five random fields. Image processing was performed using the IMARIS 9.1 software package (Bitplane, Zurich, Switzerland) for modelling in two and three dimensions [55 (link)]. The CLSM acquisitions were analyzed by the plug-in COMSTAT2 associated with the ImageJ software to determine biofilm structural parameters such as biovolume (µm³ µm⁻²) and average thickness (µm) [56 (link)].