These images were then used to determine percentage viability and biofilm coverage using pixel counting with the aid of Adobe Photoshop. Three random representative images were taken from each block used for FISH and Live/Dead staining. The 3D images were created from 1 μm z-stacks slices of varying heights (depending on the height of the biofilm) and were constructed using Zeiss 3D imaging software. SEM analysis During co-culture experiments blocks (2 mm wide) were

removed from the reactors at 72 and 144 hour time points and fixed immediately for SEM analysis. SEM fixation involves the use ARS-1620 order of 3 solutions. Solution 1 contains 0.043 g lysine (L-lysine free base Sigma L-5501) dissolved in 2 ml of 0.1 M cacodylate buffer. Solution 2 contains 0.4 ml 25% glutaraldehyde, 1.0 ml 0.2 M cacodylate buffer and 0.6 ml distilled water. Solutions 1 and 2 were mixed together thoroughly immediately before use. Samples were left in this for 10 minutes then transferred to solution 3 which is 2.5% glutaraldehyde in 0.1 M cacodylate buffer for further sample processing as described in Jacques & Graham [47]. Samples for SEM Selleck PX-478 were visualized using JEOL JSM- 6400F microscope (10 kV, 3000 V) and EIKO IB-5 sputter coater using

platinum. COMSTAT analysis of biofilms Z-stacks generated using the CLSM were further analysed using COMSTAT to determine roughness coefficient and mean biofilm thickness. Through COMSTAT a fixed threshold was applied to the images to provide Staurosporine purchase a 0 or 1 value to image pixels. One represents areas containing biomass while 0 is considered as background [48]. The thickness function is the maximum thickness over a given location which does not take into account any pores or voids within the biofilm. The thickness distribution is then used to calculate the biofilm roughness and mean biofilm thickness. Roughness coefficient provides an indication of how the thickness of the biofilm varies and also provides an indication of biofilm heterogeneity [48]. Acknowledgements This study was supported by the Australian Research Council (grants

DP0879245) and The University of Queensland Early Career Researcher Scheme (UQ2006001877). SR is also supported by the Queensland Government (Smart State Award funding), The University of Queensland (Confirmation Scholarship). P. aeruginosa PAO1, S. H 89 nmr oneidensis MR-1 and E. faecium were kindly provided by Dr Scott Rice, Dr Kenneth H Nealson and Dr Jeanette Pham respectively. The useful comments of Rene Rozendal, Thomas Seviour, Dr Stephen Myers and Jeremy Barr are highly appreciated. Acknowledgement also to Dr Keshab Sharma for technical assistance with MATLAB and COMSTAT. Electronic supplementary material Additional file 1: CLSM top view cropped image of S. oneidensis biofilm (Figure 2) (63×) providing a close-up of the nonviable cells using Live/Dead (Baclight) stain. Additional File 1 is a more detailed confocal image of the S. oneidensis biofilm.