Method of biofilm analysis were as described in previous studies (Ha et al., 2007 (link); Singhal et al., 2012 (link); Drilling et al., 2014 (link); Paramasivan et al., 2014b (link); Rajiv et al., 2015 (link)). Two random 1 × 1 cm mucosal sections from each sinus were sampled. Each sample was briefly immersed in phosphate buffered solution to wash off planktonic cells and stained with LIVE/DEAD BacLight stain (Life Technology, Mulgrave, VIC, Australia) as per manufacturer's instructions. Biofilm biomass was assessed using confocal scanning laser microscope (LSM 710, Zeiss, Germany). Within each sample 3 of the areas with highest biofilm presence had axial Z stacks recorded to construct a 3D virtual image of the overlying tissue mucosa and biofilm, making a total of 6 Z-stack images per sinus. Eighty individual images of each representative area were taken as one Z stack image (Image properties: line average 4, 512 × 512 pixels, Z-stack 80 steps). The COMSTAT2 computer software (Lyngby, Denmark) was utilized to quantify biofilm biomass in each Z-stack (Heydorn et al., 2000 (link); Klinger-Strobel et al., 2016 (link)).
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