Biofilm Volume Estimation via Microscopy

Biofilms prepared on glass substrates were examined using Nikon A1R confocal microscope and the volume was estimated using BiofilmQ software, as previously described [23 (link),29 (link)]. Briefly, biofilms were stained with Syto9 dye (Invitrogen) in dark for 15 min and imaged every 1.74 μm along the biofilm height using a DIC 20× objective. Series of images were recorded on a minimum of three replicate samples. The total volume of biofilms in the assay was then calculated by extrapolating the value obtained for the image to the total surface area of one side of the glass slide. Thus, the data presented assumes that glass slides were fully covered with biofilms and that the obtained images are representative enough to allow extrapolation to the total surface area of the glass substrates.