After incubation for one or two 48-h periods, titanium disks coated with biofilms were gently washed with PBS and stained with a fresh mixture of SYTO-9 and propidium iodide (BacLight live/dead bacterial viability kit, Molecular Probes, Eugene, United States) based on the manufacturer’s instruction. And observed by confocal laser scanning microscopy (CLSM, FV10i-LIV, Olympus, Canada) (Yu et al., 2021 (link); Liu et al., 2022 (link)). The wavelength of excitation/emission light detected for SYTO-9 and propidium iodide was set 480/500 nm and 490/635 nm, respectively. For each group, three disks were selected, and five areas for each disk were scanned from the bottom to the top of the biofilm with a 2 μm step. An Imaris 7.2 software (Bitplane, Switzerland) was employed to convert the two-dimension images into three-dimensional volume stacks. The obtained green and red fluorescence intensity was analyzed as the total volume of live and dead bacteria, and the dead bacteria proportion was calculated using red fluorescence in relation to total fluorescence (green + red) (Guo et al., 2021 (link)).
Free full text: Click here
