Fluorescence staining combined with CLSM was used to visualize the changes in microbial communities during the formation of biofilms. The biofilms adhering to the stainless-steel slices during different time periods were collected using a sterile knife. The treated biofilms were tested using a LIVE/DEAD® BacLight™ Bacterial Viability Kit by following the manufacturer’s instruction. An anti-quenching reagent ProLong™ Gold was used after fluorescence staining. Different light path combinations for CLSM were chosen to observe the survival ratios of bacteria and the structure of the biofilm.
Quantification of biofilm parameters was processed with the COMSTAT software using the CLSM images [76 (link)]. Of the available parameters, we selected total biomass, average thickness, and roughness coefficient to evaluate the biofilms [77 (link)]. The abundances of live and dead biomass were evaluated based on the fluorescence intensity. An NIS-Elements Viewer (Nikon, Japan) was used to produce three-dimensional (3-D) transmission-fluorescence images of biofilms. Optical sections were 1 µm apart on the Z-axis. The length and width of the 3-D box were both 118 µm and the thickness was 25 µm.
Quantification of biofilm parameters was processed with the COMSTAT software using the CLSM images [76 (link)]. Of the available parameters, we selected total biomass, average thickness, and roughness coefficient to evaluate the biofilms [77 (link)]. The abundances of live and dead biomass were evaluated based on the fluorescence intensity. An NIS-Elements Viewer (Nikon, Japan) was used to produce three-dimensional (3-D) transmission-fluorescence images of biofilms. Optical sections were 1 µm apart on the Z-axis. The length and width of the 3-D box were both 118 µm and the thickness was 25 µm.