For bright-field images of bacterial pellicles and colonies, Axio Zoom V16 stereomicroscope (Carl Zeiss) was used, which was equipped with a Zeiss CL 9000 LED light source, a PlanApo Z 0.5× objective, and AxioCam 591 MRm monochrome camera (Carl Zeiss, Jena).
Confocal laser scanning microscopy (CLSM) was conducted using a Leica Microsystems Confocal Microscope SP8. For surface-attached biofilms imaging, bacterial cultures were incubated in high content imaging plate (Corning, New York) to form mature biofilms as described above. Afterward, biofilms were washed with sterilized ddH2O twice to remove non-attached aggregates. CLSM images were obtained using a 63×/1.4 OIL objective. Fluorescent excitation was conducted with the argon laser at 488 nm and the emitted fluorescence was acquired at 484–536 and 567–654 nm for GFP and mKate, respectively. Z stack series of biofilms were obtained with 1 μm steps and stacked images were merged using ImageJ software. For frequencies determination of strains in each CLSM image, ImageJ was used to convert the stacks into binary images and the threshold was set above 0. Afterward, the generated stacks were activated using the biofilm selection and the total pixel volumes for each stack were retrieved using the “stacks statistics” function120 (link).
Confocal laser scanning microscopy (CLSM) was conducted using a Leica Microsystems Confocal Microscope SP8. For surface-attached biofilms imaging, bacterial cultures were incubated in high content imaging plate (Corning, New York) to form mature biofilms as described above. Afterward, biofilms were washed with sterilized ddH2O twice to remove non-attached aggregates. CLSM images were obtained using a 63×/1.4 OIL objective. Fluorescent excitation was conducted with the argon laser at 488 nm and the emitted fluorescence was acquired at 484–536 and 567–654 nm for GFP and mKate, respectively. Z stack series of biofilms were obtained with 1 μm steps and stacked images were merged using ImageJ software. For frequencies determination of strains in each CLSM image, ImageJ was used to convert the stacks into binary images and the threshold was set above 0. Afterward, the generated stacks were activated using the biofilm selection and the total pixel volumes for each stack were retrieved using the “stacks statistics” function120 (link).