Cells were grown from a single colony isolated from LB plates, transferred to LB broth, and grown to a mid-logarithmic phase in 2-ml of LB (∼3 h at 37 °C). Cells were added to the 2 mL liquid MSgg in 24 well plates (Thermo Scientific). The cultures were then grown at 30 °C for 72 h in the dark. The pellicles were imaged at 24 h and 72 h. Cells were either grown in the presence or absence of CM as indicated in each corresponding figure legends. The pellicles were imaged using stereomicroscope (Zeiss), using Objective Plain 0.5 × FWD 134 mm lens at 10× magnification. Captured images were processed using Zen software (Zeiss).
For CFU count, pellicles were harvested at 24 h or 72 h by scrapping off the entire pellicle plus pipetting the entire underlying cell suspension, and suspending in PBS (phosphate-buffered saline). To separate the cells a BRANSON digital sonicator, was used at an amplitude of 10% with pulse of 5 s. This sonicated cells suspension was transferred to a Griener 90 well plate (Sigma-Aldrich) and a serial dilution ranging from 10−1 to 10−7 was performed in PBS. From these dilutions 20 µl of samples was transferred on a LB plate. Plates were incubated at 30 °C overnight and CFU were counted next day.
For CFU count, pellicles were harvested at 24 h or 72 h by scrapping off the entire pellicle plus pipetting the entire underlying cell suspension, and suspending in PBS (phosphate-buffered saline). To separate the cells a BRANSON digital sonicator, was used at an amplitude of 10% with pulse of 5 s. This sonicated cells suspension was transferred to a Griener 90 well plate (Sigma-Aldrich) and a serial dilution ranging from 10−1 to 10−7 was performed in PBS. From these dilutions 20 µl of samples was transferred on a LB plate. Plates were incubated at 30 °C overnight and CFU were counted next day.