Quantitative Analysis of Biofilm Formation

A static biofilm growth system was conducted in 96-well plates (Nunclon®, Roskilde, Denmark). Bacterial suspensions with absorbance measurements at 660 nm (A₆₆₀) of between 0.05 and 0.13 were incubated in LB media for 24 hours at 37 °C and 5% CO₂ with vigorous shaking. The biofilm was subjected to two washes with 0.9% saline. Biofilms located at the bottom of the micro-wells were analysed using an Olympus confocal laser scanning microscope (CLSM) with 10 × lenses and 488/510 and 545/610 nm excitation/emission filters. Signals were produced by bacteria harbouring the pMRP9-1 plasmid. GFP produced and localized to live cells was also detected, similar as in previous studies2 21 (link). All signals were calculated using Olympus FLUOVIEW FV300 application software (Tokyo, Japan). The biofilm formed on the sides of microwells at the liquid-air interface and this region was specifically analysed using crystal violet as a control (Supplementary Data Figure S3).