Quantitative Biofilm Formation Assay

Quantitative biofilm formation assays were carried out as in our previous work [5 (link)]. Overnight EAEC cultures, grown in LB medium, were sub-cultured (1 in 100) into 5 ml of DMEM (Dulbecco’s Modified Eagle Medium) high glucose (Sigma) and incubated at 37°C for 1 hour with shaking. 150 µl of each culture was pipetted into a microtiter plate in triplicate, which was sealed with a Breath Easy gas permeable membrane (Sigma), and then incubated overnight statically at 37°C. After ~16-17 hrs the spent media was removed and 150 µl of 0.1% (w/v) crystal violet was added to each well and left at 4°C for 30 minutes. The crystal violet solution was removed, the plate washed thoroughly with water, excess liquid removed and 150 µl of ethanol/acetone solution (80 ml ethanol and 20 ml acetone) was added. The plate was left on a shaker for 30 minutes at room temperature and the absorbance was measured at 595 nm by a Labsystems Multiskan MS plate reader (Thermo Fisher Scientific Inc).