Biofilm Formation Assay with Resveratrol and Colistin

Biofilm formation assays were performed in 96-well polystyrene microtiter plates, as previously described (12 (link)) with minor modifications. The single colony on the blood plate was shaken overnight in 3 mL of fresh LB broth medium at 37°C. Then, the culture was adjusted to 0.5 McFarland with sterile normal saline and further diluted by 1:100 in LB broth and dispensed in a 96-well microtiter plate with 32, 64, or 128 μg/mL resveratrol and 1 μg/mL colistin either alone or in combinations. The 96-well plates were incubated at 37°C for 24 h. Then the cell suspension was removed, and the plates were washed twice with 1× phosphate-buffered saline (PBS) (Sigma–Aldrich, Milan, Italy) and inverted to dry at the room temperature. Next, 200 μL of 1% crystal violet (CV) solution (Beijing Solarbio Biotechnology Co., Ltd., China) was added to the wells for 15 min. After staining, CV was removed, and the wells were washed thrice with 1× PBS. The plate was dried naturally at room temperature, and the bound CV was solubilized by adding 200 μL of ethanol–acetone (96:5 vol/vol) solution. The absorbance of CV was read at 595 nm on a microplate reader (Multiskan FC). All tests were performed in triplicate.