Microtiter Dish Biofilm Formation Assay

The quantification of biofilm formation was performed following a previous established protocol (O’Toole, 2011). Briefly, bacterial cells were grown overnight in LB broth (5 ml) at 37°C/200 rpm. Cells were diluted 1:100 in fresh LB without NaCl for stimulates biofilm production. One hundred microliter of this dilution was added per well in a 96-well polystyrene microtitre plate (Costar Cat. No. 3599, flat bottom with lid). Six replicate wells were prepared for each strain. Microtitre plates were incubated at 30°C for 24 h. Total bacterial growth was measured at OD₆₀₀, using a GloMax®-Muti Detection System (Promega). The planktonic cells were then discarded, and the plate was washed three times with water. The remaining biofilm was fixed with 200 μl per well of methanol (100%) and stained with a 0.2% solution of crystal violet in water. After incubation at room temperature for 10 min, the plates were rinsed three times with water. The dye was solubilized by adding 125 μl of 33% acetic acid to each well and incubated the microtiter plate at room temperature for 15 min. Finally, the OD₅₆₀ was determined with the microplate reader. The amount of formed biofilm is reported as the ratio of the OD₅₆₀/OD₆₀₀ values (Oropeza et al., 2015).