Biofilm Inhibition Assay and Biofilm Degradation Assay

Biofilm inhibition and degradation by CFS were investigated by crystal violet (CV) assay [24]. To this purpose, the strains were treated with CFS and GSE at 1/2 × MIC based on the MIC related to each strains.

To examine the biofilm inhibition effects of CFS and GSE, bacterial suspension (50 μl) treated with CFS or GSE (50 μl) were transferred to a 96-well polystyrene plate and incubated at 37°C for 24 h; the control was treated with YM broth. Following incubation, cell suspensions were removed, and the wells were washed twice with 150 μl of distilled water (DW). The biofilm cells were dried at 37°C for 20 min and then stained using 1% CV solution (150 μl) for 30 min. The CV solution was removed, and the plate was washed twice with cold water. The biofilm cells were treated with dissolving solution (150 μl; 30% methanol and 10% acetic acid) to measure the optical density (OD) of the CV solution at 570 nm using a microplate reader (Molecular Devices, USA).

To examine the biofilm degradation effects of CFS and GSE, bacterial suspensions (100 μl) were transferred to a 96-well polystyrene plate and incubated at 37°C for 24 h. Following incubation, the bacterial suspensions were removed and treated with CFS or GSE (100 μl). The control was treated with YM broth. After incubation at 37°C for 24 h, biofilm quantification was performed as described above.

The biofilm inhibition and degradation rate (%) was calculated using the following equation:

Biofilm inhibition and degradation rate (%) = (1-OD_treatment/OD_control) × 100