*4.7. Effects of Cadex on Biofilm*

#### 4.7.1. Biofilm Mass Assay

The biofilm mass was assayed by CV staining according to the protocol of Cardoso et al. [51] with some modifications. Briefly, biofilms were grown on a flat bottom sterile 96-well plate (Greiner, Frickhausen, Germany) in which the cultured medium was removed. To each well, 0.2 mL of 0.2 M phosphate buffer was added three times to clean the unattached biofilms, which were left to air dry and fixed for 60 min. Then 0.2 mL of 1% CV was added to each well for 5 min. Following the staining step, the CV solution was removed and the biofilms were cleaned and dried, after which 0.2 mL of 95% ethyl alcohol was added to re-solubilize the dyed biofilms. The CV solutions were obtained and transferred to a new 96-well plate and the optical density of the content was measured using a microtiter plate spectrophotometer (Bio-Rad) at 595 nm.
