The optical density (OD) method for biofilm assessment was
modified from Stepanovic et al. (2003). Crystal Violet was used to
stain whole biofilms due to its ability to bind to negatively chargedmolecules, including nucleic acids and acidic polysaccharides
(Burmolle et al., 2006). After bacterial adhesion, surfaces were
removed from the test solutions and rinsed 3 times with 10 ml
sterile distilled water. The coupons were then placed in sterile Petri
dishes and allowed to air dry on a clean bench. All operations
relating to coupons were done by using sterile forceps. Stainless
steel coupons were then aseptically transferred to 10 ml of 0.1%
Crystal Violet solution to stain adherent cells. Excess Crystal Violet
solution was gently rinsed three times with sterile distilled water,
10 ml for each time. Stained cells were then dissolved in 10 ml of
99.5% ethanol solution, shaken by vortex mixer and sonicated for
1 min using Bransonic Ultrasonic Cleaner. The OD of the obtained
solution was measured at 500 nm by a spectrophotometer