For biofilm quantification, a micro method based on a protocol
previously reported was employed[29]. Biofilms formed after 24 h
incubation of bacterial cultures prepared as described in the previous paragraph, were stained with 20lL of an aqueous solution of crystal violet (0.1%, w/v) for 20 min. After washing with water,
the liquid was discarded from the wells and the material that
remained fixed to the polystyrene (containing bio-film) was
washed with PBS (thrice). Crystal violet bound to biofilm was
removed from each well employing 200lL absolute ethanol during
30 min at 37C with shaking. Absorbance (560 nm) of ethanol
solutions of crystal violet was determined using a microtitre plate
reader (Power Wave XS2, Biotek, VT, USA).