2.4. Biofilm formation
The ability to form a bacterial biofilm was tested on
polyvinyl chloride 96-well flat-bottomed plates and the
biofilm was quantified by measuring the optical density
(OD) after dyeing it with crystal violet (Sigma-Aldrich, UK)
followed according to a previously described protocol[19].
This method measures the bacterial cells which adhere to
the bottom of the microtiter plate wells even after repetitive
cycles of washing. Strains from an overnight culture were
inoculated with the tested organism in the presence and
the absence of sub-MIC concentrations of the extract.
Briefly, fresh bacterial suspensions were prepared in
tryptone soya broth from overnight cultures and adjusted
to OD600. Individual wells were filled with 0.1 mL aliquots
of the diluted culture. Following overnight incubation at
37 °C without agitation, plates were gently washed with
phosphate buffered saline (pH 7.4) and stained with 100 μL
of 0.1% crystal violet for 30 min at room temperature. Excess
crystal violet was removed by washing, and biofilm was
then quantified by measuring the corresponding OD570 nm of
the supernatant following the solubilisation of crystal violet
in 0.15 mL of 95% ethanol. For each clinical strain tested,
biofilm assays were performed in triplicate and the mean
biofilm absorbance value was determined.