The effect of SEO and NEs on established biofilms was evaluated after 24 h bacterial growth in polystyrene 96-well plates at 37 °C. After this incubation time, the supernatant was removed by aspiration, and replaced with medium with SEO or NEs added at sub-inhibitory concentrations and 0.02% of Tween-80. After incubation with the substances for 24 h a 37 °C, unattached bacterial cells were removed, wells were rinsed twice with PBS, and stained with crystal violet (2% w/v), as previously described. Absorbance was measured at 570 nm with a microplate reader (Bio-rad Benchmark, Hercules, CA, USA).

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