The effect of LGEO in inhibiting the formation of biofilms was examined on microtiter plates as described in the published literature with modifications (Mireles et al., 2001; Djordjevic et al., 2002). An overnight culture of the S. Heidelberg isolates grown in LB were centrifuged (3,600 x g for 15 min at 4°C), washed with PBS, resuspended, and diluted in LB. Then, 200 µL of the diluted culture (∼6.0 log10 CFU) was added to sterile 96-well polystyrene microplate wells (Catalog no. 89089-578; Ref. 655184; Greiner Bio-One, VWR) along with either 0.0075%, 0.15%, or 1% (vol/vol) LGEO. Positive controls with only the pathogen and negative controls with only the broth were also included. The outer rows and columns of the well plate were filled with sterile PBS to prevent evaporation from the central wells; the plates were then sealed with Parafilm (American National Can, Greenwich, CT, United States) and incubated for 96 h at 41°C. Biofilm formation and planktonic S. Heidelberg were quantified spectrophotometrically using a 96-well microplate reader with absorption measured at 600 nm. After incubation, the remaining medium was removed from the wells to a separate microtiter plate, and absorbance was measured to quantify planktonic or biofilm-unassociated S. Heidelberg. Wells of the original plate was gently rinsed with sterile deionized water 3 times and allowed to dry at room temperature for 15 min before the addition of 200 µL of 1% crystal violet (Item no. ES802E, Catalog no. 89133-283; Azer Scientific, Morgantown, PA) for 20 min. The stained wells were then rinsed 3 times with sterile deionized water, allowed to dry for 15 minutes, and destained with 200 µL of ethanol (99%). The destaining solution was transferred to a new microtiter plate, and the level of crystal violet present quantified by optical density (OD600) measurement. The experiment was replicated 6 times for each strain.

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