Effect of LGEO on Pre-Formed S. Heidelberg Biofilm

The effect of LGEO against pre-formed biofilms was investigated on 48-well polystyrene cell culture plates with a modified procedure (Upadhyay et al., 2013). Bacterial culture was prepared similar to that for the biofilm inhibition assay, except the inoculum was diluted with sterile PBS instead of LB. The wells containing 1 mL LB were then inoculated with 100 µL of the S. Heidelberg strains (∼6.0 log₁₀ CFU). The outer rows and columns of the well plate were filled with sterile PBS, and the lid was secured to the plate using Parafilm before incubation for 96 h at 41°C. After biofilm formation, the remaining medium was removed and treated with either 0.0075%, 0.15%, or 1% (vol/vol) LGEO in the wells for 2 h. The wells were subsequently scraped with sterile pipet tips for 5 min per well, and surviving S. Heidelberg were enumerated on XLD plates. Appropriate controls were kept along with the treatments; the experiment was conducted in duplicates and replicated 4 times.