Bacteriological agar

The ability of QAL to inhibit the QS-mediated swarming motility of P. aeruginosa was assessed by the soft agar method. Briefly, swarming soft medium (2.5 mg/mL NaCl; 30 mg/mL Na₂HPO₄; 15 mg/mL KH₂PO₄; 0.2% glucose, all from Sigma; 0.5% bacteriological agar; 0.5% casamino, all from Oxoid; and 1 mM MgSO₄ Sigma) was poured into 60 mm plates in the absence and presence of 0.155, 0.31, and 0.62 mg/mL QAL and allowed to dry at room temperature for ~2 h prior to inoculation. Each plate was inoculated with 2.5 μL of a liquid culture grown for 18 h in LB broth. The plates were incubated face up at 37 °C for 24 h. The motility spread was measured with a micrometer and the results were expressed in mm; each experiment was carried out in triplicate.

Vero cells were cultured in 6-well plates until 100% confluent. The virus was treated with 75 µM EGCG or EGCG-S for one hour. The untreated virus served as the control. Virus dilutions ranging from 10⁻³ to 10⁻⁷ were prepared. Cells were infected with the diluted virus for one hour and the unadsorbed virus was aspirated. Plaque assay method is a modification of protocol previously reported in Adams et al. [42 (link)]. After one hour of incubation, cells were overlaid with plaque media consisting of bacteriological agar containing 3X Eagle medium (Gibco Invitrogen Corporation, Grand Island, NY, USA), 1.5 mL of 5% sodium bicarbonate (Gibco Invitrogen Corporation, Carlsbad, CA, USA), 0.5 mL FBS, 0.1 mL DEAE-dextran (ICN Biomedicals Incorporated, Aurora, OH, USA), 0.1 mL penicillin/streptomycin (Cambrex, Walkersville, MD, USA), with 0.05 mL gentamicin and 0.6% bacteriological agar (Oxoid Limited, Baskingstoke, Hampshire, England). Both solutions were placed in a 41 °C water bath, combined 1:1, and cells were overlaid with 3 mL of plaque media.
After 72 h of incubation at 5% CO₂, cells were stained with crystal violet and plaques were counted followed by the calculation of mean and standard deviation.

Two bacterial strains were used in our investigations: Pantoea agglomerans 299 R::MRE-Tn7-145 and Sphingomonas melonis Fr1::MRE-Tn5-145. Both bacterial strains were grown overnight on nutrient agar plates (13 gL⁻¹ Lysogeny broth and 15 gL⁻¹ bacteriological Agar, Oxoid) containing 20 mgL⁻¹ of gentamicin (AG Scientific) at 30 °C^{54 (link)}. The bacteria was then harvested using a sterile inoculation loop and was suspended in 5 mL of sterile phosphate buffer saline (PBS pH 7.4, P4417, Sigma-Aldrich). The bacteria was then washed by centrifugation at 1150 RCF for five minutes at 10 °C. The supernatant was discarded, and the bacteria was suspended in fresh phosphate buffer, and washed for a second time – following the aforementioned process. After washing for a second time the bacteria were suspended in PBS to an OD_{600 nm} of 0.2.