96 well clear polystyrene microplate

Three classes of antibiotics: β-lactams (ampicillin, cefotaxime), aminoglycosides (kanamycin), and tetracycline, were tested in this study. Antibiotic stock solutions (25x–100x) were prepared according to the manufacturer’s instructions and stored at -20°C. Working solutions in the range of 0.2–102.5 mg/L for ampicillin, kanamycin, cefotaxime and 410 mg/L for tetracycline were used in this study (Dias et al., 2015 (link); Shang et al., 2015 (link)). Ampicillin, cefotaxime, and kanamycin working solutions were prepared immediately before use and diluted in ddH₂O to the desired concentrations (Baselga-Cervera et al., 2019 (link)). Each antibiotic concentration was mixed with 5 ml F. diplosiphon cells adjusted to OD750 nm. Assays were performed in 96-well clear polystyrene microplate (Corning^®Inc., NY) and cultures grown under conditions mentioned above. Three replicate treatments were maintained and the experiment was repeated twice. In order to minimize the effects of light scattering, every other well was left blank. Plates were sealed with a Breathe-Easy sealing membrane (Sigma-Aldrich, MO, Lot#MKCP8263) to prevent evaporative water loss and decrease the risk of contamination.

Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were determined as recommended in CLSI guidelines31 . The bacterial suspensions of 1.0 × 10⁶ CFU per/well were seeded in 96-well plates (Corning^® 96-well Clear Polystyrene Microplates) in presence of BBZ and antibiotics used in the study at concentrations 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 μg/mL and incubated at 37 °C for 24 h. MIC values were scored as the minimal concentration at which no visible growth of bacterium were observed and were detected by Tecan Micro-plate Reader at 600 nm.
MBC values were determined by plating 100 μL of each clear well from the MIC micro-broth plate that had been incubated for 24 h at 37 °C on Mueller - Hinton agar plates without antibacterial compound for overnight at 37 °C. MBC endpoints were defined as the lowest dilution of compound which resulted in 99.9 % killing of the bacterial cells from the starting inoculum32 (link).

Minimal inhibitory concentration (MIC) was determined by broth microdilution method according to CLSI methods¹⁹ and through E-test^{20 (link)}. The bacterial suspensions of 1.0 × 10⁶ CFU per/well were seeded in 96-well plates (Corning® 96-well Clear Polystyrene Microplates) at an increasing concentration range of 0.25–128 µg/mL (0.4–206.4 μM) of PPEF and BPVF, incubated at 37 °C for 24 h. MIC values were scored as the minimal concentration at which no visible growth of bacterium was observed and detected by Tecan Microplate Reader at 600 nm. The absolute MIC value was scored as the minimal concentration that inhibited growth to 90% at 24 h. The E-test was used for the detection of MIC of the standard drug^{20 (link)}.

Agarose (Molecular Biology Grade, GENEDIREX, Inc.) was dissolved in phosphate-buffered saline (PBS) 1X, pH 7.4 (Thermo Fisher Scientific, Massachusetts, USA), and autoclaved. Before coating, Agarose gel was molten by a microwave oven. Then, 200 ul of liquid Agarose was dispensed into the well of the U-bottom 96-well plate (Corning® 96-well Clear Polystyrene Microplates), and all amount was quickly aspirated away. This will leave an instant thin-layer coating along the U-shape well. The cells were seeded into each well straight away after plate coating, without centrifugation, and allowed 2 days of culture at 37 °C, 95% humidity, and 5% CO₂.