96 well polystyrene plate

Biofilm inhibitory effect was measured using crystal violet as previously described [20 (link)]. Briefly, S. aureus cells were inoculated into LB medium (for the MSSA strain) or LB supplemented with 0.2% glucose (for the MRSA strain) at an initial OD₆₀₀ of 0.05 in a total volume of 300 µL in 96-well polystyrene plates (SPL Life Sciences, Pocheon, Korea). Cells were then cultured with or without phorbaketals (0, 10, 20, 50, and 100 µg/mL) for 24 h without agitation. Biofilm cells were stained with 0.1% crystal violet for 20 min, washed three times with distilled water, and dissolved in 95% ethanol, and absorbance at 570 nm (OD₅₇₀) was measured using a Multiskan EX microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Cell growth in 96-well plates was also monitored by measuring absorbance at 620 nm (OD₆₂₀). Results represent the means of at least 12 replicate wells.

V. parahaemolyticus was inoculated in 96-well polystyrene plates (SPL life science, South Korea) without shaking at 30°C for 24 h with or without the indoles. The biofilms were stained with 100 μL of pre-warmed PBS containing CFSE stain (carboxyfluorescein diacetate succinimidyl ester) (Invitrogen, Molecular Probes, Inc., Eugene, OR, United States) for 20 min at 37°C (final concentration, 5 μM) and washed three times with PBS. The static biofilm plates were visualized by excitation using Ar 488 nm (emission wavelengths 200–550 nm). The cells were visualized by confocal laser microscopy (Nikon Eclipse Ti, Tokyo, Japan) using a 20× objective. DMSO was used as the control. Color confocal images were visualized using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in three independent cultures were chosen for microscopic analysis (Sethupathy et al., 2020 (link)).

The inoculum of V. parahaemolyticus was grown in 96-well polystyrene plates (SPL life sciences, Pocheon-si, South Korea) without shaking at 30 °C for 24 h in the presence or absence of CNMA and its selected derivatives (50 µg/mL). The formed biofilms were stained with 100 μL of pre-warmed PBS containing CFSE (carboxyfluorescein diacetate succinimidyl ester) (Invitrogen, Eugene, OR, USA) for 20 min at 37 °C and were visualized by CLSM (Nikon Eclipse Ti, Nikon Instruments, Tokyo, Japan), as described by [37 (link)]. At least 10 random positions in each of the three independent cultures were chosen for microscopic analysis [38 (link)]. Biofilm formation was quantified using COMSTAT biofilm software [39 (link)] to determine the biomasses (µm³/µm²), mean thicknesses (µm), and substratum coverage (%).

A static biofilm formation assay was performed on six bacterial strains (MSSA 6538, MSSA 25923, MRSA MW2, S. epidermidis, Pseudomonas aeruginosa PAO1, and Escherichia coli O157:H7) in 96-well polystyrene plates (SPL Life Sciences, Korea), as previously reported48 (link). Briefly, cells were inoculated into LB medium (total volume 300 μl) at an initial turbidity of 0.05 at 600 nm. Antibiofilm agents were added at different concentrations at inoculation and cultured for 24 h without shaking at 37 °C. To quantify biofilm formation, biofilms were stained with crystal violet for 20 min, dissolved in 300 μl of 95% ethanol, and absorbances were measured at 570 nm (OD₅₇₀). Cell growths in 96-well plates were also measured at 620 nm (OD₆₂₀). Biofilm formation and static cell growth results are presented as the averages of two independent cultures of twelve replicate wells.

Escherichia coli BW25113 and S. aureus cells were cultured in 96-well polystyrene plates (SPL Life Sciences, Korea) without shaking with or without 5-iodoindole. Biofilms were stained with carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Molecular Probes, Inc., Eugene, OR, USA) (Weston and Parish 1990 (link)). Planktonic cells were removed by washing with PBS three times, and static biofilms were visualized by excitation using an Ar laser 488 nm (emission wavelengths 500–550 nm) under a confocal laser microscope (Nikon Eclipse Ti, Tokyo) using a 20× objective (Kim et al. 2012 (link)). Color confocal images were constructed using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in two independent cultures were chosen for microscopic analysis.
To quantify biofilm formation, color confocal images (20 image stacks) were converted to gray scale using ImageJ. COMSTAT biofilm software (Heydorn et al. 2000 (link)) was used to determine biomasses (μm³ μm⁻²), mean thicknesses (μm), and substratum coverages (%). Thresholding value was fixed for all image stacks, and at least 4 positions and 20 planar images per position were analyzed.

To investigate the MIC of GSE (ES Food, Korea), the double broth dilution method was used [14 (link)]. CFS and GSE were dissolved in YM broth and YM broth with 0.1% Tween 80, respectively, and serially diluted two-fold. S. aureus were cultured in TSB at 37°C for 24 h and then diluted in TSB to obtain a final concentration of 105 colony-forming units (CFU)/ml. Bacterial cultures (50 μl) in TSB supplemented with 50 μl of CFS or GSE in YM broth were transferred to 96-well polystyrene plates (SPL, Korea), which were incubated at 37°C for 24 h. After incubation, the lowest concentrations of CFS and GSE that could inhibit visible S. aureus growth were defined as MICs.