Varioskan flash spectral scanning

Metabolic activity was assessed in using the alamarBlue™ assay (Invitrogen, USA) according to the manufacturer's protocol. In brief, after 14 and 21 days of culture, cells were washed with Hanks’ balanced salt solution (HBSS) and alamarBlue™ solution (10% alamarBlue™ in HBSS) was added. After 4 h of incubation at 37°C, absorbance was measured in triplicate at excitation wavelength of 550 nm and emission wavelength of 595 nm using Varioskan Flash spectral scanning multimode reader (ThermoFisher Scientific, UK). Cell metabolic activity was normalized to cell number. Nuclei were counted to obtain cell number.

Biofilm formation by P. aeruginosa PAO1 was quantitatively assessed as previously reported [25 (link)]. Briefly, 100 µl of bacterial culture diluted in M63 medium [26 ] to an initial optical density at 600 nm (OD₆₀₀) of 0.02 was transferred into each well of a 96-well polystyrene plate (Costar 3599; Corning Life Sciences, Corning, NY, USA). To check for inhibition of biofilm formation, QSI supernatants at dilutions of 1%, 3% and 5% v/v were added to wells containing the P. aeruginosa cultures. To identify any bioactive compounds, 0.5% organic or 5% aqueous extracts obtained as described above were added to diluted PAO1 cultures. An untreated P. aeruginosa PAO1 culture was used as a negative control, and a P. aeruginosa PAO1 culture mixed with 10 µM of a known QSI, furanone, was used as a positive control. Cells were incubated at 37°C for 48 h without shaking. Total biofilm formation was measured using crystal violet staining [27 (link)], and the optical density was read at 595 nm using a Varioskan Flash spectral scanning multimode reader (Thermo Scientific, Pittsburgh, PA, USA). Three repeated trials to evaluate the biofilm inhibitor assay were performed, and in each experiment the data point was the average of at least 12 replicate wells.