Fluorostar omega spectrometer

Biofilm formation assays were performed according to previously described method¹²¹ , with some alterations. 4–5 representative colonies from overnight streak plates on MH agar were resuspended in MH broth to equal CFUs (5 × 10⁵ cells/mL). These were inoculated into 96-well polystyrene microtitre plates (100 μl/well) as individual cultures and as a mixed co-cultures (at various ratios of the two strains) and plates were incubated for 18 h at 37 °C with and without 512 µg/mL cefotaxime. Quantification of the biofilm was carried out by measuring the amount of crystal violet stain retained in the wells (at an absorbance of 550 nm) with a Fluorostar Omega spectrometer (BMG Labtech, Offenburg, Germany). The results represent the average of three independent experiments, each with at least three technical replicates. Growth was quantified at OD_600nm and the biofilm OD_550nm to growth OD_600nm ratio was calculated to determine the level of biofilm formed normalized to the total number of cells per sample.

Aliquots (50 μl) of the co-isolated bacteria from the overnight cultures, were inoculated into fresh MH medium with shaking to achieve the cell density of OD₆₀₀: 0.6, respectively. The KP6870155 was then inoculated into Millicell hanging insert (Merck) containing a sole carbon source. The insert only allowed the passage of small-secreted metabolites but not bacterial cells. AB6870155 was directly inoculated into 6-well plates containing the single carbon source that cannot be used by it and subsequently combined with inserts containing KP6870155. The assembled 6 well plates were incubated at 37 °C for 24 h with shaking. Fluorostar Omega spectrometer (BMG Labtech) was used to determine the growth of AB6870155. Each carbon source was assessed in triplicates.