Lab tek chamber slide system 8 well permanox slides

Biofilms were grown either in 96-(for biofilm biomass quantification) or in 24-well (for gene expression analysis) microplates made of polystyrene plastic (Thermo Fisher Scientific Inc.). For confocal microscopy analysis, biofilms were grown in Lab-Tek ® Chamber Slide™ System 8 Well Permanox ® Slides (Thermo Fisher Scientific Inc.). Briefly, overnight cultures were adjusted with TSB to an optical density at 640 nm (OD 640 ) equivalent to ∼2 × 10 8 CFU/mL and diluted 1:100 in TSB supplemented with 0.4% (w/v) glucose (TSB G , Thermo Fisher Scientific Inc.), iron-enriched TSB G (TSB G containing different FeCl 3 concentrations) and iron-depleted TSB G (TSB G containing different Bip concentrations). pH measurements of the different culture media tested were performed with Model 15 ph meter (Denver Instrument, NY USA). Bip was added to the medium and allowed to stand for at least 30 min prior inoculation. Subsequently, the diluted bacterial suspension was placed into the plates and incubated at 37 °C with shaking at 120 rpm (ES-20 Shaker-Incubator) for the appropriate period (from 6 to 24 h).