Black 96 well microtiter plate

In order to evaluate the effects of benzydamine on Candida capacity to adhere to plastic and to form a biofilm, fungal cells (2 × 10⁶/ml in cF12 medium) were treated for 1, 5, or 15 min with 4 different concentrations of benzydamine (6 mg/ml (0.6%), 3 mg/ml (0.3%), 1.5 mg/ml (0.15%), and 0.75 mg/ml (0.075%)), with the 3 MoWs, or with cF12 medium alone (used as a control). After treatment, fungal cells were washed, dispensed in a black 96-well microtiter plate (PerkinElmer Life Sciences, Cambridge, UK) (0.1 ml/well, in triplicate), and placed at 37 °C with 5% CO₂. Incubation was carried out for 90 min (for adhesion assessment) or for 24 h (for biofilm formation assessment).

A stationary phase culture of a given Bifidobacterium strain was centrifuged twice (5,000 g, 5 min, room temperature). Culture supernatant was neutralized (pH 7.0) with 5 M NaOH to exclude any possible pH effects, filter sterilized and subsequently diluted to final concentrations of 20% (v/v) with sterile deionised milliQ water. AI-2 levels were determined in a V. harveyi BB170 assay as described previously [38] (link). Briefly, an overnight culture of the reporter strain was diluted 1∶5000 into fresh sterile, double concentrated MB medium and 100 µl of this cell suspension was added to the wells of a black 96-well microtiter plate (Perkin Elmer). Subsequently, 100 µl of the appropriate sterile supernatant dilution was added to the wells, the microtiter plates were incubated at 30°C and bioluminescence was measured after 5 hours using the EnVision Multilabel Reader (Perkin Elmer). Bioluminescence was expressed as the fraction of bioluminescence measured in the positive control reaction.