M eller hinton broth

The most active berry extract to inhibit biofilm development and to treat the mature MRSA biofilm from both previous experiments was used in this experiment. As such, 4 ml of Müeller-Hinton broth (Sigma Aldrich, United States) with or without (positive control) each extract was distributed in a well from a 12-well polypropylene plate (Thermo Fisher Scientific, USA) along with 200 µL of a 10⁶ CFU/ml from each MRSA strain in saline per well. These were then incubated at 37°C and 5% CO₂ for 24 h. The bacterial viability was periodically measured at 1, 3, 6, 12 and 24 h, respectively, by taking a sample of 100 µL from each well and mixing it with 100-µL Müeller-Hinton broth supplemented with 20% of alamarBlue to reach a final concentration of 10% alamarBlue in a well from a Nunc™ 96-well polypropylene MicroWell™ plate. The plate was incubated at 37°C at 90 rpm for 30 min. Next, fluorescence was measured using an excitation wavelength of 560 nm and an emission wavelength of 590 nm following incubation. After the last measurement at 24 h, CFU per milliliter was estimated using the drop-plate method (Esteban et al., 2008 (link)) on mannitol salt agar plates. This experiment was performed in triplicate for each strain (n = 3).