2.6. Antibiofilm Activity

The plates were assembled in a process similar to the MIC test, and after 24 h the antibiofilm activity was assessed through the methods mentioned and described below. After 24 h incubation, the plates were washed with sterile water (200 μl/well) to remove the loosely adhered and air-dried cells. The adhered cells of the biofilm were fixed in the plate wells by addition of 200 μl of methanol for 15 min. Afterwards, the methanol was removed and 200 μl of a 1% violet crystal solution was added for 15 min. Subsequently, the plates were washed and air dried again, after which 200 μl of ethanol (96%) was added to each well which were left shaking for 5 min and then read on a microplate reader at 595 nm [35].

After the incubation period, the culture medium was removed and the plates were subjected to three washes with distilled water. Next, 200 μl of a 0.9% saline solution was aliquoted into the wells, and the plate was incubated in a sonic bath (LK-D32 Ultrasonic Bath) operating at 50 kHz for 10 min. The liquid from the wells for each concentration teste was pooled to bring the volume to 1 ml, after which a 20 μl aliquot was withdrawn and subjected to serial dilution in 180 μl volumes of 0.85% saline solution (10⁻¹ to 10⁻⁷). In a Petri dish containing TSA, three 10 μl aliquots were cultured for each concentration. These plates were incubated for 24 h at 37°C, after which the resulting colonies were counted [35].