Phytomenadione

Inhibition of biofilm formation was determined in 96-well, flat-bottomed plates [28 (link)], to which 100 µL of cell suspension in RPMI-1640 was added (1 to 5 × 10⁶ cells/mL, adjusted to a turbidity equivalent to 0.5 McFarland scale), as well as 100 µL of the drug (EO and/or clotrimazole), at concentrations of 4 × MIC, 2 × MIC, 1 × MIC, 0.5 × MIC and 0.25 × MIC. The culture was incubated at 35 °C for 48 h. Then, non-adherent cells were removed, and the wells were washed three times with PBS (10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4). Then, 100 µL of MTT solution (5 mg/mL; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) plus 10 µL of phytomenadione (1 µM; 2-methyl-3-[(E,7R,11R)-3,7,11,15-tetramethylhexadec-2-enyl]naphthalene-1, 4-dione; Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The plate was incubated at 35 °C for 24 h in the dark. Subsequently, the supernatant was removed, and 100 µL of DMSO was added to each well, and the plate was incubated at 35 °C for 15 min, protected from light. Then, 80 µL of solvent was removed from each well and transferred to another plate, and the reading was performed at 490 nm [29 (link)]. Growth and sterility controls were included for each plate in the experiment. The tests were performed in triplicate.