Quantifying ROS Production in S. aureus

The production of ROS by the S. aureus strain exposed to EOs was performed using a peroxynitrite indicator, 20–70-dichlorodihydrofluorescein diacetate (DCFH-DA) (SigmaAldrich, UK), which can detect a broad range of ROS [70 (link)]. The adjusted bacterial culture (0.5 McF) was treated with different concentrations of EOs and their combination (corresponding to MIC/2, MIC, 2 × MIC, and 4 × MIC), in presence of DCFH-DA at a final concentration of 5 mM in 0.85% saline, and incubated at 37 °C aerobically for 24 h. Untreated bacterial culture was served as a negative control. The fluorescence emission of DCFH-DA was measured at 525 nm using a Tecan microtiter plate reader with an excitation wavelength of 485 nm [71 (link)]. Experiment was carried out in triplicate.