Determination of minimum inhibitory concentration and minimum fungicidal concentration using broth dilution method

The method used in this study for determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of CEOs was adapted from Adukwu et al. (2012) with slight modifications. Briefly, Sabouraud Dextrose broth (SDB, Oxoid Ltd., UK) was prepared with a 0.5% (v/v) concentration of Tween 20 (Sigma, USA), used as the EO emulsifier. Both CBEO and CLEO were diluted twofold into SDB with Tween 20 to create a concentration range between 4 and 0.03% (v/v). The amount of 180 μL of the diluted CEOs was transferred into the corresponding wells of a 96-well microdilution plate with 20 μL of yeast suspension. Microplate wells of EO dilutions without yeast, and SDB and yeast suspension, were used as negative controls, and the plates incubated at 30 °C for 48 h. After the appropriate incubation time, the optical density (OD) of each well was recorded at 595 nm using the microplate reader (infinteF200Pro, Tecan, Switzerland). The MIC was defined as the lowest CEO concentration that produced inhibition of yeast growth.

MFC was measured by transferring 20 μL from each treated well of CEO concentration from 4% (v/v) to 0.03% (v/v) to labelled SDA plates followed by incubation at 30 °C for 48 h. The MFC was defined as the lowest CEO concentration that inhibited growth of the yeast or permitted less than three CFUs to occur, resulting thus in 99.9% fungicidal activity (Espinel-Ingroff et al. 2002).