A new micro-agar dilution method to determine the minimum inhibitory concentration of essential oils against microorganisms

Essential oils (EOs) from plants have attracted wide attention due to their unique flavors and antimicrobial, insecticidal, antioxidant, and anti-inflammatory properties. Antimicrobial activity, the main reason for their widespread use in the food industry, can be determined in vitro by the minimum inhibitory concentration (MIC), which is a key step to evaluate the antimicrobial potential of EOs. However, EOs are lipophilic and insoluble, resulting in the difficulty of accurately measuring their MIC values. In this study, in addition to analyzing the problems in the MIC determination of EOs using the common micro-broth dilution method or the agar method, a new solution called micro-agar dilution method was proposed. As the miniaturization of agar dilution method, this novel method could reduce the dosage of EOs by 16.3 times and medium/PBS by 3.3 times, respectively, and showed better reliability and accuracy than the typical methods. Additionally, the limit of solvents should be evaluated before use if used. Notably, this research could also provide a guide for the MIC determination of other insoluble antibacterial substances.