Protocol for determining minimum inhibitory concentrations of essential oils against bacterial pathogens using broth microdilution

Essential oils (EOs) are widely investigated for antimicrobial activity, yet minimum inhibitory concentrations (MICs) lack cross-study comparability due to the absence of a harmonized reference assay. Here, we present a protocol to determine MICs of EOs against aerobic bacterial pathogens using a broth microdilution technique. We describe steps for incorporating EO dispersion using Tween 80 and preparing a standardized bacterial inoculum. We detail procedures for establishing two-fold dilution series in 96-well plates and visually determining the MIC endpoint.