Validation of a method of broth microdilution for the determination of antibacterial activity of essential oils

The aim of the present study was to adapt and optimize a broth microdilution method and compare it to the agar dilution method for the evaluation of activity of essential oils from medicinal plants against Gram-negative bacteria. Based on bibliographic research, active and not active oils were selected. The sensitivity and specificity were established as parameters for validation. The comparison between both methods was made using contingency analysis tables, based on the observed frequencies. For both methods, the minimum inhibitory concentration was determined against Escherichia coli strains, in an essential oil concentration range between 0.03 and 0.48% (v/v).

A stable emulsion formation was achieved with the addition of Tween 80 and constant agitation, guaranteeing the continuous contact of oil with bacteria (critical step in the microdilution method). The statistical analysis of results obtained with both methods presented a good sensitivity and specificity (100% in both cases), which let us correctly discriminate between active and non-active oils. The values obtained for the minimal inhibitory concentration were independent of the technique used. Finally, the obtained results show that the validated microtechnique allows important diminishment of time and resources for investigations dealing with essential oils or lipophilic extracts evaluation.