Assessment of Antimicrobial Activity of Fourteen Essential Oils When Using Dilution and Diffusion Methods

Abstract

The lack of solubility and the high degree of volatility of essential oils present significant problems to determining the biological effects of these oils. The activity of 10 essential oils and 4 essential oil blends against a Gram-positive bacterium, Staphylococcus aureus., and a yeast, Candida albicans., was compared using a dilution assay and two diffusion methods. The tube dilution assay, using a 0.2% agar solution to provide a stable homogeneous dispersion of oils, was used to measure minimum inhibitory concentrations (MICs). The relative merits of using p.-iodonitro tetrazolium dye (INT) or optical density (OD) in measuring MIC of essential oils also were evaluated. All 14 oils were active against both microorganisms. Thyme, mountain savory, and Turkish oregano were the most active oils against S. aureus. with MICs of 0.31–0.42 µl/ml. Thyme, Turkish oregano, and mountain savory also were the most active against C. albicans. with MICs of 0.31–0.42 µl/ml. The MIC values as determined by INT or OD methods are strongly correlated for both microorganisms, and both give an accurate estimation of MIC. In the disk diffusion assay, thyme, Turkish oregano, Melissa, mountain savory, and the Exodus II (E2) blend were most active against S. aureus.. In the hole-plate assay, the same oils were active against S. aureus. except for Melissa. For C. albicans., thyme, Melissa, mountain savory, Turkish oregano, rosewood, E2 blend, and the T1 blend were most active in the disk diffusion assay. The same oils were active against C. albicans. in the hole-plate diffusion assay except for the T1 blend. Of the methods tested, the tube dilution assay best addresses the solubility and volatility concerns of essential oils. We favor the determination of MIC using INT because of several eliminated steps. Although there were differences in the way in which some essential oils responded in the two diffusion methods, the correlations between the disk and hole-plate diffusion methods were high and yielded comparable results. However, results from the dilution assay were weakly correlated with agar diffusion results. Diffusion assays are useful as a qualitative assessment of biological activity of essential oils but are not appropriate in assessing quantitative effects.