Evaluation of common antibacterial screening methods utilised in essential oil research

The use of essential oils as therapeutic agents has become increasingly popular; however, research into the antimicrobial activity of these products has been hampered by the lack of standardized and reliable screening methods. The lack of standardized methods also makes direct comparison of results between studies impossible. In this study, the most commonly used antimicrobial assays cited in the literature were evaluated for reliability and their ability to accurately assess, and directly compare, essential oil antimicrobial activity. The methods used were disc diffusion, well diffusion, agar dilution and broth dilution. This study revealed that the disc diffusion, well diffusion and agar dilution methods were unreliable and produced inconsistent results. This was largely due to problems related to achieving stable dispersion of the oils in aqueous media, diffusion of lipophilic constituents in aqueous media and varying methods for determining numbers of viable bacteria remaining after the addition of the oil. An optimized broth dilution method, using 0.02% Tween 80 to emulsify the oils, was developed and shown to be the most accurate method for testing the antimicrobial activity of the hydrophobic and viscous essential oils. When evaluated against a range of oils, this method provided the most reliable and correct results and allowed direct comparison of the antibacterial activity of the test oils, irrespective of viscosity and hydrophobicity.