[The optimization of methods utilized for testing the antibacterial activity of essential oils]

The antibacterial activity of essential oils: Lavandulae aetherleum, Limonis aetherleum and Melaleucae aetherleum was determined against Staphylococcus aureus Gram (+) and Escherichia coli Gram (-) bacteria, using four methods: agar diffusion (paper disk and agar wells) and broth dilution (turbidimetric and rezazurin reduction assay). This study revealed that the sensitivity of the agar diffusion techniques was much lower than the broth dilution methods. This was mainly due to partitioning of the oil components in the agar according to their affinity with water. This limitation could be lessened by the selection of suitable oil emulsifier/solvent, which itself not influence the bacterial growth. By initial oil solubilization with 96% ethanol (1:1), the increase of agar dilution methods sensitivity could be achieved. The broth microdilution methods, using 0.5% Tween 20 or 0.15% agar to emulsify the oils, were optimized and shown to be most accurate for testing their antimicrobial activity. By introducing resazurin, as an MIC endpoint indicator, the sensitivity of these methods was further enhanced.