SCREENING METHODS: Disk diffusion method(2)

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diffusion method(2)

A paper disk (6 or 8 mm) containing a known volume of the test substance is then placed in the center of the agar and the dish incubated for 24 h or more. At this time the “cleared” zone (zone of inhibition) surrounding the disk is measured and compared with zones for standard antibiotics or literature values of isolated chemicals or similar extracts. On the other hands, data from this assay is typically presented as mean size of zone of inhibition (with or without standard deviation), or a ranking system such as “+”, “++”, and “+++” to indicate levels of activity. Few authors also provide levels of activity (slight, moderate, strong) without any reference to standardized criteria.

One of the major criticisms of this method is that it relies on the ability of the extract to diffuse through agar and any component of the extract that does diffuse away from the disk will create a concentration gradient, potentially creating a gradient of active antibacterial compounds. All of the antibacterial testing methods use an aqueous base for dispersion of the test substance, either via diffusion in agar or dispersion within nutrient broth, consequently assays using extracts with limited solubility in aqueous media (e.g. essential oils) may not reflect the true antibacterial activity. There is also no consensus on the best agar to use for these assays.

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