SCREENING METHODS: Broth dilution methods(1)

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Difficulties with partitioning of hydrophobic compounds in agar a more accurate method was developed known as broth dilution method. In this method, bacteria are grown in test-tubes in a liquid media in the presence of the test substance (plate-3) (Panda et al., 2009a,b,c). At regular time intervals (e.g. every 10 min or every hour) a sample is removed and the bacterial count (CFU) determined by serial dilution. In contrast to the single data point (e.g. 24 h incubation) utilized in disk diffusion and agar dilution assay, the broth dilution method allows much finer evaluation of the antibacterial events over time. In addition to this, this method has other advantages includes recovery from the effects of the test substance and proportion of organisms killed at a given time point can be determined.

However, the method is also time and resource intensive and can be impractical where very large numbers of test substances are to be screened. As with other testing methods incorporation of hydrophobic compounds and essential oils into the aqueous media is problematic, and as there is no solid phase to trap these compounds they rapidly separate from the media and form a layer across the surface of the media.
platwe3Screening Methods in the Study-6
Plate 3: Photograph of broth dilution method (A-G: decreasing concentration of extract, H-control)

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