SCREENING METHODS: Agar cup method (Cont.)

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Agar cup method (Cont.) During the evaluation of antimicrobial activity of medicinal plant extracts by agar dilution method, several researchers used different solvents (Prudent et al, 1995; Pintore et al., 2002), different volumes of inoculums (Juven et al., 1994; Prudent et al., 1995), various inoculation techniques, e.g. dotting (Pintore et al, 2002) or streaking (Farag et al, 1989). Despite these variations, the MICs of medicinal plant extracts determined by agar dilution generally are in approximately the same order of magnitude (Farag et al., 1989; Prudent et al., 1995; Pintore et al., 2002). A criticism of this method is that when a scoring system is used it is difficult to compare one set of results with another. This method suffers from several other limitations, including many that have been discussed previously: (a) use of larger volumes of test substance than in other methods, (b) confounding antibacterial actions from volatiles, (c) difficulty of achieving stable emulsions of essential oils in agar and (d) restriction on the maximum concentration that can be used before the agar becomes too dilute to solidify properly. However this technique is much more difficult with the extract deals with essential oil and other hydrophobic plant extracts. Many researchers have thought they had incorporated their essential oil into nutrient broth or other media but after an hour of the experiment, the oil had separated out and was floating on top of the media. Griffin (2000), in their work on tea tree oil found that at concentrations above 2% v/v the oil separated from the agar substrate and was seen as droplets on the agar surface. The most commonly utilized method to overcome this problem is the use of surfactants such as Tween-20, Tween-80, and alkyl dimethyl betaine (ADB). Several authors have described the use of these products and the effect on antibacterial activity. The results of their studies show that surfactants can interfere with calculation of MIC values and the growth of some test organisms (Hammer et al, 1999). However, it has also been demonstrated that it is possible to use very small quantities of Tween (<0.5% v/v) to emulsify the essential oil in media and thus avoid the effects on organism growth (Griffin, 2000; Hood et al, 2004). Hammer et al. (1999) also showed that inclusion of organic matter such as bovine serum albumin in the agar also affected the antibacterial activity of tea tree oil.

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