**2.7 Minimum inhibitory concentration (MIC) assays**

Based on the preliminary reports all the medicinal plants were identified to have potent antimicrobial activity and Minimum Inhibitory Concentrations (MIC) of the extracts was determined according to Elizabeth, (2001). A final concentration of 0.5% (v/v) Tween-20 (Sigma) was used to enhance crude extract solubility. A series of two fold dilution of each extract, ranging from 0.2 to 100 mg/ml, was prepared. After sterilization, the medium was inoculated with 3μl aliquots of culture containing approximately 105 CFU/ml of each organism of 24 hours slant culture in aseptic condition and transferred into sterile 6 inch diameter petridish and allowed to set at room temperature for about 10 minutes and then kept in a refrigerator for 30 minutes. After the media solidified a number 3-cup borer (6mm) diameter was properly sterilized by flaming and used to make three to five uniform cups/wells in each petridish. A drop of molten nutrient agar was used to seal the base of each cup. Different plant crude extracts ranging from 0.2 to 100mg/ml were added to the cups/wells of each petridish and the control plates without plant extract. Inhibition of organism growth in the plates containing test crude extracts was judged by comparison with growth in blank control plates. The MICs were determined as the lowest concentration of extracts inhibiting visible growth of each organism on the agar plate. Similarly the MICs of methanol extracts were determined against all other microorganisms.
