**3.3 Determination of the minimal inhibitory concentration**

The minimal inhibitory concentration (MIC) can be determined as the lowest concentration at which no growth occurs and is determined as follows: PDB medium will be prepared and sterilized in universal bottles, each containing 10 mL medium. Different amounts of the tested extract will be added to the broth medium to give the following concentration: 0.3125 to 100 mg/mL. To each flask 0.5 mL of Tween-80 will be added as emulsifying agent. The flasks will be inoculated with 0.5 mL fungal conidial spore suspension containing 105 spores per mL from a 120-h-old culture and will be incubated at 28 C for 5 days. The MIC value is determined as the lowest concentration of plant extract in the broth medium that inhibited visible growth of the test fungal strains. Each assay should be carried out in triplicate. The MIC test will be quantified the antifungal activity of plant extract.

#### **3.4 Determination of minimum fungicidal concentration**

The hyphal growth inhibition test can be used to determine the antifungal activity of the plant extract against fungal strains as previously described Picman et al. (1990). Briefly, dilutions of the test solutions dissolved in vehicle will be added to sterile melted PDA at 45 C to give final concentrations of 100, 10, 1, 0.8, 0.6, 0.4, 0.2, and 0.1 mg/mL of plants extracts. The resultant solution will be thoroughly mixed and approximately 15 mL will be poured onto the petri plate. Plugs of 1 mm of fungal mycelium cut from the edge of actively growing colonies will be inoculated in the center of the agar plate and then incubated in a humid chamber at 25 C. Control cultures will be received an equivalent amount of vehicle. Three replicates will be used for each concentration. Radial growth is measured when the control colonies almost reached 1.5 cm. Results will be expressed as the percentage of hyphal growth inhibited (Gamliel et al., 1989). Concentration response curves will be prepared in which the percentage of hyphal growth inhibition is plotted against concentration mg/mL. The concentration required to give 50% inhibition of hyphal growth IC50 will be calculated from the regression equation. Miconazole can be used as a positive control.
