The agar diffusion test method appl

The agar diffusion test method applied by Anejaand Joshi [20] was used to observe theantimicrobial activity of both oil and ethanolicextract. The microbial suspension were swabbedand spread on nutrient agar. The plates werecultured in 0.1 mL (105-106cells/mL) andsubsequently 8 mm wide wells were bored withinthese agar plates using a sterile cork borer. Theeach wells were aseptically filled with 100 μL ofethanolic extract (250, 500 and 1000 μg/mL) and20 μL of essential oil was placed on theinoculated agar. The plates were incubatedovernight at 37 oC for bacteria and at 20-22 oCfor 5 days for yeast cultures. Microbial growthwas determined by measuring the diameter ofzone of inhibition. For each strain, a negativecontrol was maintained where DMSO was usedin place of extract. In order to determine thesensitivity of gram negative and gram positivebacteria, Gentamycin (20 g/disc) was chosen asstandard, while Nystatin (50 g/disc) was selectedas Standard for yeasts (Table 2). Eachexperiment was carried out three times andmean values are presented.Minimum ınhibitory concentration (MIC)assayTube dilution method was used to determine MICof the extracts. The essential oil and extract ofCyperus fuscus were dissolved in 10 %dimethylsulfoxide (DMSO). The highestconcentration (1000 μg/mL) was prepared at thebeginning. Then serial two-fold dilutions wereprepared resulting the concentrations rangingfrom 31.25 to 1000 μg/mL in 15 mL sterile testtubes containing nutrient broth and 100 µL of thebacterial suspension containing 108 CFU/mL ofrespective test organisms. The tubes wereincubated at 37 ºC in an incubator for 24 h forbacteria and 22 oC for 48 h for yeast. A tubecontaining nutrient broth without extract wasused as control. The least extract concentrationinhibiting the growth of the test organisms wasconsidered as MIC (Table 2).Statistical analysisA completely randomized experimental designwas used with three replications in factorialarrangements. One-way analysis of variance wasalso used. Tukey HSD multiple comparison testswere used to find out which group originated thedifference between the groups. The normalityassumption in the analyses was tested byKolmogorov-Smirnov test. Statistical analyseswere performed using SPSS (Version 20.0,SPSS Inc, USA) statistical package program. Inthe analyses, significance level was set at p <0.05.