MATERIALS AND METHODS
The study area
The study area is found in Gondar town which is located in the North-Western part of Ethiopia mainly in the Amhara Region. It is placed about 738 Km from Addis Ababa, the capital city of Ethiopia. Geographically Gondar has located 120 35′ 07” North latitude and 370 26′ 08” East longitudes and altitude range from 2000?2200m above sea level (David R, 2011).
Collection of Croton macrostachyus stem bark
Croton macrostachyus stem bark was collected from behind Tewodros campus student cafe, University of Gondar, Ethiopia. The plant material was transported to the Microbiology Laboratory of Biotechnology Department, the University of Gondar to assess the antibacterial activity of the chloroform, methanol, and water extracts of C. macrostachyus stem bark against both clinical and standard strains of E. coli and S. aureus.
Preparation of Croton macrostachyus stem bark extracts
This was carried out as previous illustrated, with slight modifications, by Predrag et al., 2005. The freshly collected stem bark was cut into pieces and shade dried at room temperature for 10 days and milled using an electric blender. The powder was transferred into closed containers. Each of the powdered plant material was extracted with chloroform, methanol, and water. Twenty-five gram of powdered sample was mixed in a conical flask with 100ml of solvents of chloroform, methanol, and water separately for 48 Hrs, until absolute extraction of the bioactive material. After 48 Hrs each of the extracts was filtered quickly through gauge and then by a more delicate filtration through Whatman no1 filter paper. The filtrates were then placed in a rotary evaporator in order to remove the extraction solvents and collected in a sterile container for extra use. Extracts were kept at 4o C to maintain the antibacterial property before they were used for agar well diffusion and broth dilution assay.
Each plant extract was then suspended in DMSO (Oxoid) to the concentration of 500 mg/ml to find out the zones of inhibition. For the minimum inhibitory concentration measurements the stock solution (500 mg/ml) was serially diluted to give the concentrations of 250 mg/ml, 125 mg/ml, 62.5 mg/ml, 31.25 mg/ml, 15.63 mg/ml, 7.81 mg/ml, 3.90 mg/ml, and 1.95 mg/ml.
Determination of antibacterial activity
Antibacterial activity of the extracts was analyzed using agar well diffusion assay according to the technique described by Taye et al. (2011). Numerous plates of Mueller-Hinton agar were made and labeled according to the extract and by the name of the bacteria, including a negative control (DMSO) and positive control (Chloramphenicol). A bacterial suspension in sterile normal saline was prepared with reference to the 0.5 McFarland Standards. The turbidity of the bacterial suspension was compared with 0.5 McFarland standard solutions, followed by culture of 100 µl of the bacterial suspension on Mueller-Hinton agar plates using a sterile glass rod spreader and allowed to remain in the incubator for 15 minutes to remove excess moisture. On each plate, equidistant wells were made with a 6 mm diameter sterilized cork borer, 2 mm from the edge of the plate. One hundred microliters of each plant extract (500 mg/ml) was aseptically introduced into a respective agar well. This was followed by incubation at 37o C for 24-48 Hrs. The inhibition zone around each well was measured in millimeters (mm) and the assay was carried out three times for each extract to obtain consecutive results.
Determination of Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC)
The MICs of plant extracts was determined using a broth dilution method as described by Bauer et al. (1966). The extract stock solution (500 mg/ml) was serially diluted in a broth to give the concentrations of 250 mg/ml, 125 mg/ml, 62.5 mg/ml, 31.25 mg/ml, 15.63 mg/ml, 7.81 mg/ml, 3.90 mg/ml, and 1.95 mg/ml. One hundred microliters of inoculums (1.5×106 CFU/ml) were then inoculated in to each tube. The growths inhibition was observed after 24 Hrs of incubation at 37o C. The presence of growth was evaluated by comparing turbidity of culture containing test tubes with the negative control. The lowest concentration, at which there was no turbidity, was regarded as MIC value of the extract.
The MBC was determined by sub-culturing samples from the wells with concentrations above the MIC value on new Mueller Hinton agar plates. The MBC was regarded as the lowest concentration of the extract related to no bacterial culture. Each assay was carried out in triplicate.
The data was analyzed using Statistical Package for Social Sciences Software (SPSS) version 20 software. The inhibition zones of the extracts on the pathogens were compared using one way ANOVA.