MATERIALS
AND METHODS

 

The
study area

The
study area is found in the town of Gondar which is located in the North Western
part of Ethiopia mainly in the Amhara Region. It is placed
about 738 Km from Addis Ababa, capital city of Ethiopia. Geographically
Gondar is 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 material was transported to the Microbiology Laboratory at
the Department of Biotechnology, University of Gondar to
evaluate 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. aureas.

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 complete extraction of the bioactive material. After 48 Hrs each of the
extracts was filtered rapidly 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 dissolved in DMSO (Oxoid) to the concentration of 500
mg/ml to determine 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 antimicrobial activity

Antimicrobial
activity of the extracts was analyzed using agar well diffusion assay according
to the technique described by Taye et al. (2011). Several 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. 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 incubator for 15 minutes.On eachplate,
equidistant wells were made with a 6 mm diameter sterilized cork borer, 2 mm from
the edge of the plate. One hundred micro liter 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 MIC and MBC

The
MICs of plant extracts were 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 micro liters 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 e concentration of the extract related with no bacterial culture.
Each assay was performed in triplicate.

Data
analysis

The
data was analysed 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.