A Study On In-Vitro Antimicrobial Effects Of Some Selected Plants On Staphylococcus Aureus Isolated From Bovine Clinical Mastitis
T TOLOSA, H WAGAYE, F REGASSA
crude extracts, in-vitro antimicrobial effects, medicinal plants, staphylococcu aures, zone of inhibition
T TOLOSA, H WAGAYE, F REGASSA. A Study On In-Vitro Antimicrobial Effects Of Some Selected Plants On Staphylococcus Aureus Isolated From Bovine Clinical Mastitis. The Internet Journal of Veterinary Medicine. 2009 Volume 8 Number 1.
A study on in-vitro antimicrobial effects of some selected plants on
The conventional drugs used for the treatment of mastitis are of limited in types in developing countries in general and in Ethiopia in particular due to this and other factors causal agents have showed variable degree of resistance.
In Ethiopia, traditional healers use a number of plants for the treatment of bovine mastitis. The efficiency of some of these plants/herbs have been tested against a range of causative agents of mastitis. In-vitro study conducted by Sahle  indicated that
Materials and Methods
Description of the study area
A study of in-vitro antimicrobial effect of phytopreparations was carried out in Debrezeit between November 2007 and May 2008. Debreziet town is located at 47 km south-east of Addis Ababa. The area has an altitude of 1,850 meters above sea level with an average annual rain fall of 866 mm. It has a bimodal rainy seasons; a main rain season extends from the month of June to September and a short rainy season from March to May. The annual average minimum and maximum temperature is 11 0C and 26 0C, respectively. Day length is fairly constant throughout the year (12-13 hrs) with about 6 hours of sunshine during the rainy season and 8 to10 hours for the rest of the year. Humidity is about 50.9 % .
An experimental study on in-vitro antimicrobial efficacies on selected plants was conducted between November 2007 and May 2008 in AAU, Faculty of Veterinary Medicine, Debrezeit.
Herbal Materials Used for the Study
Bacterial Organisms used for the Study
One species of bacterium,
Plant Collection and Pre-extraction Preparation
The plants were chosen based on the results showed by previous workers on the leaf of the plants [3, 5, 6].
Preparation of Crude Extracts for in-vitro Experiment.
50 gm of each of the powdered herb leaves of the three plants were macerated in 80% methanol in a flask separately and mixed using a magnetic stirrer. Then after it is allowed to stand for 3 days at room temperature, each sample was then strained using a tea strainer to remove solids. The resulting filtrate was then further filtered using filter paper to obtain a solution free of solids (Fig 4). The solution was then concentrated in a rotary evaporator to remove the methanol. The plant extracts of the three plants were then taken out and put in evaporating dishes and kept in a dry oven at 40 0C to remove the remaining solvent for 24 hours. The residues left after straining and filtration and were macerated again in the same way. The procedure was repeated in the same way for third time to have sufficient amount of extracts. The resulting concentrated extracts were weighed, transferred and labeled with the respective plant names and stored at + 40 until tested for antimicrobial activity. The flow chart from collection to extraction is shown below.
Preparation of Antimicrobial Discs from Herb Extracts for in -vitro Experiment
Six serial dilutions with different concentrations (20 %, 10 %, 5%, 2.5 % and 0.625 %) of each plant extract were prepared using Dimethylsulfoxide (DMSO) as described by Olila
Antimicrobial Sensitivity Test
The antimicrobial test was conducted using agar disc diffusion method.
The turbidity of the bacterial suspension was adjusted by comparing with 0.5 McFarland turbidity standards. The standard and the test suspension were placed in a 10ml sized tests tubes and compared against a white back ground with contrasting black lines until the turbidity of the test suspension equates to that of the turbidity standard. Adjustments of the turbidity were made by adding saline or colonies depending on the degree of turbidity. A sterile swab was dipped in to the standardized suspension of the bacteria and excess fluid was expressed by pressing and rotating the swab firmly against the inside of the tube above the fluid levels. The swab was streaked in the three directions over the entire surface of the agar with objective of obtaining uniform inoculations, and a final sweep with the swab was made against the agar around the rim of the Petri dish. The inoculated plates were allowed to stand for not more than 15 minutes and the discs were placed on the agar surface using a sterile forceps. Each disc was gently pressed with the point of the sterile forceps to ensure complete contact with the agar surface .
Muller-Hinton agar (38gm) (Biotech UK) medium was used for antimicrobial sensitivity test, and was mixed with 1 litter of distilled water, boiled to dissolve completely and autoclaved at 1210c for 15minutes. The medium was later dispensed in to 90mm sterile agar plates and left to set. The agar plates were incubated for 24 hours at 370 C to confirm their sterility. When no growth occurred after 24 hours, the plates were considered sterile and used for antimicrobial sensitivity tests.
Barium solution was used as standard to determine the bacterial concentration was prepared as 1% solution in 1% H2SO4 solution. The preparation was kept in dark for the preparation of bacterial suspension. Colonies were picked from the culture under study and placed in 4ml sterile physiological saline and the culture was standardized by comparing with McFarland solution.
The appropriate crude extract impregnated discs and conventional discs were applied at spaces of 24mm apart from center to center and 15mm away from the edge of the plates. This was made no later than 15minutes after the inoculums has been seeded. The plates turned upside down, labeled and incubated at 370C for 24 hours. Diameter of zone of inhibition was measured using a ruler in millimeters and results were recorded as susceptible, intermediate or resistant by comparing with standard values for each conventional antibiotic disc .
Descriptive statistical methods were used for data analysis and results were presented as percentages, tables and graphs for illustration.
Antimicrobial Susceptibility Testing
Effects of Herbal Preparations
Each plant extracts of the three plant species were tested at different concentrations levels (20%, 10%, 5%, 2.5%, 1.5%, 2.5%, 1.25%, and 0.625%) to see their inhibitory effects against
zone of inhibition in mm for each plant extracts at doubling concentrations ranging from 0.625% to 20% (Table 1). The inhibiting zone increased with increasing concentrations of the extracts for the two plant species (
In this study an in-vitro antimicrobial efficacy test of three herbal preparations was performed on
Of the plant types tested for their efficacy in this study, the leaf part of
In this study a direct relationship between concentration and zone of inhibition was observed. Therefore, in all cases of the test plants with antimicrobial activity, there was a dose dependent inhibition on the tested bacteria showing greatest activity at highest concentrations of the crude extracts. A wider zone of inhibition with increasing concentrations of methanol extracts of leaf of
In this study the leaf of both