Asian Pac J Trop Biomed 2014; 4(10): 816-820 816

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Asian Pacific Journal of Tropical Biomedicine

journal homepage: www.elsevier.com/locate/apjtb

Document heading doi:10.12980/APJTB.4.201414B16 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved. 襃 In vitro antibacterial activity of extracts of and Ricinus communis against Escherichia coli and methicillin resistance Staphylococcus aureus

1 1 2* Bereket Abew , Samuel Sahile , Feleke Moges 1Department of Biology, Faculty of Natural and Computational Sciences, University of Gondar, Ethiopia 2Department of Medical Microbiology, College of Medicine and Health Sciences, University of Gondar, Ethiopia

PEER REVIEW ABSTRACT

Peer reviewer Objective: Zehneria scabra Z. scabra To eRicinusvaluate communisthe antibactR.er icommunisal activities of the cEscherichiarude leave scoli ext rE.ac tscoli of Staphylococcus ( ) ( ) ( ) Dr. Berhanu Andualem, Associate aureus S. aureus and S. aureus against , ( ) Professor, Department of Biotechnology Methods: and methicillin resistancZ.e scabra . R. communis College of Natural and Computational The crude powdered of and were extracted successively Sciences, Ethiopia. by organic solvents in increasing polarity [benzene, chloroform:acetone (1:1), 70% alcohol and + Tel: 251918700027 distilled water]. TheE. a ncolitibacterial susceptibS.ili taureusy of th e crude leaves extracts of were testeE.d acoligainS.st (ATCC 25922) (ATCC 2923) E-mail: [email protected] saureustandar d strains of S. aureus and and clinical isolates of , Comments Results:and mZ.et hscabraicillin resistR.an ccommunise using agar well diffusion method. S. aureus In and leaf extracts, the most sensitive standard strain was (14 00 1 20) (15 90 2 13) This paper is very interesting and has with an inhibition zone of . 依Z.. scabra mm and . 依 . mm, respectively. The minimum (MIC) 1 95 wide application in biotechnology inhibitory concentration values of extracS.ts aureusagainst test organisms ranged from . 3 250 1 4 where many researchers can follow mg/mL for extract in clinical and E.sta ncolidard strains of R. t ocommunis mg/mL for extract and MIC the screening and extraction of natural in clinical and standard strains of . The values of S. aureusextracts against test 1 95 2 3 250 products for the discovery of noble organisms ranged from . mg/mE.L fcoli.or extract and standard strains of to mg/mL 1 MIC compounds. Therefore, this study is for extract in clinical isolate of Most of the minimR.um communis bactericidal concentration and helpful to initiate other researchers in values of extracts were almost similar particularly in , or minimum Z.ba cscabratericidal the area of interest. Conclusions:concentration e qual to one dilution factor less than MIC value of the extracts mainly in . Details on Page 819 The potency of plant extracts against test organisms were depend on different organic solvents used. Clinical isolate of bacterial pathogens showed less zones of diameter compared to the standard strains. Gram-positive had wide inhibition zones than Gram-negative bacteria. Further studies should be carinri evitrod out to isolate the pure compounds and standardization of the methods of plant extracts for an testing. KEYWORDS Antibacterial, Extracts, Inhibition zone, MBC, MIC

1. Introduction incorrect use of synthetic drugs results in side’ effects and other problems[2]. Large percentage of the world s population In recent years, there has been growing interest in does not have access to conventional pharmacological alternative therapies and the therapeutic use of natural treatment, and folk medicine and ecological awareness products, especially those derived from [1]. This suggests that natural products are harmless. However, the interest in drugs of plant origin is limited due to several use of these substances is not always authorized by legal re.geasons, namely, conventional medicine can be inefficient authorities dealing with efficacy and safety procedures, ( . side effects and ineffective therapy), abusive and/or and many published papers explained the lack of quality *Corresponding author: Feleke Moges, Department of Medical Microbiology, College Article history: of Medicine and Health Sciences, Post Box 196, University of Gondar, Ethiopia. Received 9 Jan 2014 Tel: +251918778160 Received in revised form 12 Feb, 2nd revised form 19 Feb, 3rd revised form 28 Feb 2014 E-mail: [email protected] Accepted 5 Jun 2014 Foundation Project: Supported by University of Gondar under teaching and learning Available online 16 Jul 2014 program (UoG/Budget code: 6417). Bereket Abew et al./Asian Pac J Trop Biomed 2014; 4(10): 816-820 817 in the production, trade and prescription of phytomedicinal water and air dried at room temperature under shade for products. So, formulation of plants for standardization and 10 d and powdered using wooden-made pestle and mortar. regulation of phytomedicinal products is the most alternative The powdered materials were sieved and stored in air tight way[3]. container until use. In Ethiopia, medicinal plants have been used as traditional A 100 g of air-dried plant powdered leaves were measured medicine to treat number of human and animal ailments by by electronic balance (CY510) and then placed in a 2 000 mL the local people from time immemorial. About 80% of the clean round bottomed flask at room temperature caped with population in Ethiopia uses traditional medicine, mainly aluminum foil. After that, 1 000 mL benzene was added to the [4] Zehneria scabra Z. scabra Ricinus hcommuniserbal planR.ts communis. ( ) and flask which contained powdered leaves material [1 part test ( Z. scabra) are some of the traditionally used material and 10 parts of solvent (w/v)]. The mixture was kept “medicinal pl”ants. , vernacular name is called on a rotary shaker at 200 r/min for 24 h. The macerate was Hareg Resa in Amharic, is a climbing or trailing herb first filtered through double layer muslin cloth then through belongs to the that can go up to 10 m. Old Whatman No. 1 filter paper and it was assigned as extract stems become woody with corky-ridged bark. It inhabits 1. The residue was taken again and mixed with chloroform/ forest and on forest margins, riverine fringes and exotic acetone with ratio 1:1 in a volume of 1 000 mL, and the filtrate plantations across 900-2 100 m above sea level with a was assigned as extract 2. The same procedure was followed widespread in tropical Africa, South AfricZ.a, Ascabrarabia, India, using 70% alcohol and distilled water to obtain extract 3 and Java and the Philippines[5]. In Ethiopia, is one extract 4, respectively. These successive cold maceration of the traditional medical plants commonly used for the methods were done with increasing order of their polarity[13]. treatment of alopecia, wound and eczema, burn remedy and The filtrates we°re allowed to concentrate under rotary vapor ( ) skin rashes as part of a poly-herbal preparation, the ash RE 2000 at 40° C, weighed and stored in sterilized air tight [6] R. communis and wash prepared from pounded leaves . Gulo container at 4 C for further analysis. popularly called castor bean in English and in 2.3. Test organisms Amharic. It is widely spread as a wild plant through East and North Africa. In Ethiopia, castor plant is important to E. coli S. aureus treat tooth ache, cold, dysentery and itachy, fetal membrane Standard strains of (ATCC 25922) and [7-9] retention and rabies in different preparationZ. scabra. R. (ATCC 2923) were collected from Biomedical and Laboratory communis Ethno-botanical studies revealed that and Sciences ResearcE.h coliCenter, UniveS.rs iaureusty of Gondar while are being used in the treatment of pathogenic clinical isolates of , MRSA and were collected organisms in the traditional health care system in Ethiopia. from Ethiopian Health and Nutrition Research Institute, However, very little work has been done to evaluate their Addis Ababa. The microorganisms were transported to [10] efficacy in scientificin w vitroay . Therefore, this study was Microbiology L° aboratory by using nutrient agar slant and aimed to evaluate theZ. scabra antibacR.ter icommunisal activity of crude preserved at 4 C for further use. leaf extractions of anEscherichiad coli E.ag acoliinst 2.4. Standard antibiotics Staphylococcusstandard and cl iaureusnical is oS.la taureuses of ( ), S. aureus ( ) including methicillin (MRSA) ( ) resistance . Ciprofloxacin in the form of powder havingµ a broad spectrum property was used as a positive control (5 g/mL). 2. Materials and methods 2.5. Inocula preparation

2.1. Study area The inocula preparation was carried out by growth methods[14,15]. The test organisms from nutrient agar slant The study was conducted at University of Gondar, Gondar were transferr°ed into a nutrient agar medium to get a pure town. It is located in the North West Ethiopia of Amhara colony at 37 C for 24 h. Three to five pure colonies were region. The town has an elevation of 2 080 m above sea selected and transferred into a sterile test tube containing level with a mid-altitude c°limate and an average annual 5 mL of sterile nutrient broth and the solutions were mixe°d 30 37 ma°ximum temperature of C and minimum temperature of by a vortex mixer. The broth cultur5e wa6 s incubated at C 16 C[11]. until it reached the turbidity of (10 -10 CFU/mL) which was 2.2. Plant material equivalent to 0.5 McFarland standards (usually about 8 h) and turbidity was adjusted visually by comparing the test. 2.2.1. Sampling, sample collection and identification 2.6. Agar well diffusion method

Through random sampling, Z.he ascabralthy and diR.se acommunisse free of the leaves of a wild growing and Susceptibility tests were performed by agar well diffusion were collected from gardens in Gondar town by scissors and method using Muller-Hinton agar[16,17]. A sterile cotton samples were kept in plastic bags. The plants were identified swabs were dipped into the adjusted suspension by pressing and confirmed using standard manuals by plant taxonomist. and rotating the swabs firmly against the inside of the tube 2.2.2. Preparation of crude extract above the fluid level. The swab was then evenly streaked Z. scabra R. communis over the entire surface of the Muller-Hinton agar plate The leaves of and were transported repeatedly by rotating the plate approximately 600 each to Microbiology Laboratory, Department of Biology, Faculty time and the rim of the agar was swabbed to obtain uniform of Natural and Computational Sciences, University of inoculums. On each plate, six equidistant wells (one in the Gondar. The plant materials were washed using distilled center and five wells at the corner) were made with a 6 mm Bereket Abew et al./Asian Pac J Trop Biomed 2014; 4(10): 816-820 818 Z. scabra S. aureus diameter sterilized cork borer, 2 mm from the edge of the extracts of was E. [ (9coli.95依 1.79) mm] followed [15] MRSA (7 80 1 24) (3 50 0 97) plate . µ by [ . 依 . mm] and [ . 依 . mm]. Five of the holes were aseptically filled witi.eh 50 L of SimR.il acommunisrly, the most suS.s caureuseptible standard strains for extracts different concentrations of each plant extracts ( . 100 mg/ of E. coli wa s (ATCC 2923) [(15.90依2.13) mm], mL, 200 mg/mL, 300 mg/mL, 400 mg/mL and 500 mg/mL) followed byS. aureus(ATCC 25922) [(15.20依1.53) mm] and clinical sequentially, and at the center, ciprofloxacin was added as a isolaE.tes coli of [(9.90依1.98) mm], MRSA [(9.50依2.12) mm] (7 75 0 91) ( 1) positive control. The amount of control was the same as th° e Tableand 1 [ . 依 . mm] Table . P 4 C tested sample on the wells. etri plates were placed at Z. scabra for 2 h to allow diffusion of the extract into the agar and then Zone of inhibition against test organisms of leaf extract fractions of ° R. communis incubated at (37依0.1) C for 24 h. At the end the inhibition and . zones formed were measured to the nearest millimeters and Diameter of zone of inhibition (mm) leave type Organisms the experiment was performed in duplicate. Experiments IZE Ciprofloxacin Z. scabra E. coli* that gave contradicting results were done for the third time 3.50依0.97 26.75依0.33 E. coli** for an easy decision. 6.85依0.91 31.25依0.19 2.7. Determination of minimum inhibitory concentration MRSA 7.80依1.24 21.00依0.32 S. aureus* (MIC) 9.95依1.79 22.00依0.22 S. aureus** 14.00依2.00 28.00依0.49 R. communis E. coli* Z. scabra R. communis 7.75依0.91 28.00依0.16 MIC of crude extracts of the and E. coli** [18] 15.20依1.53 33.75依0.25 were performed using two fold broth dilution methods . * ( ) MRSA 9.50依2.12 21.25依0.33 The extract solution 500 mg/mL was serially diluted with S. aureus* 1 2 1 4 1 8 1 16 1 32 1 64 1 128 1 256 9.90依1.98 24.00依0.49 nutrient broth as : , : , : , : , : , : , : and : S. aureus** to bring 250 mg/mL, 125 mg/mL, 62.5 mg/mL, 31.25 mg/ 15.90依2.13 28.25依0.50 * ** mL, 15.63 mg/mL, 7.81 mg/mL, 3.95 mg/mL and 1.95 mg/ Data are expressed as mean依SEM. : Clinical isolates; : Standard strains; µ S. aureus. mL concentrations, respectively and 20 L of a standard IZE: Inhibition zone of extract; MRSA: Methicillin resistant suspension of the test organism was added to each concentration of the extract. Two test tubes containing 3.2. The MIC values of Z. scabra and R. communis extracts nutrient broth without antimicrobial agent were added in against test organisms each test. One of these tubes was inoculated with the test organism; the other was left uninoculated and served as a Z. scabra contro°l for media sterility. The broth plates were incubated The MIC value of plant extracts of against the at 37 C for 24 h. The lowest concentration, at which there test bacteria ranged from 1.95 S.m gaureus/mL (extract 3 for both was no turbidity, was regarded as MIC value of the extract. clinical and standard stains of ) to 250.00 mg/mL 2.8. Determination of minimum bactericidal concentration (exE.tra ccolit 1 and extract 4 for both clinical and standard strains ) ( 2) MIC (MBC) of , respectively, Table . The values of extract 3 ranged from 1.95 mg/mL to 62.5 mg/mL with the least MIC values compared to other crude leaf extract fraction. Extract MBC is the lowest concentration of an antibiotic required 4 had MICS. v aaureuslues ranged from 15.62 mg/mL (on standard [18] MBC ) 250 00 ( to kill a microorgµanism . The were determined by sE.tr acoliins of to . mg/mL on standard strains of ) sub-culturing 20 L of the test dilutions from° MIC tubes on where as MIC values were negligible to other strains to fresh nutrient agar plates incubating at 37 C for 24 h. The (particularly on clinical isolates). The overall trend showed lowest concentration that killed the entire bacterial colony that the MIC values of Gram-positive bacteria were lower on the plates was recorded as MBC. than Gram-negative bacteria (Table 2). 2.9. Statistical analysis Table 2 Z. scabra The MIC values of leaf extract fractions against test organisms using One-way analysis of variance (ANOVA) was used for two fold broth dilution methods. Plant extracts (mg/mL) determination of the antimicrobial susceptibility test of Organisms phyto-chemical extracted by different solvents in the Extract 1 Extract 2 Extract 3 Extract 4 E. coli* V 250.00 62.50 62.50 - specific concentration. alues were expressed as mean E. coli** 依SEM by using SPSS version 16 software and presented as 125.00 31.25 31.25 250.00 P * 0 05 MRSA 62.50 15.62 7.81 - tables. values less than . were taken as statistically S. aureus* [19] 15.62 3.91 1.95 31.25 significant . S. aureus** 3.91 1.95 1.95 15.62 * ** 3. Results : Clinical isolates; : Standard strains; -: No MIC value. R. communis 3.1. Agar well diffusion assay of the crude leaf extracts of Z. As indicted from Table 3, leaf extract showed scabra and R. communis MIC value ranging from 1.95 mg/mL to 250.00 mg/mL. The most effective extract that inhibited the growth of bacteria S. aureus was extract 3 with value ranged from 1.95 mg/mL to 62.5 mg/ Among the standard test strains, (ATCC 2923) was mL. Extract 4 inhibited all S.sta aureusndard strains in low MIC values theE. m ocolist susceptible bacteria [(14.00依2.00) mm], followeZ.d rangeE.d colifrom 3.91 mg/mL ( (ATCC 29S.2 3aureus) to 7.81 mg/ scabraby (ATCC 25922) [(6.85依0.91) mm] to the extracts of mL ( (ATCC 25922). Standard strain of (ATCC . Among clinical isolates most susceptible strains for 2923) was the most sensitive with MIC value of 1.95 mg/mL to Bereket Abew et al./Asian Pac J Trop Biomed 2014; 4(10): 816-820 819 E. coli extracts 2 and 3 followed by S.st aaureusndard strain of (ATCC zonS.e aureusof inhibition (14.00依2.00) mm against standard strain 25922) and clinical isolate of (1.95 mg/mL) to eE.xt rcoliact of (ATCC S.29 2aureus3) followed by (9.95依1.79) mm against 3. The most resistant bacteria were clinical isolate of clinical isolate of . The least diameter of zone of (E.62 .coli5 m g/mL) at extract 2 and 3 followed by clinical isolate of inhibitionE. r ecolicorded was (3.50依0.97) mm against clinical (250 00 ) 1 ( 3) Table 3 . mg/mL at extract Table . isolate of . Similarly, other study conducted by Anand et al. R. communis indicated that ethanol, mZ.et hscabraanol, ethyl acetate and The MIC values of leaf extract fractions against test organisms aqueous extracts of theS. sh aureusoot of from the standard using two fold dilution methods. bacterial pathogen of showed maximum zone of [20] Plant extracts (mg/mL) inhibition (50.00依0.11) mm , while in a study conducted by Organisms [21] Z. scabra Extract 1 Extract 2 Extract 3 Extract 4 Bruck , methanol leaves extracts of had no effect E. coli* 250.00 62.50 62.50 - on the tested microbes except at higher concentration low E. coli** S. aureus 7.81 3.91 1.95 7.81 activity against standard strain of (6 mm). These * MRSA 62.50 15.62 7.81 - variations in zone of inhibitions may be due to the difference S. aureus* 62.50 3.91 1.95 31.25 in concentration of active pricnciple; type of solvent used for S. aureus** [20] 3.91 1.95 1.95 3.91 extraction and type of bacterial strains tested . * ** R. communis : Clinical isolates; : Standard strains; -: No MIC value. Crude leaves extracts of demonstrated 3.3. The MBC values of Z. scabra and R. communis extracts mS.a aureusximum zone of inhibition against standard strains E.of against test organisms coli (ATCC 2923) was (15.90依2.13) mm, followed by S. aureus(ATCC 25922) [(15.20依1.53) mm], clinical isolates of Z. scabra [(9.90依1.98) mm] and MRSA [(9.50依2.12) mm]. TheE. l ecoliast MBC value of fraction 3 from leaf extract of was inhibition zone was recorded from clinical isolate of ranged from 1.95 mg/mL against clinical and standard strains (7.75依0.91) mm. A similar study conducted by Kensa and S. aureus E. coli E. coli of to 62.5 mg/mL againstZ. c lscabrainical isolate of . Yasmin showed that the most sensitive strainet w alas The second important extract in leaf was extract (11.3 mm)[22]. While other reports by Chukwuka . showed S. aureus R. communis E. coli[23] 2 ranged from 3.91 mg/mL against standaE.rd colistra in that leaf extract did not inhibit . The to 62.5 mg/mL against clinical isolate of followed by difference might be related to many factors such as age of eS.x taureusract 1 that ranged from 7.81 mg/mL against standardE. st rcoliain the plant, plant part used, solvent concentration, tested to 500.00 mg/mL against clinical isolate of strains used and extraction procedures followed[21]. (Table 4). The results of theS. p raureusesent study showed that standard Table 4 bacterial strain of was the most susceptible Z. scabra bacterium, which may be attributed to the absence of outer The MBC of leaf extract fractions against test organisms. membrane of the organism that makes it more accessible Plant extracts (mg/mL) Organisms Z.to pscabraermeation bR.y acommunisctive principles of the leaves extracts of Extract 1 Extract 2 Extract 3 Extract 4 [18] P E. coli* and . romising inhibition zone 500.00 62.50 62.50 - MRSA E. coli** was also obtained against which shows that herbal 250.00 31.25 31.25 250.00 * preparations are important alternatives forZ. d scabrarug resistaR.nt MRSA 62.50 15.62 7.81 - S. aureus* bacteria pathogens. Crude leaves extracts of and 15.62 7.81 1.95 31.25 communis S. aureus** have shown an interesting profile of antibacterial 7.81 3.91 1.95 15.63 * ** activity against standard bacterial strains more than clinical : Clinical isolates; : Standard strains; -: No MBC values. isolates and Gram-positive strains are more sensitive to the R. communis extracts. Therefore, further study is needed to isolate the leaf extract fraction showed greater effects pure compounds from these crude extracts. in inhibiting bacterial growth in this study, with MBC values ranged from 1.91 mS.g aureus/mL of extract 3 against standard and Conflict of interest statement clinical isolates of E. t ocoli 250 mg/mL of extract 1 aE.ga icolinst clinical isolate strains of . StanS.da aureusrd strain of was the most sensitive strains next to (3.91 mg/mL We declare that we have no conflict of interest. 3) ( 5) Tablefrom e5xtract Table . R. communis Acknowledgements The MBC of leaf extract fraction against test organisms. Plant extracts (mg/mL) Organisms Extract 1 Extract 2 Extract 3 Extract 4 This study was partially funded by University of Gondar, E. coli* 250.00 125.00 62.50 - Faculty of Natural and Computational Science, School of E. coli** 15.62 7.81 3.91 15.62 Graduate Studies, and Department of Biology. The financial * MRSA 62.50 15.62 7.81 - assistance received from University of Gondar under S. aureus* 62.50 7.81 1.95 125.00 teaching and learning program (UoG/Budget code: 6417) was S. aureus* 31.25 1.95 1.95 62.50 greatly acknowledged. * ** : Clinical isolates; : Standard strains; -: No MBC values. Comments

4. Discussion Background

Z. scabra In developing countries, about 80% of the population uses In this study, leaf extracts of showed maximum medicinal plants as traditional medicine to treat infectious Bereket Abew et al./Asian Pac J Trop Biomed 2014; 4(10): 816-820 820 Z. scabra R. communis overview of traditional medicine practices and policy in Ethiopia. diseases. and are being used for Ethiop J Health Dev 20 2006; : 127-134. treatment of pathogenic organisms in the traditional health Bull T [5] A schwanden C. Herbs for health, but how safe are they? care system. his study aimeZ.d tscabrao evaluate aR.n tcommunisibacterial World Health Organ 79 2001; : 691-692. activity of crude extract from and Flora of Ethiopia and Eritrea [6] E dwards S, Tadesse M, Hedberg I. . against clinical and standard strains. Addis Ababa: The National Herbarium; 1995, p. 27. Research frontiers [7] T eklehaymanot T, Giday M. Ethnobotanical study of medicinal Z. scabra R. communis plants used by people in Zegie Peninsula, Northwestern Ethiopia. In this study, leaf extracts of and J Ethnobiol Ethnomed 3 2007; : 12-15. shoS.w eaureusd maximum zone of inhibition against standard strain of . Promising inhibition zone were also obtained [8] Y irga G, Teferi T, Gidey M, Zerabruk S. An ethno veterinary MRSA survey of medicinal plants used to treat livestock diseases in against which shows that medicinal plant extracts are Afr J Plant Sci Seharti-Samre district, Northern Ethiopia. 2012; important alternatives for dZ.ru scabrag resistant bR.ac tcommuniserial pathogens. 6 Crude leaves extracts of and have (3): 113-119. shown an interesting profile of antibacterial activity against [9] Y ineger H, Yewhalaw D, Teketay D. Ethnomedicinal plant knowledge and practice of the Oromo ethnic group in standard bacterial strains more than clinical isolates. Gram- J Ethnobiol Ethnomed 4 positive were more sensitive than Gram-negative strains. Southwestern Ethiopia. 2008; : 11. [10] G iday M, Asfaw Z, Woldu, Z. Ethnomedicinal study of plants Related reports J Ethnopharmacol used by Sheko ethnic group of Ethiopia. 2010; 132 - : 75 85. The antimicrobial effect of plant extracts against test National Atlas of Ethiopia [11] E thiopian Mapping Authority. . 1st ed. organisms were depend oetn dalifferent solvents used. Similar study reported by Anand ., 2012 indicated that ethanol, Addis Abeba: Ethiopian Mapping Authority; 1988. [12] J eet K, Tomar S, Thakur N. Antipyretic activity of whole aerial mZ.e tscabrahanol, ethyl acetate and aqueous extracts of the shoot of Argyreia nervosa Int J Pharm Pharm Sci 4 part from . 2012; : 1-2. S.sh aureusowed maximum zone of inhibition for standard strains of . [13] L alitha MK. Manual on antimicrobial susceptibility testing. 2004. [Online] Available from: http://www.google.com.hk/ Innovations and breakthroughs url?sa=t&rct=j&q=Manual%20on%20Antimicrobial%20 S. aureus Susceptibility%20Testing&source=web&cd=1&ved=0CCoQFjA Promising inhibition zone were obtained against Z. MRSA A&url=http%3a%2f%2fwww%2eijmm%2eorg%2fdocuments%2fA scabraand wR.hic communish shows that medicinal plant extracts from and are important alternatives for drug ntimicrobial%2edoc&ei=V3wZU8mXJIKOkwWzrIHADw&usg= resistant bacterial pathogens. AFQjCNEzTSnwiSIbkuc6SWnCiiupflK0hw&bvm=bv.62578216,d. aGc&cad=rjt [Accessed on 20th January, 2014] Applications [14] W iegand I, Hilpert K, Hancock RE. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) In recent years, there has been growing interest in Nat Protoc 3 of antimicrobial substances. 2008; (2): 163-175. alternative therapies and the therapeutic use oZ.f n scabraatural [15] B ritish Society for Antimicrobial Chemotherapy. Methods prodR.uc tcommuniss, especially those derived from plants. and have antimicrobial components, therefore for antimicrobial susceptibility testing. Birmingham: BSAC; looking for its formulation and components of the extract 2010. [Online] Available from: http://bsac.org.uk/wp-content/ may lead to the production of important antibiotic alternative uploads/2012/02/Version_9.1_March_2010_final-v2.pdf [Accessed on 15th January, 2014] for the treatment of infectious diseases. Solanum surattense . J [16] S heeba E. Antibacterial activity of Burm. F Peer review Sci Eng Tech 6 2010; (1): 1-4. This paper is very interesting and has wide application [17] U ddin N, Rahman A, Ahmed NU, Rana S, Akter R, Masudul AM, Chowdhury A. Antioxidant, cytotoxic and antimicrobial in biotechnology where many researchers can follow Eclipta alba Int J Biol Med Res properties of ethanol extract. the screening and extraction of natural products for the 1 2010; (4): 341-346. discovery of noble compounds. Therefore, this study is Biostatistics and microbiology: a survival manual helpful to initiate other researchers in the area of interest. [18] P aulson DS. Bozeman: Bioscience Laboratories Inc.; 2008, p. 222. [19] A nand SP, Doss A, Jeyachandran R. Antagonistic microbial References Zehneria scabra Int J screening of shoot extracts of (L.F.) Sonder. Res Ayurveda Pharm 3 2012; (1): 109-111. [1] G hosh L, Gayen JR, Sinha S, Pal S, Pal M, Saha BP. Antibacterial [20] B ruck M. Studies on extracts of some medicinal plants Rumex nepalensis Phytother Res efficacy of Spreng. roots. 2003; traditionally used for dermatological disorders in Ethiopia 17 (5): 558-559. [dissertation]. Addis Ababa: Addis Ababa University; 2004. [2] V aghasiya Y, Chanda S. Screening of some traditionally used [21] K ensa VM, Yasmin SS. Phytochemical screening and Klebsiella Ricinus communis Plant Sci Feed Indian plants for antibacterial activity against antibacterial activity on L. pneumoniae J Herb Med Toxicol 3 1 . 2009; (2): 161-164. 2011; (9): 167-173. [3] A mit L, Vikas G, Vaibhav T, Vikash K, Siddhartha G. [22] C hukwuka KS, Ikheloa JO, Okonko IO, Moody JO, Mankinde Bersama Phytochemistry and pharmacological activities of TA. The antimicrobial activities of some medicinal plants on englerina Int Res J Pharm 1 Escherichia coli Adv Appl Sci Guerke - an overview. 2010; : 89-94. as an agent of diarrhea in livestock. Res 2 [4] K assaye KD, Amberbir A, Getachew B, Mussem Y. A historical 2011; (4): 37-48.