Stokes disc diffusion technique varies from kirby bauer disc diffusion in the use of both control and test strain on a same plate. Mar 31, 2019 evaluation of antimicrobial activity was performed using a disc diffusion method conc. The activity was quantitatively assessed on the basis of inhibition zone and their activity index was also calculated along with minimum inhibitory concentration mic. In vitro screening of five local medicinal plants for. Enumerate the methods used for antimicrobial susceptibility testing ast 6. Antibacterial and antifungal activities from leaf extracts of. Angeliki kourmouli 0 2 3 marco valenti 0 2 3 erwin van rijn 0 2 3 hubertus j.
In this test, a number of small, sterile filter paper disks of uniform size 6 mm that have each been impregnated. This prompted the development of a disk diffusion procedure. Antimicrobial activity of frankincense of boswellia serrata. The interpretation of resistance and susceptibility to antimicrobials is determined through in vivo testing of blood and urine to calculate the obtainable level of a given antimicrobial that results in resolution of an infection. Principles of assessing bacterial susceptibility to.
Klebsiella pneumoniae was not inhibited by both aqueous and ethanol leaf extracts at the concentrations used for this study. Disc diffusion in agar is a common method of testing the sensitivity of bacteria to antibiotics. In order to eliminate or minimize variability in this testing, bauer et al. It is our hope that this document will be useful to our clients, the practicing veterinarians. Hospital proposed a single disk method for antimicrobial susceptibility. Can disc diffusion susceptibility tests assess the. Disc diffusion method was used for antibacterial activity. Disc diffusion or the kirby bauer test is one of the classic microbiology techniques, and it is still very commonly used. It uses antibiotic discs to test the extent to which bacteria are affected by those antibiotics. The antibacterial activity was expressed as the mean zone of inhibition diameters mm produced by the plant extract. Automation may force the method out of the diagnostic laboratory but in this country as well as in the smaller. If the zones of adjacent antimicrobial disks overlap, the zone diameter can be determined by measuring the radius of the zone.
It relies on the diffusion of the antibacterial agents into the agar surrounding the disks. In vitro antimicrobial activity of some medicinal plants. It is essential that the reports are relevant, timely interpreted correctly to ensure quality control. Variations of the stokes test are still the most commonly used method for. The antimicrobial activities of the extracts and fractions were determined by the disk diffusion and minimum inhibitory concentration mic methods. Jun 01, 20 kirbybauer disk diffusion susceptibility test for antibiotics, disinfectants and antiseptics the disk diffusion technique is used to determine the antibacterial activity of antibiotics and disinfectants. Modified kirbybauer disc diffusion test method is a reference method which could be used as a routine technique to test the sensitivity of the isolate in the clinical laboratory.
Antimicrobial activity of aqueous and ethanol leaf. Disc diffusion, agar dilution and broth microdilution were performed following clsi guidelines for antimicrobial preparation, inoculum preparation, inoculation, incubation and data interpretation clinical and laboratory standards institute 2008a, 2006, b. The antibacterial activity was assayed by measuring the diameter of the inhibition zone formed around the well nccls, 1993. The antimicrobial activity was determined using agar well diffusion method and micro dilution method summarized in table 12. Kirbybauer disk diffusion susceptibility test protocol, an alternate method for measuring zones. Secondly, the method used to assess antimicrobial activity, and the choice of test organisms, varies between publications janssen et al. Laboratory manual of standardized methods for antimicrobial sensitivity tests for bacteria. The qc procedure developed for rast demonstrated that inhibition zones were systematically different compared with standard disc diffusion tables s9 to s. All aspects of the kirbybauer procedure are standardized to ensure consistent and accurate results. Procedures for testing the activity of antimicrobial agents, 308. Eucast rapid disc diffusion method for antimicrobial.
Pdf on the theory of the disk diffusion test researchgate. Alexomycin, apramycin, tiamulin, tilmicosin, and tylosin were tested by broth microdilution against various national committee for clinical laboratory standards ncclsrecommended qc organisms staphylococcus. The antimicrobial activity of the oil was evaluated via disc diffusion method against five standard human pathogens gram positive. Theoretical analysis of antibiotic diffusion data by the disc method is built on the assumption that antibiotics diffuse freely and the diffusion limiting factor is hydrodynamic viscous drag. The disk diffusion test, or agar diffusion test, or kirbybauer test disc diffusion antibiotic susceptibility test, disc diffusion antibiotic sensitivity test, kb test, is a test of the antibiotic sensitivity of bacteria. Measure from the center of the antimicrobial disk to a point on the circumference of the zone where a distinct edge is. The use of disc diffusion susceptibility tests to determine the antibacterial activity of engineered nanoparticles enps is questionable because their low diffusivity practically prevents them from penetrating through the culture media. Multidrug resistant mdr isolates were defined as those isolates that are resistant to three classes of antibiotics 17.
Disc diffusion method is better in my point of view, disc paper you can buy it from himedia ready made. Anumber of methods, including the kirbybauer disc diffusion susceptibility technique, have been used to determine the antibacterial activity of enps oberdorster et al. These data and data from two clinical trials initiated by eucast to. The eucast disk diffusion test is based on mh media and disks of a good quality. Escherichia coli nctc6571 and staphylococcus aureus nctc10418 were. J nanopart res can disc diffusion susceptibility tests assess the antimicrobial activity of engineered nanoparticles. A laboratory test which determines how effective antibiotic therapy is against a bacterial infections. The disk diffusion test, or agar diffusion test, or kirbybauer test discdiffusion antibiotic susceptibility test, discdiffusion antibiotic sensitivity test, kb test, is a test of the antibiotic sensitivity of bacteria. These data and data from two clinical trials initiated by eucast to validate the rast method to be published separately were used to define specific qc targets and ranges for the rast. Jun 02, 2011 kirbybauer disc diffusion method antibiotic susceptibility testing skill based learning dr. The antibacterial effect of some medicinal plant extracts. Kirbybauer disk diffusion susceptibility test protocol asm. The microbroth dilution method has become more widely used due to its ability to become automated, however, the kirby bauer method may be able to detect subpopulations of resistant bacteria that would be visualized as inner colonies in the zone of diffusion. The results showed that cupfhs have higher antibacterial activity compared to pfhas.
The antibacterial and antifungal activities of extracts 5, 25, 50, 100, 250. Kirby bauer method and the liquid media based microbroth dilution method. The rate of diffusion through the agar is not as rapid as the rate of extraction of the antimicrobial out of the disk, therefore the concentration of antimicrobial. Disc diffusion method disc diffusion method for antimicrobial susceptibility testing was carried out according to the standard method by bauer et al. The linear functions between izds in millimeters of the disk diffusion method and mics in micrograms per milliliter of the broth microdilution method or etest were characterized by a relatively strong correlation coefficient r. Evaluation of antimicrobial activity of different solvent. A stock solution of extract was prepared by dissolving 0. Kourmouli 1 faculty of civil engineering and geosciences, delft university of technology, 2628cn delft. In 1955, stokes described a disc diffusion procedure whereby the zone diameter.
Testing an antibiotic using a disk diffusion assay kirby bauer. A disc of blotting paper is impregnated with a known volume and appropriate concentration of an antimicrobial, and this is placed on a plate of susceptibility testing agar uniformly inoculated with the. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. Antimicrobial activity based on the broth dilution. Procedure muller hinton agar plates were prepared and the test microorganisms were inoculated by the spread plate method. Bsac disc diffusion method for antimicrobial susceptibility testing version 4 january 2005 all enquiries to jenny andrews at. Regards article antibacterial activity of annona squamosa l. The antibacterial activities of extracts were evaluated using the disk diffusion method as well as well diffusion method. Compare dilution and diffusion methods and know the basic techniques of agar disk diffusion, broth dilution and agar dilution methods 7. Disk diffusion is based on the determination of an inhibition zone proportional to the bacterial susceptibility to the antimicrobial present in the disk. Introduction antimicrobial susceptibility test is very important for treating infectious diseases and monitoring antimicrobial resistance in various pathogens.
The plates are incubated and the zones of inhibition around each disc are measured. Comparison of broth microdilution, etest, and agar disk. We have tested the closeness of this relationship by comparing 20,000. Uses of antibiotic sensitivity testing antibiotic sensitivity test. Kirbybauer disk diffusion susceptibility test protocol. This multicenter study proposes antimicrobial susceptibility mic and disk diffusion methods quality control qc parameters for seven compounds utilized in veterinary health. Preliminary screening of antibacterial activity using. Antimicrobial susceptibility testing ast types and. Chapter15 antimicrobial activity the general methods of antimicrobial assay arc, 1. Evaluation of diffusion and dilution methods to determine the. M51 method for antifungal disk diffusion susceptibility testing of nondermatophyte filamentous fungi and corresponding supplement m51s the details of the data necessary to establish breakpoints, qc parameters, and how the data are presented for evaluation are described in clsi document m23. It is important to note, however, that the substances normally. The disk diffusion test, or agar diffusion test, or kirbybauer test is a test of the antibiotic. Lack of growth of the test organism is observed by clear areas surrounding the disks in a region called the zone of inhibition.
It is calibrated to eucast clinical breakpoints using broth microdilution for mic determination. Antimicrobial activities of different extracts were evaluated by the agar well diffusion method murray. The antimicrobial begins to diffuse into the surrounding agar. The kirbybauer disc method this method is also called the agar diffusion method or the disk diffusion method. Disk diffusion is based on the determination of an inhibition zone. The diffusion of the antimicrobial agent into the seeded culture media results in a gradient of the antimicrobial. Agar disk diffusion method the testing method most frequently used is the standardized filter paper disk agar diffusion method, also known as the nccls national committee for clinical laboratory standards or kirbybauer method. Antimicrobial susceptibility test was performed for all microbial isolates by modified kirby bauer disc diffusion method following the clinical and laboratory standards institute clsi guideline. Modified kirbybauer disc diffusion method for antimicrobial. A microorganism is judged sensitive or resistant according to the diameter of the zone of inhibition of cultural growth, which is then correlated statistically with the minimal inhibitory concentration mic. The procedure followed is simply that a filter disk impregnated with an antibiotic is applied to the surface of an agar plate containing the organism to be tested and the plate is incubated at 37c for 2448 hours. The procedure followed is simply that a filter disk impregnated with an antibiotic is applied to the surface of an agar plate containing the organism to. Disc diffusion technique kirbybiir method materials and methods test organisms and inoculums.
Quality control guidelines for disk diffusion and broth. As the etest and the disk diffusion method are based on the same principle, also a higher variation between the results was found for the same antimicrobial agents concerning the disk diffusion method. Bauer kirby antibiotic susceptibility testing procedure duration. Antimicrobial activity of essential oils and other plant. Because of convenience, efficiency, and cost, the disc diffusion method is probably the most widely used method for determining antimicrobial resistance around the world. Of particular importance is the concept of efficacy ratios. The disc diffusion method of ast is the most practical method and is still the method of choice for the average laboratory. Antibiotic susceptibility testing by a standardized single disk method. Test methods in detecting antimicrobial resistance there are several antimicrobial susceptibility testing methods available today, and each one has their respective advantages and disadvantages.
Determination of antimicrobial activity culture of the bacterial organism was aseptically introduced and evenly spread using sterile l rod on the surface of sterile mueller hinton agar m173m1084, himedia plates. Media are supplemented with 12% sodium chloride nacl if intended for. The agar used is meullerhinton agar that is rigorously tested for composition and ph. Lecture 9 antimicrobial susceptibility testing disk. In this study, we investigate the ability of such a test, namely the kirbybauer disc diffusion test, to determine the antimicrobial activity of au and ag. Similarly to the procedure used in disk diffusion method, the agar plate surface is inoculated by spreading a volume of the microbial inoculum over the entire agar surface. The antibacterial activity of pfhas and cupfhs from canola and palm kernel oils were investigated against escherichia coli e. Agar well diffusion method is widely used to evaluate the antimicrobial activity of plants or microbial extracts 32, 33. The disc diffusion method for antibiotic susceptibility testing is the kirbybauer method.
The bioautographic and diffusion methods are known as qualitative techniques since these methods will only give an idea of the presence or absence of substances with antimicrobial activity. Chloramphenicol disc was used as a positive control. Both the diffusion and dilution methods have been widely used for many years to accurately measure antibacterial activity and are routinely used in antibacterial susceptibility testing. The qc values for the reference ast methods bmd and standard disc diffusion were within published ranges. Antibacterial screening methods for evaluation of natural. The disk diffusion method is performed using muellerhinton agar mha, which. Efficacy ratios can be used to calculate which antimicrobial drug has the highest predicted efficacy or activity against a given bacterial isolate. The agar disc diffusion test is the most convenient and widely used. Antibacterial activities and antibacterial mechanism of polygonum. A method frequently used to screen plant extracts for antimicrobial activity is the agar disc diffusion technique morris et al. Escherichia coli and pseudomonasa aeruginosa and the fungi candida albicans. This publication led the world health organization to form a committee in 1961 to lay the groundwork for the development of a standardized procedure for single antimicrobial disk susceptibility testing 7. The various methods used are disc diffusion, well diffusion, agar dilution and broth dilution.
Beaumont 0 2 3 olgaioanna kalantzi 0 2 3 andreas schmidtott 0 2 3 george biskos 0 2 3 0 a. The disc diffusion method was originally described in 1966, and is well standardized and has been widely evaluated. The disk diffusion technique is used to determine the antibacterial activity of antibiotics and disinfectants. In this test, wafers containing antibiotics are placed on an agar plate. Bsac disc diffusion method for antimicrobial susceptibility. Test methods in detecting antimicrobial resistance. Antimicrobial and antifungal susceptibility testing resources. Antibacterial and antifungal activities from leaf extracts. Methods for in vitro evaluating antimicrobial activity. Antibacterial activity of leaf extracts of baeckea. Determination of mic by agar diffusion method youtube. Zone size observed in a disk diffusion test has no meaning in and of itself 7. In the present study, the microbial activity of hydroalcohol extracts of leaves of cassia fistula linn. Pdf the procedure for disk diffusion susceptibility testing has been worked out for.
Assay of antibiotic by agar diffusion method duration. The inoculum for the disk diffusion method is prepared using a suitable broth. Disc diffusion or the kirbybauer test is one of the classic microbiology techniques, and it is still very commonly used. In vitro antimicrobial activity of different solvent extracts. Bsac disc diffusion method for antimicrobial susceptibility testing version 2.
Similarly to the procedure used in diskdiffusion method, the agar plate surface is inoculated by spreading a volume of the microbial inoculum over the entire agar surface. Stokes disc diffusion technique is not as highly standardized as the kirbybauer technique and is used in laboratories particularly when the exact amount of antimicrobial in a disc cannot be guaranteed due to difficulties in obtaining discs and storing them. Oct 28, 2016 antimicrobial susceptibility testing disk diffusion methods 2. Techniques used for the determination of antimicrobial resistance. Antimicrobial susceptibility testing disk diffusion methods. A comparison of disc diffusion and microbroth dilution. Disc diffusion techniques are used by most laboratories to test routinely for antimicrobial susceptibility.