Laboratory methodologies for bacterial antimicrobial susceptibility testing 4 oie terrestrial manual 2012 i disk diffusion, ii broth dilution, iii agar dilution. It uses antibiotic discs to test the extent to which bacteria are affected by those antibiotics. Broth micro or macrodilution is one of the most basic antimicrobial susceptibility testing methods. Because oxa48 confers highlevel temocillin resistance, a disc diffusion assay using temocillin as well as ba and dpa inhibition.
However, the disc diffusion method must be visually read and does not provide quantitative data. Methods for in vitro evaluating antimicrobial activity. Have detailed theoretical knowledge on how to perform the main methods in a laboratory 8. Arrange the wells so there are four per plate, as far apart as possible.
The purpose of the kirbybauer disk diffusion susceptibility test is to determine the sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to various antimicrobial compounds in order to assist a physician in selecting treatment options for his or her patients. Antimicrobial potentials of catharanthus roseus by disc. Principles of assessing bacterial susceptibility to. Comparison of disc diffusion assay with the clsi reference method m27a2 for testing in vitro posaconazole activity against common and uncommon yeasts. As more antimicrobial compounds were discovered, it was predicted that infectious diseases would be eliminated through the use of these antimicrobials 7. The agar used is meullerhinton agar that is rigorously tested for composition and ph.
For the disk diffusion assay, each group should swab ten muellerhinton agar plates from the control culture. The extent of diffusion of the antimicro bial agent into the agar is. Using a disk diffusion assay to introduce statistical methods. This method is based on the principle that antibioticimpregnated disk, placed. The diskdiffusion agar method tests the effectiveness of antibiotics on a specific.
Request pdf antimicrobial potentials of catharanthus roseus by disc diffusion assay the present research work investigates the in vitro antimicrobial activity of different solvent extracted. The disk diffusion test, or agar diffusion test, or kirbybauer test is a test of the antibiotic. However, for oxa48 like carbapenemases, no specific inhibitor is available. We have tested the closeness of this relationship by comparing 20,000 simultaneous. Using a forceps, place each specific antibiotic paper disk presoakedyou dont need to do anything on the gramnegative plate as shown on the next page. 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. Jan 01, 2020 the eucast disk diffusion test is based on mh media and disks of a good quality. The kirbybauer disc method this method is also called the agar diffusion method or the disk diffusion method. Jan 17, 2016 assay of antibiotic by agar diffusion method duration. Disc diffusion assay all the glassware and media used were sterilized in an autoclave at 121c for 20 min. When disc zone diameters were interpreted according to the casfm, there were seven 3. Staphylococcus aureus atcc 25923 and escherichia coli atcc 25922, by two variants of the agar diffusion method well and disc, two variants of the bioautographic method direct and indirect and by microdilution assay. Kirbybauer disk diffusion susceptibility test protocol, inoculation of the muellerhinton agar plate.
Comparison of disc diffusion to reference agar dilution mics figures 1 and 2 present the scattegrams of 200 enterobacteriaceae isolates tested by agar dilution and disc diffusion methods for colistin. This diffusion will place the chemical in the agar only around the disc. Disk diffusion by the kirbybauer method is a standardized technique for testing rapidly growing pathogens. Disk diffusion method for susceptibility testing of neisseria. The inhibition zones were reported in millimeter mm. Repeat this, inoculating ten more muellerhinton agar plates from the experimental culture. Enumerate the methods used for antimicrobial susceptibility testing ast 6. However, the agar diffusion method can be used for determination of mic values provided the necessary reference curves for conversion of inhibition zones into mic values are available. After streaking the muellerhinton agar plate as described in step 3, rim the plate with the swab by running the swab around the edge of the entire the plate to pick up any excessive inoculum that may have been splashed near the edge. Laboratory protocols level 2 training course susceptibility.
States had adopted the disk diffusion method for determining susceptibility of bacteria to antimicrobials. The disc diffusion assay kirbybauer method was used to screen for antibiotic activity. T he solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. The procedure involves preparing twofold dilutions of the antimicrobial agent e. Bsac disc diffusion method for antimicrobial susceptibility. Using a 10 ul glass syringe hamilton or a micropipette, apply the sample in 8 ul aliquots to the. Antimicrobial susceptibility testing can be used for drug discovery, epidemiology and prediction of therapeutic outcome. Bacterial suspensions were put into chaerunisaa, anis yohana et al j.
In section 4, place the disk containing the control antibiotic. Pdf on the theory of the disk diffusion test researchgate. In addition, the disc diffusion test is also relatively easy to setup and inexpensive. The behaviour of hydrolytic enzyme in gels as compared to that of its behaviour in solution is quite different. In addition, this resistance mechanism has high potential. Agar diffusion tests are often used as qualitative methods to determine whether a bacterium is resistant, intermediately resistant or susceptible.
This can be done by a number of techniques, which include the disc diffusion method, the broth dilution assay and the etests. Lecture 9 antimicrobial susceptibility testing disk diffusion. Testing an antibiotic using a disk diffusion assay kirby bauer. Swabbing should be done by following the nccls protocol 2, 6. Kirbybauer disk diffusion susceptibility test protocol. When a 6mm antimicrobial disc is placed on a mullerhinton agar plate, immediately water is absorbed into the disc from the agar. Advantages of a standardized disk diffusion based assay for evaluating the antifungal susceptibility of dermatophytes include the ease of use, reproducibility, accuracy, and low cost 1, 3, 10, 15, 16, 22. The disk diffusion method is performed using muellerhinton agar mha, which. British society for antimicrobial chemotherapy bsac to actively support the eucast disc diffusion method for antimicrobial susceptibility testing in preference to the current bsac disc diffusion method from january 2016, the bsac standing committee for antimicrobial susceptibility testing, with the support of council, will. To set up an micmbc assay, 1 first prepare 2fold serial dilutions of the test compounds up to 7 and one quality control qc antibiotic in a microdilution plate.
Pdf comparison of disc diffusion assay with the clsi. Kirbybauer disc diffusion assay lenzmeier research laboratory page 2 analysis of antibiotics. The study of this behaviour is required to better understand the enzyme. Preparation of ginger extracts in soybean oil 10 grams of ginger powder was mixed with 40 ml soybean oil.
First developed in the 1950s, it was refined and by w. The pathogenic organism is grown on muellerhinton agar in. Bsac disc diffusion method for antimicrobial susceptibility testing version 2. However, those 20 mbl producers all showed synergy with dpa and were therefore interpreted as. Inoculate the plate with the test organism by streaking the swab in a backandforth motion very close together as you move across and down the plate. Media are supplemented with 12% sodium chloride nacl if intended for. The bacterial strains escherichia coli atcc 25922 and staphylococcus aureus atcc 25923 were used as model test strains for gramnegative and grampositive bacteria, respectively. Jul 28, 2010 however, no agarbased susceptibility testing method has been standardized for the testing of dermatophytes. The kirbybauer test for antibiotic susceptibility, called the disc diffusion test, is a standard that has been used for years. Disc diffusion susceptibility methods introduction when a filter paper disc impregnated with a chemical is placed on agar the chemical will diffuse from the disc into the agar.
Currently, no agarbased susceptibility testing method has been standardized for testing dermatophytes. Antimicrobial susceptibility testing by the disc diffusion method is discussed. Agar diffusion assay, 66 agar disc diffusion, 73 agglutininlike sequences als, 97 antibiotic resistance, 94, 96 antibodiespeptides, 110 antifungal activity, 98100 antimicrobial peptides amps, 44, 45 biological pro. Antimicrobial susceptibility testing by the kirbybauer disc diffusion. Dip a sterile cotton swab into the appropriate liquid culture. The activity pattern of pectinase in gel matrix was studied. Use only gentle suction to remove the plugs of agarose, since you want uniform wells. A comparison of disc diffusion and microbroth dilution. All aspects of the kirbybauer procedure are standardized to ensure.
Antibacterial activities of pfhas and cupfhs were evaluated using well diffusion method on muellerhinton agar mha. The uses and limitations of disc diffusion in the antibiotic. We describe a newly developed agarbased method employing disk diffusion assay to test the susceptibility of 47 isolates of dermatophytes against 8 antifungals. Comparison of disc diffusion assay with the clsi reference. The antimicrobial in the disc diffuses into the surrounding agar. Kirbybauer disk diffusion susceptibility test protocol created. The standard medium for disk diffusion and mic testing of neisseria gonorrhoeae that of the national committee for clinical laboratory standards was tested to establish zone correlations for the mic breakpoints currently used in sweden. Accurate determination of bacterial susceptibility to antibiotics is essential to the successful management of bacterial infections and to the comparative analysis of antimicrobial agents. Agarbased disk diffusion assay for susceptibility testing of dermatophytes e. Tuesday, 08 december 2009 author jan hudzicki information history the publication on penicillin by alexander fleming in 1928 is a milestone in the history of medicine. Agarbased disk diffusion assay for susceptibility testing of.
Agar plug diffusion method is often used to highlight the antagonism between microorganisms 34, 35, and the procedure is similar to that used in the disk. Bauer, then standardized by the world health organization in 1961. Kirby bauer method and the liquid media based microbroth dilution method. Kirbybauer disk diffusion susceptibility test protocol american. The disk diffusion method readily permits detection of inducible. This medium is prepared according to manufacturers instructions, dispensed in tubes at 45 ml and sterilized.
Jan 17, 2017 in this video, autumn kraft, a graduate student in the micro department, shows us how to properly perform the disc diffusion assay in the laboratory. It is calibrated to eucast clinical breakpoints using broth microdilution for mic determination. A disc diffusion assay for detection of class a, b and oxa48. Disc diffusion in agar is a common method of testing the sensitivity of bacteria to antibiotics. The eucast disk diffusion test is based on mh media and disks of a good quality. Antimicrobial activity evaluation and comparison of. Jun 01, 20 kirbybauer disc diffusion assay lenzmeier research laboratory page 2 analysis of antibiotics. 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. Testing an antibiotic using a disk diffusion assay kirby bauer method.
The inoculum for the disk diffusion method is prepared using a suitable broth. The disc diffusion method for antibiotic susceptibility testing is the kirbybauer method. Disc diffusion or the kirbybauer test is one of the classic microbiology techniques, and it is still very commonly used. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. Class a and b carbapenemases in enterobacteriaceae may be detected using carbapenemase inhibition tests with boronic acid derivatives ba and dipicolinic acid dpaedta, respectively. Radial diffusion assay for tannins miami university. Our results show that the method is reproducible, is simple, and could be used to determine the antifungal susceptibility of. Bauer kirby antibiotic susceptibility testing procedure. As more antimicrobial compounds were discovered, it was predicted that infectious diseases would be. Ghannoum1 center for medical mycology, department of dermatology, school of medicine, case western reserve university and university hospitals case medical center, cleveland, ohio,1 and department of microbiology, university of. We concluded that the wellvariant of the diffusion method was more sensitive than the disc variant. Emilia canton, javier peman, ana espinelingroff, estrella martinmazuelos, alfonso carrillomunoz, jose pedro martinez, comparison of disc diffusion assay with the clsi reference method m27a2 for testing in vitro posaconazole activity against common and uncommon yeasts, journal of antimicrobial chemotherapy, volume 61, issue 1. Because of convenience, efficiency, and cost, the disc diffusion method is. 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.
To evaluate the suitability of disc diffusion dd assay for testing posaconazole activity and to corroborate its activity against recently isolated yeasts by the clsi reference microdilution m27. 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. Compare dilution and diffusion methods and know the basic techniques of agar disk diffusion, broth dilution and agar dilution methods 7. Original article comparison of disc diffusion, etest and agar. Because of convenience, efficiency, and cost, the disc diffusion method is probably the most widely used method for. Standardisation of disk diffusion results for antibiotic susceptibility.
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