[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$foNKp74RJ7Wg1BzGJpXnqSc1kBKCrReiEg4j4pPRt_-8":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":66},[4,8,12,16,20,24,28],{"title":5,"slug":6,"path":7},"About Microbeonline.com","about-microbeonline-com","\u002Fabout-microbeonline-com\u002F",{"title":9,"slug":10,"path":11},"About Me","about-me","\u002Fabout-microbeonline-com\u002Fabout-me\u002F",{"title":13,"slug":14,"path":15},"Advertise with Us","advertise-us","\u002Fadvertise-us\u002F",{"title":17,"slug":18,"path":19},"Privacy Policy","privacy-policy","\u002Fprivacy-policy\u002F",{"title":21,"slug":22,"path":23},"Abbreviations","abbreviations","\u002Fabbreviations\u002F",{"title":25,"slug":26,"path":27},"Microbes","microbes","\u002Fmicrobes\u002F",{"title":29,"slug":30,"path":31},"Books","recommended-books","\u002Frecommended-books\u002F",{"type":33,"data":34},"blog",{"slug":35,"title":36,"description":37,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":40,"lastUpdatedDate":41,"draft":42,"category":43,"image":38,"body":44,"faq":45,"tags":64,"related":65},"antimicrobial-susceptibility-testing-procedure-modified-kirby-bauer-method","Modified Kirby-Bauer Disc Diffusion Method: Procedure, Reading Rules & Common Errors","The full modified Kirby-Bauer procedure — inoculum prep, disc placement, and the three exceptions to standard zone reading that catch most students off guard, including the β-lactamase \"heaped edge\" rule.",null,"Acharya Tankeshwar","2013-08-27","2026-06-20",false,"general-microbiology","Modified Kirby-Bauer disc diffusion test method is a reference method that could be used as a routine technique to test the susceptibility of a bacterial isolate in a clinical laboratory. The disc diffusion method was originally described in 1966, is well standardized, and has been widely evaluated.\n\n## Why This Matters\n\nDisc diffusion is the method most clinical labs actually run, day to day — it doesn't need the equipment automated MIC systems require, which is exactly why it remains the CLSI-recommended routine method in resource-limited settings as much as in well-funded ones. That also means small protocol deviations don't stay theoretical — they change real reports.\n\n**The Warm Incubator Problem:** the procedure below notes that incubating above 35°C invalidates oxacillin\u002Fmethicillin results. That's not a minor footnote. **An incubator running even slightly warm can make a methicillin-resistant *S. aureus* (MRSA) isolate falsely appear oxacillin-*susceptible*** and a falsely reassuring report can lead to a β-lactam being prescribed for an infection it won't touch. The protocol's precision exists because of consequences exactly like this one.\n\n![Kirby Bauer Disc Diffusion Method - Kirby Bauer Disc Diffusion Method (Image source: Ref.1)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FKirby-Bauer-Disc-Diffusion-Method.png)Figure: Kirby Bauer Disc Diffusion Method (Image source: Ref.1)\n\n## Procedure for Modified Kirby Bauer method\n\n![Swabbing Pattern for Susceptibility Testing - Image 1: Swabbing pattern to ensure proper inoculation of the organism.](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FSwabbing-pattern.jpg)Figure: Image 1: Swabbing pattern to ensure proper inoculation of the organism.\n\n 1. Prepare the inoculum from the primary culture plate by **touching with a loop the tops of each of 3 – 5 colonies of similar appearance**, of the organism to be tested and transfer this growth to a tube of saline. If the inoculum has to be made from a pure culture,  suspend a loopful of the confluent growth similarly.\\\n    \\\n    *Note: Multiple similar looking colonies should be picked to minimize the possibility of testing a non representative colony such as picking a susceptible colony only and missing the resistant mutants dispersed in other colonies.*\n 2. Compare the tube with the **0.5 McFarland turbidity standard (approx cell density 1.5 x10^8 CFU\u002Fml)** and adjust the density of the test suspension to that of the standard by adding more bacteria or more sterile saline.\\\n    \\\n    Remember: Proper adjustment of the turbidity of the inoculum is essential to ensure that the resulting lawn of growth is confluent or almost confluent.\n 3. Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess inoculum by pressing and rotating the swab firmly against the side of the tube above the level of the liquid.\n 4. Streak the swab all over the surface of the medium three times, rotating the plate through an angle of 60 ° after each application. Finally, pass the swab around the edge of the agar surface. The swab should follow as it is drawn across the plate (as shown in the *figure). #Discard the swab into an appropriate container.*\n 5. Leave the inoculum to dry for a few minutes (at **least 3 to 5 minutes,** but no more than 15 minutes) at room temperature with the lid closed.\n 6. Place the appropriate antimicrobial-impregnated disks on the surface of the agar (*antimicrobial disks can be purchased from any reputable suppliers*)\n 7. Antimicrobial discs can be placed on the inoculated plates using a pair of sterile forceps. It is convenient to **use a template** to place the discs uniformly or a sterile needle-tip may also be used to place the antibiotic discs on the plate. Alternatively, an **antibiotic disc dispenser (as shown in image-2)** can be used to apply the discs to the inoculated plate.\n 8. Disks should not be placed closer than 24 mm (center to center) on the Mueller Hinton agar plate.  Ordinarily, no more than 12 disks should be placed on a 150-mm plate or more than 5 disks on a 100-mm plate. #*avoid placing disks close to the edge of the plate as the zones will not be fully round and can be difficult to measure.*\n 9. **Agar depth matters as much as disc spacing.** Plates poured too thin or too thick will skew zone sizes regardless of how carefully everything else is done. See [Mueller-Hinton Agar](https:\u002F\u002Fmicrobeonline.com\u002Fmueller-hinton-agar\u002F) for the exact depth specification and plate volumes by plate size.\n10. Each disc should be **gently pressed** down to ensure complete contact with the agar surface and do not fall when the plate is inverted during incubation. #*Do not push the disc into the agar.*\n11. The plates should be placed in an incubator at **35 °C** within 30 minutes of preparation. Temperatures above 35 °C invalidate results for oxacillin\u002Fmethicillin. #*Do not incubate in an atmosphere of carbon dioxide, this will decrease the pH of the agar and result in errors due to incorrect pH of the media.*\n12. After overnight incubation, the diameter of each zone (including the diameter of the disc) should be measured and recorded in mm. The results should then be interpreted according to the **antimicrobial susceptibility interpretation chart.**\n\n## Measurement of the zone of inhibition\n\nThe measurements can be made\n\n- with a ruler on the under-surface of the plate without opening the lid.\n- If the medium is opaque, the zone can be measured by means of **a pair of calipers**.\n- A template may be used to assess the final result of the susceptibility tests.\n\n![Using antibiotic disc dispenser - Image 2: Using antibiotic disc dispenser](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FAntibiotic-disc-dispenser.jpg)Figure: Image 2: Using antibiotic disc dispenser\n\nThe endpoint of inhibition is judged by the naked eye at the edge where the growth starts, but there **are three exceptions:**\n\n1. With sulfonamides and co-trimoxazole, slight growth occurs within the inhibition zone; such growth should be ignored.\n2. When β-lactamase-producing staphylococci are tested against penicillin, zones of inhibition are produced with a heaped-up, clearly defined edge; these are readily recognizable when compared with the sensitive control, and regardless of the size of the zone of inhibition, they should be reported as resistant.\n3. Certain *Proteus* species may swarm into the area of inhibition around some antibiotics, but the zone of inhibition is usually clearly outlined and the thin layer of swarming growth should be ignored.\n\n![Measuring AST Plate - Measuring zones of inhibition.  Gray shading represents a confluent lawn of bacterial growth.\nThe white circle represents no growth of the test organism.](\u002FMeasuring-AST-plate.jpg)\\## Results\n\nResults can be read after 18 hours of incubation. Following incubation, measure the zone sizes to the nearest millimeter (mm) using a ruler or caliper; include the diameter of the disk in the measurement.\n\n**Interpretation and Reporting of antimicrobial susceptibility results**\n\n1. Using the published **CLSI guidelines**, determine the susceptibility or resistance of the organism to each drug tested. Note that there are different charts for different organisms.\n2. For each drug, indicate on the recording sheet whether the zone size is **susceptible (S), intermediate (I), or resistant (R)** based on the interpretation chart. Zone sizes are not reported to physicians.\n\n## Limitations\n\nIf performed precisely according to standard protocol, disk diffusion method yields data that can reliably predict the *in vivo* effectiveness of the drug in question but it has a few limitations too:\n\n1. Does not provide accurate information about the [minimum inhibitory concentration (MIC)](\u002Fminimum-inhibitory-concentration-mic-broth-dilution-method-procedure-interpretation\u002F)\n2. Does not provide reliable results with some antibiotic\u002Forganism combinations, such as for penicillin G in *Neisseria meningitidis* and *S. pneumoniae*.\n\n## Standard Kirby-Bauer vs. the Stokes Method\n\nBoth are disc diffusion methods reading the same kind of zone — the difference is how each controls for variability. Standard Kirby-Bauer relies on tightly standardizing every variable independently: inoculum density, agar depth, disc potency, incubation temperature. The [Stokes method](https:\u002F\u002Fmicrobeonline.com\u002Fstokes-disc-diffusion-method-principle-procedure-interpretation-results\u002F) takes a different approach — it runs a known control strain on the *same* plate as the test organism, so the comparison is made directly against a reference rather than against a fixed mm chart. That makes Stokes more forgiving of small batch-to-batch variation in media or disc potency, which is part of why it remains popular in labs without tightly controlled reagent supply chains.\n\n## Learning & Remembering\n\n**Clinical story 1 — The Warm Incubator Problem:** see \"Why This Matters\" above — how a few degrees of incubator drift can turn a resistant MRSA isolate into a falsely reassuring oxacillin-susceptible report.\n\n**Clinical story 2 — The Heaped Edge:** Most resistant zones are simply smaller. But when β-lactamase-producing staphylococci are tested against penicillin, the zone can look deceptively close to normal size — except for a telltale heaped-up, sharply defined edge where the enzyme has degraded the drug right at the boundary. CLSI guidance is explicit: report this as resistant *regardless of zone size*, because the edge — not the diameter — is the tell here.\n\n**One sentence that captures it:** A zone of inhibition only means what the protocol says it means — change the temperature, the agar depth, or the inoculum, and the same isolate can report as a different organism entirely.\n\n**Exam facts**\n\n| Question | Answer |\n| --- | --- |\n| Minimum disc spacing (center to center)? | 24 mm |\n| Max discs on a 150 mm plate \u002F 100 mm plate? | 12 \u002F 5 |\n| Standardized agar depth? | \\~4 mm |\n| Incubation temperature? | 35°C — above this invalidates oxacillin\u002Fmethicillin results |\n| Drying time before applying discs? | 3–5 minutes minimum, no more than 15 |\n| Name the three exceptions to standard zone-edge reading | Sulfonamide\u002Fco-trimoxazole haze (ignore), β-lactamase-producing staph vs. penicillin (heaped edge = resistant regardless of size), *Proteus* swarming (ignore the thin swarm layer) |\n| What does disc diffusion NOT provide? | An exact MIC value |\n\n**References and further reading**\n\n1. CLSI. *M02—Performance Standards for Antimicrobial Disk Susceptibility Tests*, 14th ed. Clinical and Laboratory Standards Institute; current edition.\n2. Cappuccino, J. G., & Welsh, C. T. *Microbiology: A Laboratory Manual*, 11th ed. Pearson; 2016.",[46,49,52,55,58,61],{"question":47,"answer":48},"Why does incubating above 35°C invalidate oxacillin\u002Fmethicillin results?","Temperatures above 35°C can cause a methicillin-resistant Staphylococcus aureus (MRSA) isolate to falsely appear oxacillin-susceptible, leading to a misleading report and potentially ineffective treatment if a β-lactam is prescribed.",{"question":50,"answer":51},"Why is a heaped-up zone edge reported as resistant even if the zone looks otherwise normal-sized?","In β-lactamase-producing staphylococci tested against penicillin, the enzyme degrades the drug right at the zone boundary, creating a heaped-up, sharply defined edge. CLSI guidance is to report this as resistant regardless of the overall zone diameter.",{"question":53,"answer":54},"Why does agar depth matter for disc diffusion accuracy?","CLSI specifies a uniform Mueller-Hinton agar depth of approximately 4 mm. Agar poured too thin lets antibiotic diffuse further than intended, producing falsely large zones; too thick restricts diffusion and produces falsely small ones.",{"question":56,"answer":57},"What's the difference between standard Kirby-Bauer and the Stokes method?","Standard Kirby-Bauer relies on tightly standardizing inoculum density, agar depth, disc potency, and incubation temperature independently. The Stokes method instead runs a known control strain on the same plate as the test organism, comparing results directly rather than against a fixed chart — making it more forgiving of batch-to-batch variation.",{"question":59,"answer":60},"Why are sulfonamide and co-trimoxazole zones read differently from other antibiotics?","Slight bacterial growth often occurs within the inhibition zone with these drugs even in susceptible isolates. This faint growth should be ignored when reading the zone edge.",{"question":62,"answer":63},"Can disc diffusion provide an exact MIC value?","No — disc diffusion only categorizes isolates as Susceptible, Intermediate, or Resistant. For an exact MIC value, methods like E-test or broth\u002Fagar dilution are needed.",[],[],[67,73,80,85,89,93,98,103,107,111],{"slug":68,"name":39,"description":69,"image":70,"body":71,"postCount":72},"acharya-tankeshwar","Editor-in-chief","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Ftankeshwar-acharya-author-microbeonline.jpg","***Tankeshwar Acharya, MSc (Medical Microbiology)***\n\n*Tankeshwar Acharya is an Assistant Professor in the Department of Microbiology at Patan Academy of Health Sciences (PAHS), Nepal, where he has been teaching and practicing clinical microbiology for over 14 years. He is the founder of Microbe Online, one of the leading free microbiology education resources on the web, covering bacteriology, mycology, parasitology, immunology, and clinical laboratory diagnostics written from direct experience in both the classroom and the diagnostic laboratory.*",433,{"slug":74,"name":75,"description":76,"image":77,"body":78,"postCount":79},"ashma-shrestha","Ashma Shrestha","SEO Copywriter and Science Communicator\nKathmandu, Nepal","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Fashma-shrestha.png","Ashma Shrestha holds a Master of Science in Medical Microbiology from the Institute of Science and Technology (IOST), Tribhuvan University, Nepal, where she developed a strong foundation in virology, molecular biology, and diagnostic microbiology.\n\nShe now works as an SEO Copywriter at Resolution Digital, where she combines her scientific training with research-driven content strategy. She is certified in Google Analytics and Google Business Profile (GBP), and brings a data-informed approach to science communication writing content that is not only accurate but structured to reach and serve the students who need it most.\n\nAt microbeonline, Ashma contributes articles primarily in virology and molecular biology, areas she finds most compelling for their mechanistic depth and their growing clinical relevance. Her writing reflects the same standard the site is built on: factual rigor, clear explanation of the *why* behind microbiology concepts, and content that helps students move from memorization to genuine understanding.\n\nShe is passionate about making complex microbiological concepts accessible without sacrificing accuracy; a skill that sits at the intersection of her scientific training and her professional work in content and SEO.",81,{"slug":81,"name":82,"description":83,"image":38,"body":38,"postCount":84},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":86,"name":87,"description":83,"image":38,"body":38,"postCount":88},"samikshya-acharya","Samikshya Acharya",20,{"slug":90,"name":91,"description":83,"image":38,"body":38,"postCount":92},"alisha-tripathi","Alisha Tripathi",6,{"slug":94,"name":95,"description":96,"image":38,"body":38,"postCount":97},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":99,"name":100,"description":101,"image":38,"body":38,"postCount":102},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":104,"name":105,"description":83,"image":38,"body":38,"postCount":106},"srijana-khanal","Srijana Khanal",18,{"slug":108,"name":109,"description":101,"image":38,"body":38,"postCount":110},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":112,"name":113,"description":83,"image":38,"body":114,"postCount":115},"nisha-rijal","Nisha Rijal","**Nisha Rijal** is a microbiologist and quality assurance specialist. She served for nearly 12 years as a microbiologist at the National Public Health Laboratory (NPHL), Nepal's national reference laboratory, and continues to work as a consultant microbiologist in international public health organization. ",51]