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Short answer
The Kirby-Bauer test is the standardised form of the disk diffusion method for antimicrobial susceptibility testing. Paper disks holding a fixed amount of an antibiotic sit on an agar plate evenly seeded with the test bacterium, and after overnight incubation the clear zone around each disk is measured in millimetres and read against published breakpoint tables as susceptible, intermediate or resistant.
Definition and scope
The Kirby-Bauer test, also written disc diffusion or disk diffusion, is a culture-based method that reports how a bacterial isolate responds to individual antimicrobial agents. The EUCAST manual describes disk diffusion as “one of the oldest approaches to antimicrobial susceptibility testing” that “remains one of the most widely used” in routine clinical laboratories, suitable for most bacterial pathogens and requiring no special equipment. A 2019 review in Diagnostics credits Bauer and Kirby with the standardised version, confirmed as a standard method in 1966, after earlier diffusion approaches (the Oxford cup method, early paper-disk methods) gave results that varied between laboratories.
The output is a zone diameter, not a minimum inhibitory concentration (MIC). Dilution methods and gradient strips are the routes to an MIC; see the CASRAI guide to minimum inhibitory concentration by broth microdilution for that side. For a view of how susceptibility results are pooled across a facility, see the guide to the antibiogram.
How the method works in practice
The principle is diffusion against growth. Antimicrobial leaves the disk and spreads into the agar, forming a gradient, while the bacteria seeded across the plate try to grow. Where the agent reaches an inhibitory level before the bacteria establish themselves, a clear zone forms. Because the zone depends on a race between diffusion and growth, every condition that changes either one changes the zone, which is why the method only works when it is run exactly as the standard describes. EUCAST states that the technique “must be followed without modification in order to produce reliable results.”
The standardised conditions in the EUCAST disk diffusion manual, version 13.0 (January 2025) are:
- Medium. Mueller-Hinton (MH) agar at a level depth of 4.0 ± 0.5 mm. Streptococci, Haemophilus influenzae and several other fastidious organisms use MH-F agar, which is MH with 5% mechanically defibrinated horse blood and 20 mg/L β-NAD.
- Inoculum. A saline suspension of colonies adjusted to a 0.5 McFarland turbidity standard (accepted range 0.4-0.6), roughly 1-2 × 108 CFU/mL for Escherichia coli. The CASRAI guide to the 0.5 McFarland standard explains that reference.
- Disks. The EUCAST breakpoints and quality control criteria are validated for 6-mm paper disks.
- Incubation. For most non-fastidious organisms, 35 ± 1 °C in air for 18 ± 2 hours. Streptococci, Haemophilus and similar organisms are incubated in 4-6% CO2 in air.
- Reading. Zones are measured to the nearest millimetre with a ruler or calliper (or a zone reader calibrated to manual reading) and converted to susceptible, intermediate or resistant using the current breakpoint tables.
EUCAST zone diameter breakpoints are calibrated to its harmonised European MIC breakpoints, which EUCAST publishes freely on its website. The 2019 review names the Clinical and Laboratory Standards Institute (CLSI) as the provider of the most popular other guideline set. The two bodies do not always share breakpoints, so a zone is only meaningful against the table of the standard the laboratory follows.
Agar diffusion test, agar diffusion assay and disk diffusion assay
Disk diffusion is the form of agar diffusion in which an impregnated paper disk is the source of the agent. The EUCAST manual and the 2019 Diagnostics review use “disk diffusion” and “disc diffusion” for the clinical susceptibility test and treat the Oxford cup method as an earlier diffusion technique rather than the same procedure. This guide covers the clinical disk diffusion susceptibility test and does not describe other agar diffusion uses.
Where it matters for labs and clinics: what moves the zone
The EUCAST manual is explicit about which deviations shift zone sizes and in which direction. The table below is a quality checklist for a laboratory auditing its own set-up, drawn from the manual’s sections:
| Variable | Effect on the zone | EUCAST section |
|---|---|---|
| Inoculum denser than 0.5 McFarland | Reduced zones; a lighter inoculum has the opposite effect | 3.3 |
| Plates left at room temperature after inoculation before disks go on | Organism begins to grow and zones are erroneously reduced | 4.6 |
| Plates left at room temperature after disks go on | Pre-diffusion gives erroneously large zones | 6.1 |
| Loss of potency in stored disks | Reduced zones; described as a common source of error | 5.5 |
| Agar depth above or below the limit | Smaller or larger zones respectively | 9.6 |
| Incubation beyond the recommended time | Growth within zones and false resistant reporting | 6.3.1 |
| Surface moisture on the agar or lid | Fuzzy zone edges or haze within zones | 2.3, 2.7 |
The timing rule that ties several rows together is EUCAST’s 15-15-15 minute rule: use the inoculum suspension within 15 minutes of preparation, apply disks within 15 minutes of inoculation and incubate plates within 15 minutes of disk application.
Quality control and supplies
The manual requires control strains with known zone ranges to be tested alongside clinical isolates, daily or at least four times a week for agents in routine panels, and read before clinical results are reported. Each new batch of Mueller-Hinton agar is tested and its depth measured. The manual links specific drug controls to medium faults: aminoglycosides can reveal unacceptable divalent cation content, trimethoprim-sulfamethoxazole thymine and thymidine content, and erythromycin an unacceptable pH. Disks must be stored in sealed containers with a moisture-indicating desiccant, protected from light, allowed to reach room temperature before opening, and discarded on the manufacturer’s expiry date. These are the consumables a susceptibility bench depends on: agar plates, disks, saline, turbidity standards and incubator capacity. Related reading: Petri dishes and their types and laboratory incubators. EUCAST also recommends keeping stacks to a maximum of five plates for most incubators because uneven heating can affect results.
Common misunderstandings
It does not give an MIC
The zone diameter is interpreted through breakpoints that are calibrated to MIC breakpoints, but the test itself reports a diameter and a category, not a concentration.
A larger zone does not rank drugs against each other
Each agent has its own breakpoints in the tables, so zone sizes for different drugs cannot be compared directly. The category, read against the right table, is the result.
It is not suitable for everything
The 2019 review lists limited data for some organisms (Pseudomonas, Bacillus and Corynebacterium among them) and weaker performance with slow-growing and fastidious bacteria. The EUCAST manual covers anaerobes in a separate document.
Longer incubation is not safer
Beyond the recommended time, growth inside zones can produce false resistant results. The exceptions are specific: Enterococcus with glycopeptides is read at 24 hours, and isolates with insufficient growth for some fastidious organisms are re-incubated to a total of 40-44 hours.
Zone readers do not replace calibration
Automated readers reduce operator variability, but the manual requires them to be calibrated to manual reading.
Frequently asked questions
What is the Kirby-Bauer test used for?
It reports how a bacterial isolate responds to individual antimicrobial agents, as a zone diameter that is converted to susceptible, intermediate or resistant using breakpoint tables. EUCAST describes disk diffusion as suitable for the majority of bacterial pathogens and as requiring no special equipment.
Which agar does the test use?
Mueller-Hinton agar at a level depth of 4.0 ± 0.5 mm for most organisms, and MH-F agar (MH with 5% mechanically defibrinated horse blood and 20 mg/L β-NAD) for streptococci, Haemophilus influenzae and several other fastidious organisms.
Why is the inoculum adjusted to 0.5 McFarland?
Because inoculum density changes the zone. EUCAST states that a denser inoculum reduces zones of inhibition and a lighter one has the opposite effect, so the suspension is adjusted to 0.5 McFarland (acceptable variation 0.4-0.6).
How long are plates incubated?
For most non-fastidious organisms, 18 ± 2 hours at 35 ± 1 °C in air. EUCAST advises against incubating beyond the recommended time because growth within zones can lead to false resistant results.
Is disk diffusion the same as an MIC test?
No. Disk diffusion gives a zone diameter and a category. EUCAST zone breakpoints are calibrated to its MIC breakpoints, but the test does not measure a concentration.
Sources
- EUCAST, Antimicrobial susceptibility testing: EUCAST disk diffusion method, version 13.0, January 2025 (https://www.eucast.org/bacteria/methodology-and-instructions/disk-diffusion-and-quality-control/), checked 2026-10-29. Manual PDF: https://www.eucast.org/fileadmin/eucast/pdf/disk_test_documents/2025/Manual_v_13.0_EUCAST_Disk_Test_2025.pdf.
- Khan ZA, Siddiqui MF, Park S. Current and emerging methods of antibiotic susceptibility testing. Diagnostics 2019;9(2):49. https://pmc.ncbi.nlm.nih.gov/articles/PMC6627445/, checked 2026-10-29.
Informational only. Susceptibility results for individual patients must be produced and interpreted under a laboratory’s accredited procedures and the current breakpoint tables of its chosen standard.
