Clinical Guide
Kirby-Bauer Disk Diffusion Test: Measuring & Interpreting Zone of Inhibition
A practical guide to performing the Kirby-Bauer test, measuring zone of inhibition diameters, and interpreting susceptibility using CLSI M100 breakpoints.
What Is the Kirby-Bauer Test?
The Kirby-Bauer disk diffusion test is a standardized qualitative method for antimicrobial susceptibility testing (AST). Introduced by William Kirby and Alfred Bauer in the 1960s, it remains one of the most widely used techniques in clinical microbiology because it is simple, inexpensive, and reproducible.
In this method, paper disks impregnated with a fixed concentration of antibiotic are placed on a Mueller-Hinton agar plate inoculated with the test organism. The antibiotic diffuses into the agar, inhibiting bacterial growth in a clear circular area called the zone of inhibition.
Standard Procedure (CLSI M02)
- Prepare a bacterial suspension matching a 0.5 McFarland turbidity standard.
- Within 15 minutes, swab the suspension evenly across the surface of a Mueller-Hinton agar plate (150 mm plates preferred for multiple disks).
- Allow the plate to dry for 3-5 minutes before applying disks.
- Place antibiotic disks using sterile forceps or a disk dispenser, ensuring adequate spacing (typically no more than 12 disks on a 150 mm plate).
- Invert and incubate at 35 ± 2 °C for 16-18 hours (24 hours for some organisms).
- Measure the diameter of each zone of inhibition to the nearest whole millimeter using calipers or a ruler held against the back of the plate.
Measuring Zone of Inhibition
Measure the diameter — not the radius — of the clear zone surrounding each disk, including the 6 mm disk itself. Read against a black background with reflected light. For swarming organisms like Proteus, ignore the swarm and measure to the obvious edge of growth inhibition. For sulfonamides and trimethoprim, ignore slight growth (≤20%) within the zone.
Interpreting Zone Diameters (CLSI M100)
The Clinical and Laboratory Standards Institute (CLSI) publishes zone-diameter breakpoints annually in the M100 document. Zones are interpreted as Susceptible (S), Intermediate (I), or Resistant (R) based on organism-drug specific cutoffs. Unlike MIC, larger zones indicate greater susceptibility.
| Category | Zone Diameter | Clinical Meaning |
|---|---|---|
| Susceptible (S) | ≥ S breakpoint | Organism is likely inhibited by standard dosing of the antibiotic. |
| Intermediate (I) | Between S and R | May be effective at higher doses or at sites where drug concentrates. |
| Resistant (R) | ≤ R breakpoint | Standard dosing unlikely to inhibit the organism; choose a different agent. |
For example, CLSI M100 lists ciprofloxacin against Enterobacterales as S ≥ 26 mm, I 22-25 mm, R ≤ 21 mm (5 μg disk). Always consult the current M100 edition for the organism-drug combination you are testing.
Quality Control
- Run QC strains (e.g., E. coli ATCC 25922, S. aureus ATCC 25923, P. aeruginosa ATCC 27853) daily or weekly per CLSI M100 Table 4A.
- Verify Mueller-Hinton agar depth (4 mm) and pH (7.2-7.4).
- Check disk potency, storage (-20 °C long-term, 2-8 °C working stock), and expiry.
- Reject plates with contaminated growth, poor lawn, or overlapping zones.
Kirby-Bauer vs. MIC Methods
Disk diffusion gives a qualitative S/I/R answer and is ideal for routine testing of rapidly growing organisms. MIC methods (broth microdilution, gradient strips) give a quantitative concentration value, which is essential for serious infections, intermediate results, or when calculating a BMQ (S breakpoint ÷ MIC). Many labs use disk diffusion for initial screening and MIC for confirmation or dose adjustment.
Have MIC data instead?
Use the Bactima Interpreter to convert MIC values to S/I/R and BMQ using CLSI and EUCAST breakpoints.