Kirby–Bauer disk diffusion

Principle

• An antibiotic diffuses from a paper disk into agar, forming a concentration gradient.
• The diameter of the zone of inhibition is inversely related to the MIC and is interpreted using CLSI or EUCAST breakpoints.

Specimen

• Pure, isolated colonies (18–24 h growth) of a rapidly growing organism.

Reagents and equipment

• Mueller–Hinton agar (MHA), pH 7.2–7.4, poured to a depth of 4 mm; MH with 5% sheep blood or HTM/MH-F for fastidious organisms.
• 0.5 McFarland standard (about 1.5 × 10^8 CFU/mL), antibiotic disks stored per manufacturer, calipers or ruler.

Procedure

• Prepare a saline suspension matching 0.5 McFarland (direct colony or growth method).
• Within 15 minutes, dip a sterile swab, press out excess and swab the whole plate in three directions (rotating about 60°), then the rim.
• Apply disks within 15 minutes, pressed firmly; centers at least 24 mm apart (no more than 12 on a 150 mm plate, 5 on a 100 mm plate).
• Invert and incubate within 15 minutes at 35 ± 2 °C in ambient air for 16–20 h (some drug–organism pairs, e.g. vancomycin with enterococci, need 24 h).

Results

• Measure complete inhibition zone diameters in mm from the back of the plate with reflected light (blood media: from the top, lid removed).
• Report Susceptible, Susceptible-dose dependent/Intermediate or Resistant per current breakpoints.

Quality control and pitfalls

• QC strains include E. coli ATCC 25922, S. aureus ATCC 25923 and P. aeruginosa ATCC 27853.
• Agar too deep gives falsely small zones; too thin gives falsely large zones.
• Heavy inoculum gives small zones; light inoculum gives large zones.
• Excess thymidine affects trimethoprim–sulfamethoxazole; wrong divalent cation content affects aminoglycosides with P. aeruginosa and tetracyclines.
• Not reliable for some combinations (e.g. vancomycin with staphylococci, colistin); use MIC methods.

Clinical use

• Routine, low-cost susceptibility testing to guide antimicrobial therapy and resistance surveillance.