Standard Operating Procedure: Kirby-Bauer Disc Diffusion

1. Purpose and Scope

To perform standardized disc diffusion antimicrobial susceptibility testing (AST) to guide antibiotic therapy.

2. Specimen Requirements

• Type: Pure bacterial culture (18-24 hours old).
• Organisms: Rapidly growing bacteria (not fastidious organisms).
• Rejection: Mixed cultures, contaminated isolates.

3. Safety and Precautions

• Wear appropriate PPE.
• Handle cultures in biological safety cabinet.
• Dispose of antibiotic discs and cultures properly.

4. Equipment and Reagents

• Mueller-Hinton agar plates (4 mm depth, pH 7.2-7.4).
• Antibiotic discs (stored at -20°C or 2-8°C as per manufacturer).
• Sterile cotton swabs.
• 0.85% saline or Mueller-Hinton broth.
• McFarland turbidity standards (0.5).
• Incubator (35±2°C).
• Caliper or ruler (mm), or automated zone reader.
• Forceps or disc dispenser.

5. Step-by-Step Procedure

1. Inoculum Preparation:

• Pick 3-5 well-isolated colonies of same morphological type.
• Suspend in saline or broth to match 0.5 McFarland standard (1-2 × 10⁸ CFU/mL).
• Use within 15 minutes of preparation.

1. Plate Inoculation:

• Dip sterile swab into suspension, rotate against tube wall to remove excess.
• Swab entire agar surface uniformly (rotate plate 60° and repeat 2 more times).
• Leave lid ajar for 3-5 minutes to dry.

1. Disc Application:

• Apply discs with sterile forceps or automatic dispenser.
• Place discs ≥24 mm apart (center-to-center).
• Place discs ≥15 mm from plate edge.
• Press gently to ensure contact.
• Do not move discs once placed.

1. Incubation:

• Invert plates within 15 minutes of disc application.
• Incubate at 35±2°C for 16-18 hours (ambient air).

1. Reading Results:

• Examine under reflected light.
• Measure zone diameters (including disc) to nearest mm.
• Measure from back of plate.
• For sulfonamides/trimethoprim: ignore slight growth; measure obvious inhibition.
• For swarming organisms (Proteus): ignore swarming, measure obvious inhibition.

6. Interpretation

• Compare zone diameters to current CLSI or EUCAST breakpoints.
• Categorize as:
• S (Susceptible): Infection likely treatable with standard dose.
• I (Intermediate): May be treatable with higher dose or at specific sites.
• R (Resistant): Not inhibited by achievable concentrations.

7. Quality Control

• Test QC strains weekly:
• E. coli ATCC 25922
• S. aureus ATCC 25923
• P. aeruginosa ATCC 27853
• Verify zone diameters fall within CLSI acceptable ranges.
• Check Mueller-Hinton agar depth (4 mm) and pH (7.2-7.4).
• Store discs properly; check expiration dates.

8. Reporting Results

• Report antibiotic name and interpretation (S/I/R).
• Report critical resistance immediately (MRSA, VRE, ESBL, carbapenem-resistant).
• Include comments for unusual resistance patterns.