Overview
This lecture explains the procedure and purpose of the disk diffusion (Kirby-Bauer) assay for determining bacterial sensitivity to antibiotics.
Purpose of the Disk Diffusion (Kirby-Bauer) Assay
- Used to determine the sensitivity of pathogens to specific antibiotics.
- Helps physicians select effective treatments for infections.
- Each bacterium must be tested separately for accurate results.
Materials and Preparation
- Use Mueller-Hinton agar as the recommended nutrient medium.
- Label the bottom of agar plates with your name, date, and bacterium tested.
- Inoculate the surface thoroughly to achieve a bacterial lawn.
Inoculation Procedure
- Flame the tube and culture mouth before use.
- Dip a sterile cotton swab in the broth culture and remove excess liquid.
- Swab the entire agar surface in one direction, rotate plate 90°, and repeat twice more, ensuring no gaps in coverage.
- Allow the plate to stand for five minutes before proceeding.
Placing Antibiotic Disks
- Use standardized antibiotic-impregnated paper disks for testing.
- Sterilize forceps with alcohol and flame before handling each disk.
- Place five different antibiotic disks evenly spaced on the agar surface.
- Repeat sterilization between disks to avoid cross-contamination.
Incubation and Results
- Incubate the plate upside-down at 35°C for 24-48 hours.
- Antibiotics diffuse from disks and may inhibit bacterial growth.
- Measure zones of inhibition (clear areas) with a ruler on the underside of the plate.
- Compare zone sizes with clinical standards to determine if bacteria are susceptible or resistant.
Disposal and Clean-Up
- Properly discard all used plates to be autoclaved after data recording.
Key Terms & Definitions
- Disk Diffusion (Kirby-Bauer) Method — a test to measure bacterial sensitivity to antibiotics via antibiotic-soaked disks on agar.
- Mueller-Hinton agar — standard growth medium for antibiotic susceptibility testing.
- Zone of inhibition — clear area around an antibiotic disk where bacterial growth is prevented.
- Susceptible — bacteria are effectively inhibited by the antibiotic.
Action Items / Next Steps
- Practice the procedure in the lab following sterile technique.
- Record and interpret zones of inhibition for tested antibiotics.
- Properly discard all biological materials for autoclaving.