Understanding the Disk Diffusion Method

Dec 5, 2024

Biology Lecture: Disk Diffusion Method / Kerby-Bauer Test

Introduction

  • The Disk Diffusion Method, also known as the Kerby-Bauer test, assesses the effectiveness of antibiotics.
  • Utilizes antibiotic discs, small filter paper-like discs soaked in antibiotic solutions, placed on an agar plate.
  • Aimed at determining which antibiotics are effective or ineffective against specific bacteria.

Antibiotic Resistance

  • Antibiotic resistance varies between bacterial species and strains due to genetic differences.
  • Some strains may acquire antibiotic-resistant genes, leading to differences in resistance and effectiveness.

Standardization

  • The test is highly standardized to ensure consistent results across different labs and conditions.
  • Steps must be followed precisely to compare results globally.

Method Steps

  1. Prepare Bacterial Suspension
    • Bacteria suspended in liquid culture: 150 million cells/mL concentration.
  2. Inoculate Agar Plate
    • Use Mueller Hinton agar at pH 7.2 to 7.4.
    • Form a "lawn" by evenly spreading bacteria over the plate.
  3. Add Antibiotic Discs
    • Use a dispenser to place discs on the agar surface.
    • Discs represent different antibiotics (e.g., penicillin, streptomycin).
    • Blank discs can be used for custom antibiotics.
  4. Tap Discs with Sterile Forceps
    • Ensures discs adhere properly.
    • Sterilize forceps with alcohol and flame.
  5. Incubation
    • Incubate plates upside down at 37°C overnight.
    • Prevents condensation from affecting the bacteria.
  6. Measure Zones of Inhibition
    • Use a ruler to measure the diameter of inhibition zones.
    • Record as 0 mm if bacteria grow up to the disc.
  7. Assess Sensitivity with Reference Table
    • Reference table considers antibiotic type, concentration, and inhibition zone.
    • Determines if the antibiotic is ineffective, effective, or very effective.

Zones of Inhibition

  • Larger zones indicate more effective antibiotics.
  • Complete bacterial growth up to the disc signifies resistance.

Additional Resources

  • Check out additional videos on antibiotic resistance and other diagnostic tests (e.g., catalase, coagulase, oxidase tests).

Conclusion

  • The disk diffusion method is a standardized test essential for understanding antibiotic effectiveness and resistance patterns in bacteria.