Antibiotic Susceptibility Testing (Kirby-Bauer Method)

Antibiotic susceptibility testing (AST) is an essential laboratory technique used to determine the effectiveness of antibiotics against bacterial pathogens. One of the most widely used methods for AST is the Kirby-Bauer disk diffusion method, which measures the zone of inhibition around antibiotic-impregnated disks placed on an agar plate inoculated with a bacterial culture. This technique provides crucial information for selecting the appropriate antibiotic therapy for infections and for monitoring bacterial resistance patterns.

Principle of the Kirby-Bauer Method

The Kirby-Bauer method is based on the diffusion of antibiotics from a paper disk into an agar medium, creating a concentration gradient. When the antibiotic inhibits bacterial growth, a clear zone of inhibition forms around the disk. The size of this zone is measured and compared to standard interpretative criteria to determine whether the bacterial strain is susceptible, intermediate, or resistant to the antibiotic.

Materials and Equipment

To perform the Kirby-Bauer method, the following materials and equipment are required:

• Bacterial culture (grown in broth and adjusted to standard turbidity)

• Mueller-Hinton agar plates (preferred medium for disk diffusion tests)

• Sterile cotton swabs

• Antibiotic-impregnated disks

• Forceps

• Incubator (35-37°C)

• Caliper or ruler for measuring zones of inhibition

Procedure for the Kirby-Bauer Method

1. Preparation of Bacterial Suspension:

   • Pick a bacterial colony and suspend it in sterile saline or broth.

   • Adjust turbidity to 0.5 McFarland standard using a spectrophotometer or visual comparison.

2. Inoculation of Agar Plate:

   • Dip a sterile cotton swab into the bacterial suspension.

   • Evenly spread the culture over the entire surface of a Mueller-Hinton agar plate using a lawn streak technique.

   • Allow the plate to dry for 5 minutes.

3. Application of Antibiotic Disks:

   • Use sterile forceps to place antibiotic-impregnated disks onto the agar surface at appropriate distances.

   • Lightly press each disk to ensure contact with the medium.

4. Incubation:

   • Incubate the plates at 35-37°C for 16-18 hours.

5. Measurement of Zones of Inhibition:

   • After incubation, measure the diameter of the clear zones around each antibiotic disk in millimeters.

   • Compare results to the Clinical and Laboratory Standards Institute (CLSI) guidelines to categorize bacteria as susceptible (S), intermediate (I), or resistant (R).

Interpretation of Results

The measured zone of inhibition is compared to standard reference values provided by the CLSI or EUCAST (European Committee on Antimicrobial Susceptibility Testing).

• Susceptible (S): The bacteria are inhibited by the antibiotic and are likely to respond to treatment.

• Intermediate (I): The bacteria have reduced susceptibility, and treatment may require a higher dose or combination therapy.

• Resistant (R): The bacteria can grow despite the antibiotic, indicating that the drug is ineffective for treatment.

Factors Affecting Kirby-Bauer Test Results

Several factors influence the accuracy of the Kirby-Bauer test:

1. Agar composition and depth – Mueller-Hinton agar must be 4 mm thick to ensure proper diffusion.

2. Inoculum density – Too many bacteria can reduce inhibition zones, leading to false resistance results.

3. Incubation time and temperature – Deviations can alter antibiotic activity and affect results.

4. Disk potency and storage – Antibiotic disks should be stored properly to maintain efficacy.

Applications of the Kirby-Bauer Method

The Kirby-Bauer disk diffusion test is widely used in:

1. Clinical diagnostics – Guides antibiotic selection for patient treatment.

2. Epidemiology and public health – Monitors trends in bacterial resistance.

3. Pharmaceutical research – Evaluates the efficacy of new antibiotics.

4. Quality control – Ensures standardization of antimicrobial testing in laboratories.

Advantages and Limitations

Advantages:

• Simple and cost-effective method.

• Standardized and widely accepted by organizations like CLSI and WHO.

• Applicable to a wide range of bacterial species.

Limitations:

• Does not determine the Minimum Inhibitory Concentration (MIC).

• Not suitable for all bacteria, such as anaerobic or slow-growing organisms.

• Results can be affected by environmental conditions and technical errors.