The Kirby-Bauer technique is a standard microbiological method used to determine the susceptibility of bacteria to antimicrobial agents. It involves measuring the zone of inhibition of bacterial growth around antibiotic-impregnated disks placed on an agar plate inoculated with the test organism. Here are the general steps of the Kirby-Bauer technique:
1. Preparation of Inoculum:
- A pure culture of the test bacterial strain is obtained and grown in a suitable broth culture medium to achieve logarithmic growth phase (turbidity).
- The bacterial suspension is standardized to a specific turbidity, typically equivalent to a 0.5 McFarland turbidity standard, to ensure a consistent inoculum density.
2. Inoculation of Agar Plate:
- Sterile agar plates containing Mueller-Hinton agar or another appropriate agar medium are used for the test.
- Using a sterile cotton swab, the standardized inoculum is evenly spread over the surface of the agar plate to create a confluent lawn of bacterial growth.
- The inoculated plate is allowed to dry for a few minutes to absorb excess moisture.
3. Application of Antibiotic Disks:
- Sterile antibiotic disks containing specific concentrations of antimicrobial agents are placed onto the surface of the agar plate at evenly spaced intervals using sterile forceps or a disk dispenser.
- The disks are gently pressed to ensure good contact with the agar surface without damaging the disks.
4. Incubation:
- The inoculated plate with antibiotic disks is incubated at an appropriate temperature (usually 35-37°C) for a specified period (typically 16-24 hours) to allow for bacterial growth and the diffusion of antimicrobial agents.
5. Measurement of Inhibition Zones:
- After incubation, the plates are examined for the presence of zones of inhibition around the antibiotic disks.
- Using a ruler or a digital caliper, the diameter of each zone of inhibition is measured in millimeters.
- The zone diameter indicates the susceptibility of the test organism to the specific antibiotic.
6. Interpretation of Results:
- The measured zone diameters are compared to standardized tables or guidelines, such as those provided by the Clinical and Laboratory Standards Institute (CLSI), to determine the susceptibility or resistance of the test organism to the antimicrobial agents tested.
- Organisms are categorized as susceptible, intermediate, or resistant based on the size of the inhibition zones and established interpretive criteria.
7. Quality Control:
- To ensure the accuracy and reliability of the Kirby-Bauer technique, quality control strains with known susceptibility patterns are included in each batch of testing to monitor the performance of the method and the reagents used.
It's important to follow standardized procedures and interpret results according to established guidelines to obtain reliable and clinically relevant information from Kirby-Bauer antibiotic susceptibility testing.