The disk diffusion method, commonly known as the Kirby-Bauer test, is a foundational technique in clinical microbiology for assessing the effectiveness of antimicrobial agents against bacteria. This standardized test determines whether a specific organism is susceptible or resistant to various antibiotics. By exposing a bacterial culture to antibiotic-impregnated disks on an agar plate, microbiologists observe the resulting growth inhibition. Adherence to a detailed, standardized procedure is absolutely essential for ensuring the accuracy and reproducibility of results, which directly guide patient treatment decisions.
Preparing the Materials and Inoculum
The integrity of the disk diffusion test begins with the preparation of the growth medium and the bacterial suspension. Mueller-Hinton Agar (MHA) serves as the standard medium due to its consistent composition and properties, which allow for reliable diffusion of the antimicrobial compounds. The agar’s pH is carefully maintained within a narrow range of 7.2 to 7.4, as deviations can compromise the potency of certain antibiotics. For instance, some drugs appear less active at a lower pH. The depth of the MHA in the plate is also a factor, ideally maintained at approximately 4 millimeters, which influences the rate and extent of antibiotic diffusion. MHA is specifically chosen because it is low in substances that might inhibit the action of certain antimicrobial classes, such as sulfonamides and trimethoprim. This control over the growth environment is necessary to prevent false results.
Preparing the bacterial culture involves creating a standardized inoculum, which is a suspension of the organism in a liquid, typically sterile saline or broth. This suspension must match the turbidity of a 0.5 McFarland standard, a chemical reference solution that represents a bacterial density of roughly \(1.5 \times 10^8\) colony-forming units per milliliter. Proper inoculum density is essential; a suspension that is too light can lead to falsely susceptible results, while one that is too heavy may cause zones of inhibition to be smaller, suggesting false resistance. To achieve this standardization, colonies are selected from a fresh, pure culture and suspended until the visual turbidity matches the 0.5 McFarland standard. The final, adjusted inoculum must be used promptly, typically within 15 minutes of preparation, to ensure the bacterial concentration remains stable and viable for the test.
Executing the Plating and Disk Placement
The physical application of the prepared inoculum and the antibiotic disks requires precise technique. A sterile cotton swab is dipped into the standardized bacterial suspension, and excess liquid is removed by pressing and rotating the swab firmly against the inside wall of the tube. The goal is to create a dense, uniform layer of bacteria, often referred to as a “confluent lawn,” across the entire surface of the MHA plate. The inoculation is performed by swabbing the entire agar surface in one direction, then rotating the plate approximately 60 degrees and swabbing again, followed by a third swabbing after another 60-degree rotation. This three-directional technique ensures even distribution of the organisms, which is foundational for uniform growth.
Once the plate is inoculated, the antimicrobial disks must be placed onto the agar surface quickly, ideally within 15 minutes of swabbing. This rapid placement prevents the antibiotics from pre-diffusing into the agar before the inoculum has had a chance to dry completely and the bacteria can establish growth. The disks, impregnated with a specific concentration of antibiotic, are placed using sterile forceps or a dispenser, ensuring they are evenly spaced to prevent the zones of inhibition from overlapping. Each disk must be gently pressed onto the agar to ensure complete contact. Disks should never be repositioned once they have touched the surface, as this would compromise the integrity of the initial diffusion point. The plates are inverted and transferred to an incubator. Standard incubation conditions are \(35^{\circ}\text{C}\) for 16 to 18 hours in an ambient air atmosphere.
Measuring and Interpreting Results
Following the incubation period, the plates are examined for the presence of a zone of inhibition, which is a clear, circular area around the disk where bacterial growth has been completely suppressed. This zone represents the area where the antibiotic concentration was high enough to inhibit the organism’s growth. If no clear area surrounds the disk, the organism is considered resistant. The diameter of the zone of inhibition is measured in millimeters using a ruler or a caliper, measuring from one edge of the zone across the disk to the opposite edge. It is important to measure the entire diameter, including the 6-millimeter paper disk itself. For some organisms or antibiotic combinations, only the diameter of the completely clear zone is measured, ignoring any faint or hazy inner zone.
The measured zone diameter is compared against established interpretive standards, such as those provided by the Clinical and Laboratory Standards Institute (CLSI). These standardized charts contain specific zone diameter breakpoints for each antibiotic-organism combination. Based on the measured diameter, the organism is categorized into one of three clinical classifications: Susceptible (S), Intermediate (I), or Resistant (R). A Susceptible result suggests the infection should respond to standard doses of the antibiotic. An Intermediate result indicates the antibiotic may be effective at higher doses or in body sites where the drug concentrates. A Resistant classification means the organism will not be inhibited by the antibiotic at clinically achievable concentrations, making the drug unsuitable for treatment.
Ensuring Accuracy Through Quality Control
Maintaining the reliability of the disk diffusion test requires a rigorous quality control program to ensure that all materials and procedural steps are functioning as expected. This process involves the regular testing of standardized control strains alongside the patient isolates. Specific organisms, such as Escherichia coli and Staphylococcus aureus, are used because their susceptibility to various antibiotics is well-established and monitored. When these control strains are tested, the resulting zone diameters must fall within a defined, acceptable range to validate the batch of media, the potency of the antibiotic disks, and the overall test procedure. If the control strain results are outside the acceptable limits, the test run is considered invalid, and the procedure must be investigated and repeated. Monitoring the incubator temperature to ensure it remains at the required \(35^{\circ}\text{C} \pm 2^{\circ}\text{C}\) is also a daily quality check. The stability of the antimicrobial disks is maintained through proper storage; they should be kept frozen, typically at \(-20^{\circ}\text{C}\), to preserve the potency of the antibiotic compound. Every new batch of Mueller-Hinton Agar or newly received antibiotic disks must be tested with the control strains before being used for patient samples.