The Kirby-Bauer test, officially known as the disk diffusion susceptibility test, is a standardized laboratory procedure used to determine if a specific microorganism is susceptible or resistant to various antimicrobial agents. This technique is fundamental in clinical settings because it guides healthcare providers in selecting the most effective antibiotic to treat a patient’s infection. The method relies on the principle of antibiotic diffusion into a bacterial lawn to create measurable areas of growth inhibition.
Essential Materials and Preparation Standards
The foundation of the Kirby-Bauer test rests on the use of specialized, quality-controlled materials, beginning with the culture medium. Mueller-Hinton Agar (MHA) is the standardized medium for this test because its composition allows for consistent and reliable antibiotic diffusion. The agar is a “loose” medium, which facilitates the movement of the antimicrobial agents, and it contains starch that absorbs bacterial toxins. Furthermore, MHA is formulated to be low in certain inhibitors, ensuring that the test results accurately reflect the antibiotic’s true efficacy against the organism.
Before inoculation, the bacterial suspension must be carefully standardized to ensure a uniform concentration of organisms across all tests. A pure culture of the isolated bacteria is suspended in a sterile saline solution until its turbidity, or cloudiness, visually matches the 0.5 McFarland standard. This standard represents a specific bacterial density that yields a uniform bacterial growth, often called a “lawn.” This standardization is critical for accurate results, requiring adjustment if the suspension is too dense or too light.
The other primary materials needed are the antimicrobial disks, which are small filter paper disks impregnated with a precise, known concentration of a specific antibiotic. Sterile cotton swabs are used to inoculate the agar, and forceps or a disk dispensing apparatus are required to place the disks onto the agar surface. These materials, along with the standardized bacterial suspension, must be prepared and used within a short timeframe, usually within 15 minutes of the suspension’s preparation, to maintain the organism’s viability and concentration.
The Execution Sequence
The procedural sequence begins with the inoculation of the Mueller-Hinton agar plate using the standardized bacterial suspension. A sterile cotton swab is dipped into the suspension, and excess liquid is removed by pressing the swab against the inside wall of the tube. The goal is to create a confluent lawn of growth by streaking the entire surface of the agar plate three times. The plate is rotated approximately 60 degrees after each pass to ensure complete and uniform coverage.
After the entire plate surface has been inoculated, the lid is left slightly ajar to allow the agar surface to dry for about three to five minutes, but no more than 15 minutes. This drying period permits the absorption of excess surface moisture, which prevents the antibiotic disks from floating and ensures proper drug diffusion into the medium. The antibiotic disks are then dispensed onto the dried agar surface, maintaining a minimum distance of 24 millimeters between the disks to avoid overlapping zones of inhibition.
Each disk must be gently pressed onto the agar to ensure complete contact, as the antibiotic begins to diffuse almost immediately upon contact with the moist medium. The plate is then inverted and transferred to an incubator set to a standardized temperature, typically 35°C. The incubation period is strictly controlled, usually lasting 16 to 18 hours, to allow for sufficient bacterial growth and antimicrobial diffusion before the results are read.
Analysis and Result Interpretation
Following the incubation period, the result is visible as a uniform layer of bacterial growth across the plate, interrupted by circular, clear areas around the disks where the antimicrobial agent prevented growth. This clear area is called the Zone of Inhibition, and its size is directly related to the organism’s susceptibility to the antibiotic. The diameter of each zone, including the diameter of the disk itself, is measured in millimeters using a ruler or sliding caliper.
Accurate measurement is critical and involves measuring the diameter of the clear zone from edge to edge, typically using a ruler or sliding caliper. Once the zone diameter is measured for each antibiotic, the value is compared against established tables, such as those published by the Clinical and Laboratory Standards Institute (CLSI). These standardized tables provide the specific breakpoint values for each antibiotic-organism combination, translating the measurement into a clinical category.
The final interpretation categorizes the microorganism as Susceptible (S), Intermediate (I), or Resistant (R) to the tested drug. A Susceptible result indicates that the infection should respond to the antibiotic dosage. Conversely, a Resistant result suggests the drug will likely be ineffective against the organism. The Intermediate category signifies that the antibiotic may be effective only at higher doses or in specific body sites where the drug concentrates.