The disk diffusion assay is a foundational laboratory test used to determine the effectiveness of an antimicrobial agent, typically an antibiotic, against a specific microorganism like bacteria. This technique provides microbiologists with a quick and relatively simple method to assess microbial susceptibility, which is the organism’s sensitivity to a drug. The results of this assay inform clinical decisions by predicting whether a particular antibiotic will successfully treat a patient’s infection. The simplicity and reliability of this method have made it one of the most widely used tests for initial antimicrobial susceptibility screening around the world.
Setting Up the Assay
The disk diffusion assay, often standardized as the Kirby-Bauer method, requires meticulous preparation to ensure reproducible results. The first step involves preparing a standardized bacterial suspension from a pure culture of the microorganism being tested. This suspension is adjusted to a specific turbidity, usually matching the 0.5 McFarland standard, which ensures a uniform concentration of approximately \(1.5 times 10^8\) colony-forming units per milliliter (CFU/mL) of bacteria.
The standardized bacterial suspension is then used to create a confluent “lawn” of growth on the surface of a specialized growth medium called Mueller-Hinton agar. This agar is selected because it is non-selective and non-differential, allowing a wide range of organisms to grow, and its loose consistency facilitates the proper diffusion of the antibiotic. The inoculation is performed by streaking the entire agar surface multiple times to ensure the bacteria are evenly distributed across the entire plate.
Once the bacterial lawn is established, small, sterile paper disks impregnated with a known concentration of a specific antibiotic are placed onto the agar surface. As soon as the disks touch the agar, the antibiotic compound immediately begins to diffuse radially outward, creating a concentration gradient in the surrounding medium. The plates are then incubated, typically at 35°C, for 16 to 24 hours, allowing the bacteria to grow and the antibiotic to inhibit growth where it is effective.
Measuring Antimicrobial Effectiveness
The effectiveness of the antibiotic is determined by observing the resulting “zone of inhibition,” which is a clear, circular area around the disk where bacterial growth has been prevented. The antibiotic’s concentration decreases as it diffuses further from the disk, and the edge of the zone represents the point where the drug concentration is no longer sufficient to stop the bacteria from growing. A larger zone indicates that a lower concentration of the antibiotic was sufficient to inhibit the organism, suggesting greater susceptibility.
To interpret the result, the diameter of the zone of inhibition is measured in millimeters (mm), including the diameter of the disk itself. This measurement must be compared against established interpretive criteria, known as “breakpoints,” published by regulatory bodies like the Clinical and Laboratory Standards Institute (CLSI). These breakpoints are determined by correlating the zone size with the Minimum Inhibitory Concentration (MIC) and the drug concentrations achievable in a patient’s body.
The comparison translates the measured zone size into one of three clinical categories: Susceptible (S), Intermediate (I), or Resistant (R). A Susceptible result indicates a high likelihood of therapeutic success with standard dosing. An Intermediate result suggests that the organism’s growth is inhibited, but therapeutic success is less predictable, often requiring increased drug exposure or concentration at the site of infection. A Resistant result means the bacteria are not inhibited by drug concentrations that are safe and achievable in the patient, predicting a high likelihood of treatment failure.
Clinical Relevance and Public Health
The results of the disk diffusion assay directly influence patient care by guiding clinicians in selecting the most appropriate antibiotic treatment for an infection. Identifying which antibiotics an isolated pathogen is susceptible to allows for targeted therapy, improving patient outcomes and avoiding the use of ineffective drugs. Obtaining these results quickly is a significant benefit, as it reduces the time a patient may spend on a broad-spectrum or incorrect antibiotic.
Beyond individual patient management, this assay provides data for tracking antimicrobial resistance (AMR). When numerous isolates show resistance to a drug, it signals the emergence or spread of a resistant strain within a hospital or community. This epidemiological data is compiled to inform public health policies, drive antibiotic stewardship programs, and guide the development of new infection control strategies. The ongoing use and standardization of the disk diffusion method remain an important component in the global effort to curb the threat of antibiotic resistance.