The disk diffusion assay is a laboratory procedure designed to evaluate how effectively antimicrobial agents inhibit bacterial growth. This technique is widely used to determine if a specific bacterial strain is susceptible to various antibiotics. By assessing bacterial susceptibility, the assay helps guide appropriate treatment decisions for bacterial infections.
How the Test is Performed
Performing a disk diffusion assay, often following the Kirby-Bauer method, begins with preparing an agar plate. Mueller-Hinton agar is commonly used because it supports the growth of many non-fastidious organisms and allows for consistent diffusion of antimicrobial agents. The agar is poured into Petri dishes to a uniform depth and allowed to solidify.
A bacterial suspension is prepared from a pure culture of the microorganism to be tested. This suspension is adjusted to a standardized turbidity. A sterile cotton swab is then dipped into this standardized suspension, and excess liquid is removed by pressing the swab against the inside wall of the tube.
The inoculated swab is then used to streak the entire surface of the agar plate, rotating the plate several times to ensure a uniform “lawn” of bacterial growth. After the inoculum has dried, paper disks impregnated with specific concentrations of different antimicrobial agents are carefully placed onto the agar surface. These disks must be evenly spaced to prevent overlapping zones of inhibition. The plates are then incubated at a controlled temperature for 18 to 24 hours.
Interpreting the Results
After the incubation period, the agar plates are examined for zones of inhibition around each antibiotic disk. A zone of inhibition is a clear circular area where bacterial growth has been prevented or killed by the antimicrobial agent diffusing from the disk. The presence of such a zone indicates that the bacteria are sensitive to that antibiotic, while its absence suggests resistance.
To interpret the results, the diameter of each zone of inhibition is measured in millimeters. These measured diameters are then compared to standardized interpretive charts, such as those provided by the Clinical and Laboratory Standards Institute (CLSI). These charts define specific breakpoints for each antibiotic, categorizing the bacterial isolate as “susceptible,” “intermediate,” or “resistant.”
A “susceptible” result indicates that the infection is likely to respond to treatment with the recommended dosage of that antibiotic. An “intermediate” result suggests that the antimicrobial agent may be effective in body sites where it can be concentrated, or if a higher-than-normal dosage can be safely administered. A “resistant” classification means the bacteria are not inhibited by the achievable concentrations of the antibiotic, and the treatment is unlikely to be effective.
Importance of the Assay
The disk diffusion assay is important in clinical microbiology and public health. It provides valuable information that guides clinicians in selecting the most appropriate antibiotic to treat bacterial infections. This guidance helps optimize therapeutic outcomes and minimizes the use of broad-spectrum antibiotics, which can contribute to antibiotic resistance.
Beyond individual patient care, the assay is also instrumental in monitoring patterns of antibiotic resistance over time within a community or hospital setting. This surveillance data is crucial for public health initiatives, informing policies and antibiotic stewardship programs aimed at preserving the effectiveness of existing antimicrobial drugs. The simplicity, cost-effectiveness, and broad applicability of the disk diffusion method make it a widely adopted technique globally.