Disk Diffusion Assay Guide: Accurate Results

The disk diffusion assay, also known as the Kirby-Bauer test, is a widely used method for assessing the antimicrobial susceptibility of microorganisms. This technique involves placing a paper disk impregnated with a known concentration of an antimicrobial agent on an agar plate inoculated with the test microorganism. The zone of inhibition, which is the area around the disk where the microorganism does not grow, is measured to determine the susceptibility of the microorganism to the antimicrobial agent.

Principle of the Disk Diffusion Assay

The disk diffusion assay is based on the principle that antimicrobial agents will diffuse from the paper disk into the surrounding agar, creating a concentration gradient. The microorganism will grow in the areas where the concentration of the antimicrobial agent is below its minimum inhibitory concentration (MIC), but will be inhibited in areas where the concentration is above its MIC. The size of the zone of inhibition is directly proportional to the susceptibility of the microorganism to the antimicrobial agent.

Materials Needed

To perform a disk diffusion assay, the following materials are needed:

• Agar plates (Mueller-Hinton or similar)
• Paper disks (6-8 mm in diameter)
• Antimicrobial agents (in the form of powders or solutions)
• Microorganism to be tested (in the form of a broth culture or agar slant)
• Inoculation loop or swab
• Incubator
• Measuring device (e.g. caliper or ruler)

Step-by-Step Procedure

1. Preparation of the Agar Plate: The agar plate is prepared by pouring a suitable volume of melted agar into a sterile petri dish. The agar is allowed to solidify, and then the plate is inoculated with the test microorganism using an inoculation loop or swab.
2. Application of the Paper Disk: A paper disk impregnated with a known concentration of the antimicrobial agent is placed on the surface of the agar plate, using a pair of sterile forceps.
3. Incubation: The plate is incubated at a suitable temperature (usually 35-37°C) for a specified period of time (usually 16-24 hours).
4. Measurement of the Zone of Inhibition: After incubation, the plate is removed from the incubator and the zone of inhibition is measured using a caliper or ruler. The measurement is taken from the edge of the disk to the edge of the zone of inhibition.

Interpretation of Results

The results of the disk diffusion assay are interpreted by comparing the size of the zone of inhibition to a set of established criteria. The criteria are based on the MIC of the antimicrobial agent and the susceptible or resistant status of the microorganism. The following interpretation is commonly used:

• Susceptible: A zone of inhibition of ≥20 mm indicates that the microorganism is susceptible to the antimicrobial agent.
• Intermediate: A zone of inhibition of 15-19 mm indicates that the microorganism has an intermediate susceptibility to the antimicrobial agent.
• Resistant: A zone of inhibition of ≤14 mm indicates that the microorganism is resistant to the antimicrobial agent.

Factors Affecting the Accuracy of the Disk Diffusion Assay

Several factors can affect the accuracy of the disk diffusion assay, including:

• Inoculum size: The size of the inoculum can affect the size of the zone of inhibition. A larger inoculum can result in a smaller zone of inhibition.
• Temperature: The temperature at which the plate is incubated can affect the growth of the microorganism and the diffusion of the antimicrobial agent.
• pH: The pH of the agar can affect the activity of the antimicrobial agent and the growth of the microorganism.
• Agar composition: The composition of the agar can affect the diffusion of the antimicrobial agent and the growth of the microorganism.

Troubleshooting Common Problems

Common problems that can occur during the disk diffusion assay include:

• Contamination: The plate can become contaminated with other microorganisms, resulting in a faulty zone of inhibition.
• Inadequate inoculum: The inoculum may not be sufficient to produce a clear zone of inhibition.
• Incorrect incubation temperature: The plate may not be incubated at the correct temperature, resulting in abnormal growth or diffusion of the antimicrobial agent.

To troubleshoot these problems, it is essential to follow a standardized protocol and to use quality control strains to ensure the accuracy of the results.

Conclusion

The disk diffusion assay is a widely used and effective method for assessing the antimicrobial susceptibility of microorganisms. However, it is essential to follow a standardized protocol and to use quality control strains to ensure accurate and reliable results. By understanding the principles of the disk diffusion assay and by following the step-by-step procedure, microbiologists can accurately determine the susceptibility of microorganisms to antimicrobial agents and provide valuable information for the treatment of infectious diseases.