In a disk diffusion experiment, what constitutes a proper set of controls?

In a disk diffusion experiment, what constitutes a proper set of controls?

• A. A plate with no antibiotic disks to assess baseline growth.
• B. A plate with several antibiotic disks but no solvent control to detect diffusion issues.
• C. A plate with a single antibiotic disk and no positive control to benchmark.
• D. A plate with a positive control disk containing a known susceptible agent and a negative control disk with solvent only.

Answer: (D)

The main idea is to verify both that the assay is capable of showing inhibition when it should and that nothing in the setup itself is falsely affecting growth. In disk diffusion, you want a positive control disk containing a known susceptible agent to confirm that the organism and the assay conditions allow a true inhibition zone to form if the agent works. You also want a negative control disk with solvent only to ensure that the solvent or the disk material itself isn’t inhibiting growth or altering diffusion. Together, these controls validate that any observed zones are due to the antibiotic’s activity, not experimental artifacts. Other setups miss one or both of these checks, which can lead to misinterpretation of results.