By Elliot Walsh, Updated Mar 24, 2022

Bacteria are the planet’s most prolific organisms, with over one trillion species and more than five million trillion trillion individuals. Though less than 1 % of these microbes cause disease in humans, they can range from mild stomach upset to life‑threatening infections like the bubonic plague, which killed an estimated 50 million people in the 14th century.

Antibiotics and Their Mechanisms of Action

Antibiotics selectively target bacterial structures and processes, sparing human cells. Common classes include:

• Beta‑lactams (e.g., penicillin) disrupt cell‑wall synthesis, leading to bacterial lysis.
• Macrolides (e.g., erythromycin) bind bacterial ribosomes, blocking protein synthesis.
• Quinolones interfere with DNA replication by inhibiting gyrase and topoisomerase IV.

Testing Antibiotic Resistance

Since the 1920s, bacteria have evolved resistance, prompting the development of standardized susceptibility assays. Early methods involved serial dilutions of bacterial cultures on antibiotic‑laden plates, which were time‑consuming and variable.

The Kirby‑Bauer Disk Diffusion Test

Standardized by microbiologists W. M. M. Kirby and A. W. Bauer, the Kirby‑Bauer test remains the gold standard for rapid, reliable resistance screening. The procedure is straightforward:

1. Streak a pure bacterial isolate onto an agar plate.
2. Place antibiotic‑laden disks on the surface; each disk contains a specific antibiotic.
3. Incubate the plate under optimal conditions.

As the antibiotic diffuses outward, susceptible bacteria are inhibited, creating a clear, circular area of no growth around each disk—known as the zone of inhibition. The size of this zone directly reflects bacterial sensitivity: larger zones indicate greater susceptibility; smaller zones suggest resistance.

Measuring the Zone of Inhibition

Accurate measurement is critical for interpreting results. Follow these steps:

1. Place the agar plate on a non‑reflective surface.
2. Using a millimeter ruler or caliper, position the zero mark at the center of the antibiotic disk.
3. Measure from the disk center to the outer edge of the inhibition zone (radius). Multiply by two to obtain the diameter, or measure directly across the zone (diameter).

All measurements should be recorded in millimeters (mm).

Use the CLSI reference tables to interpret the diameter relative to bacterial species and antibiotic. These charts can be found here.