A Deferred Growth Inhibition Assay to Evaluate the Effect of Bacteria-Derived Antimicrobial Compounds

Begin with a nutrient-containing agar plate and place a drop of an overnight culture of inhibitor-producing primary bacteria at the center.

Allow the culture to dry, ensuring minimum spreading of bacteria, and incubate.

Individual bacteria form distinct colonies and secrete antimicrobial compounds — inhibitor molecules that diffuse through the agar to a certain distance.

Spray a diluted culture of a competitor bacterial strain evenly onto the agar surface that contains colonies of primary bacteria.

During incubation, the antimicrobial compounds interact with the competitor bacteria and disrupt their essential cellular processes.

This results in the death of competitor bacteria in that specific area, causing a significant decline in bacterial growth.

In the area without antimicrobial compounds, competitor bacteria utilize the nutrients and form distinct colonies.

This creates a differential growth pattern with a distinct zone of inhibition. Determine the diameter of this zone and zone clarity to evaluate the effect of bacteria-derived antimicrobial compounds.