This study investigates the antimicrobial effects of inhibitor-producing primary bacteria on competitor bacterial strains using a deferred growth inhibition assay. By measuring the zones of inhibition, researchers can evaluate the efficacy of antimicrobial compounds secreted by the primary bacteria.
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.
To prepare bacterial overnight cultures, streak the competitor and inhibitor strains onto a sterile BHI agar plate and incubate at 37 degrees Celsius aerobically for approximately 18 hours. In a 28-milliliter universal bottle, inoculate 10 milliliters of sterile BHI broth with a single colony of the required competitor bacterial strain.
Incubate the culture in a universal orbital shaker at 37 degrees Celsius and 250 RPM overnight. The following day, ensure the agar plates are completely dry with no condensation on the lid to prevent the inoculation from spreading on the plate. Pipette 25 microliters of the overnight culture onto the center of the agar plates, avoiding completely depressing the pipette plunger to prevent air bubbles that spray the culture across the agar.
Allow the culture to dry at room temperature to limit the spread of the culture. Then, incubate the agar plates at 37 degrees Celsius aerobically overnight. After growing an overnight culture while working under a level 2 safety cabinet lined with absorbent paper, use BHI broth to dilute the culture tenfold to yield approximately 4 times 10 to the sixth CFU per milliliter. Then, pour the diluted culture into a sterile plastic perfume vaporizer bottle.
To carry out a deferred growth inhibition assay, spray the culture through each of the vaporizer bottles to ensure that the culture is loaded in the spray mechanism prior to spraying onto the agar. From a distance of approximately 15 centimeters above the agar, spray approximately 250 microliters of diluted culture over the entire agar surface. Incubate the plates at 37 degrees Celsius aerobically overnight.
To interpret deferred growth inhibition, measure the growth inhibition zone of the sprayed competitor strain in millimeters, subtracting the diameter of the central spot of the inhibitor producer. Finally, record the clarity score for zones of inhibition according to the text protocol.
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