Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

The overall goal of this protocol is to examine competition from one bacterial isolate on another in both a quantitative and qualitative manner. This method can help answer key questions regarding investigations into competitive exclusion within bacterial communities. The main advantage of this technique is that it reflects what occurs in a bacterial community.

Invading isolates must be able to survive the inhibitory effects of established isolates for invasion to be successful. Demonstrating the procedure will be Emma Crank, a graduate student from the hospital biology. To begin, prepare brain heart infusion or BHI agar according to the manufacturer's instructions.

Autoclave the agar at 121 degrees Celsius in 15 psi for 20 minutes to sterilize. Pour 15 milliliter aliquots of sterilized BHI agar into sterile petri dishes, and allow the agar to set. Prepare BHI broth according to the manufacturer's instructions, and transfer 10 milliliter aliquots into 28 milliliter glass universal bottles.

Then autoclave the BHI broth for 20 minutes. To prepare vaporizer bottles, begin by dismantling the bottles. Spray a 5%surface active cleaning agent through the vaporizer to eliminate internal contaminants.

Then soak the vaporizer bottle, lid, and vaporizer in the cleaning agent overnight. The following day, dry the vaporizer, lid, and bottles under the sterile airflow hood. Then use 70%ethanol to fill the bottles.

Afterwards, reattach the vaporizer and spray some of the ethanol through the vaporizer. Place the lid on the vaporizer and store it with ethanol inside until use. Prior to use, use sterile BHI broth to aseptically rinse the bottle, and spray the broth through the vaporizer.

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 two safety cabinet lined with absorbent paper, use BHI broth to dilute the culture tenfold to yield approximately four times ten 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. As shown in this figure, the assay results should be observable as a difference in the overlaid competitor strain near the centrally spotted inhibitor-producing strain.

The differences can be full inhibition, partial inhibition, no inhibition, or positive association. This graph compares the zones of inhibition between inhibitor-producing isolates, which can be the result of the amount of antimicrobial produced, the ability of inhibitor to diffuse through the medium, the type of inhibitor produced, or the resistance level of the competitor strain to the inhibitors. This example shows that if the test competitor culture is too dilute, the zone of inhibition will appear larger and with a less clearly defined edge than is typically observed.

If the inhibitor producing strain spot was not fully dried before incubation overnight, the inhibitor strain will spread and not form a completely defined spot. Alternatively, if the competitor strain is not sprayed evenly, this can lead to an uneven spread of bacteria. Finally, if the competitor strain is too concentrated, it may grow on top of the inhibitor strain and can in extreme cases lead to inversion of the competitor inhibitor strain roles.

Once mastered, this technique will take one hour split over four days per isolate pair tested. While attempting this procedure, it's important to remember to spray evenly with good replication between assays. Following this procedure, inhibition data can be overlaid onto taxonomic data about the presence or absence of a bacterial species from a community.

It is then possible to determine if inhibitory effects influence the ability of a species to colonize in an environment. After its development, this technique allows researchers in the field of community microbiomes to explore the effects of trait variance such as anti-microbial peptide prediction in community structure in competitive exclusion. After watching this video, you should have a good understanding of how to compare the ability of one bacterial isolate to competitively inhibit the growth of another in both a qualitative and quantitative manner.

Don't forget that working with aerosolized bacteria can be extremely hazardous, and precautions such as working within a level two safety cabinet and wearing appropriate PPE should always be taken while performing this procedure.