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

The following method is adapted to test competitor strains with inhibitor-producing strains.

NOTE: This protocol involves aerosol generation with bacterial cultures. This can only be performed in a contained space with the implementation of locally approved safety considerations. Spraying of the culture onto the agar plates within a level 2 safety cabinet will minimize contamination of the agar plates and the surrounding environment. This will also protect the researcher from aerosol inhalation of the sprayed bacteria, this is a major concern if the researcher is using containment level 2 organisms. Appropriate personal protective equipment must be worn. The area surrounding the petri dish is best covered with disposable absorbent tissue. Post-spray decontamination using UV light is advised.

1. Preparing the Agar Plates

1. Prepare brain heart infusion (BHI) agar in accordance with the manufacturer's instructions.
2. Autoclave the BHI agar at 121 °C, 15 Pound-force per square inch (psi) for 20 min, or according to the autoclave manufacturer's recommendations, to sterilize.
3. Pour 15 ml aliquots of sterilized BHI agar into sterile petri dishes and allow it to set.
   NOTE: Ensure every agar plate to be used within the assay originates from the same stock of agar and that there is the same amount of agar in each plate. This limits the variability in available nutrients and inhibitor diffusion between plates allowing comparability.

2. Preparing Sterile Broth

1. Prepare BHI broth in accordance with the manufacturer's instructions.
2. Add 10 ml aliquots of BHI to 28 ml glass universal bottles.
3. Autoclave the BHI broth at 121 °C, 15 psi for 20 min, or according to the autoclave manufacturer's recommendations, to sterilize.         
   NOTE: Ensure all broth used in the assay originates from the same batch of broth autoclaved at the same time to limit variation in nutrients which could cause variation in results.

3. Preparing Sterile Vaporizer Bottle(s)

1. Dismantle the vaporizer bottle.
2. Spray 5% surface active cleaning agent through the vaporizer to eliminate internal contaminants, and soak the vaporizer bottle, lid, and vaporizer in the 5% surface active cleaning agent overnight.         
   NOTE: Gloves and appropriate personal protective equipment should be used when handling surface active cleaning agents.
3. Dry the vaporizer, lid, and bottles under a sterile air flow hood.
4. Fill the bottle with 70% ethanol, reattach the vaporizer, and spray some of the ethanol through the vaporizer.
5. Put the lid on the vaporizer and store the ethanol inside until use.
6. Immediately prior to use, aseptically rinse the bottle with sterile BHI broth and spray the broth through the vaporizer.  
   NOTE: This prevents the ethanol from affecting the inoculum added to the bottle in section 6.3.

4. Preparing the Bacterial Overnight Culture(s)

1. Streak the competitor and inhibitor strains onto a sterile BHI agar plate and incubate at 37 °C aerobically for approximately 18 hr (overnight).
   NOTE: This plate produces a stock of bacteria that can be used for multiple replicates within the same week when stored at 4 °C.
2. Inoculate 10 ml of sterile BHI broth with a single colony of the required competitor bacterial strain within a 28 ml universal bottle.
3. Incubate the universal bottle at 37 °C aerobically overnight by shaking at 250 rpm in an orbital shaker.

5. Spot Culture of the Inhibitor-producing Isolate

1. Prepare the overnight culture(s) as described in section 4.
2. Ensure agar plates are completely dry, with no condensation on the lid, prior to inoculation (e.g. by incubating the plate for 60 min at 37 °C); this prevents the inoculum from spreading across the plate from the inoculation site.
3. Pipette 25 µl of the overnight culture onto the center of the agar plates. Avoid completely depressing the pipette plunger to prevent air bubbles that spray culture across the agar.
4. Allow the culture to dry at room temperature to limit the spread of the culture. Incubate the agar plates at 37 °C aerobically overnight.

6. Preparing the Spray Inoculum of the Test Competitor Strain(s)

1. Prepare the overnight culture as described in section 4. Dilute the overnight culture 10-fold in BHI broth to yield approximately 4 x 10⁶  colony forming unit (CFU) ml^-1 and a suspension of OD₆₀₀ 0.3 ±0.05.
   NOTE: A 10-fold dilution will not give 4 x 10⁶ CFU ml^-1 for all bacterial species, calculate the necessary dilution factor by spread plating serial dilutions between 1 x 10^-1-1 x 10^-6.
2. Pour the diluted culture into a sterile plastic perfume vaporizer bottle.
   NOTE: The total volume must be more than is necessary for the number of agar plates being sprayed. Approximately 250 µl per plate is suitable for a 9 cm diameter Petri dish.

7. Deferred Growth Inhibition Assay

1. 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.
2. From a distance of approximately 15 cm above the agar, spray approximately 250 µl of diluted culture (3 sprays of a typical 50 ml vaporizer) over the entire agar surface. Incubate the agar plates at 37 °C aerobically overnight. Repeat for other plates with the same number of sprays.

8. Interpreting Deferred Growth Inhibition Assay Results

1. Measure the growth inhibition zone of the sprayed competitor strain (Figure 1A–C, "X" to "X") in millimeters, subtracting the diameter of the central spot of the inhibitor-producer (Figure 1A–C, "Y" to "Y").         
   NOTE: Figure 1E shows how this data could be presented
2. Record the clarity score for zones of inhibition. 
   NOTE: This indicates the degree of inhibition observed. Examples are shown in Figure 1A–D, strains that can be used for standardization are suggested in the representative results section. The following scale can be used for clarity scoring: a clarity score of 0 to describe a zone of complete inhibition of the competitor strain's growth. A score of 1 describes a zone of almost complete inhibition of growth, with only individual colonies present in the zone of clearing. A score of 2 describes a visible zone of reduced growth, growth within this zone is confluent or near confluent but reduced in density by >50 %. A score of 3 describes only minimal growth reduction, growth is confluent and reduced by less than 50 %, and a zone is still visible and measurable. A score of 4 describes no visible inhibition of growth. A score of 5 describes that the growth of the competitor strain was increased in the proximity of the test inhibitor-producing strain. See Figure 1 for examples. These scores are subjective to the experimenter.