Source: Moran, J. C. et al., Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition. J. Vis. Exp. (2016)
This video showcases the deferred growth inhibition assay, used to investigate how antimicrobial compounds produced by one type of bacteria affect another competing bacterial strain.
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
2. Preparing Sterile Broth
3. Preparing Sterile Vaporizer Bottle(s)
4. Preparing the Bacterial Overnight Culture(s)
5. Spot Culture of the Inhibitor-producing Isolate
6. Preparing the Spray Inoculum of the Test Competitor Strain(s)
7. Deferred Growth Inhibition Assay
8. Interpreting Deferred Growth Inhibition Assay Results
Figure 1: The range of qualitative and quantitative results that can be obtained from the deferred growth inhibition assay. Results for the growth inhibition can range from (A) complete inhibition (score of 0), (B) partial inhibition (score of 1), (C) partial inhibition (score of 2) to (D) no inhibition (score of 4). Labels indicate the edges of growth of the inhibitor strains (Y) and the outermost edge of the inhibition zones (X). The size of the zone of inhibition range from 0 mm to more than 10 mm, as shown in (E), which shows the measurements for the plates photographed in A, B, C and D. Results are shown for recent staphylococcal isolates (A, C) or common lab staphylococcal isolates S. epidermidis Tü3298 (inhibitor) and S. epidermidis Rp62A (competitor) (B), and S. epidermidis Rp62A (inhibitor) and S. aureus Newman (competitor) (D).
The authors have nothing to disclose.
Brain heart infusion broth | Lab M | lab049 | Rich general purpose media |
Agar-agar | Merck Milipore | 101614 | |
Petri dishes | Sarstedt | 82.1473.001 | |
Plastic perfume vaporizer | not known | – | 10 cm high with 3.5 cm diameter clear plastic spray bottle with lid, purchased from the travel section of a supermarket |
Universal bottles | no longer in production, alternative SLS BOT5006 | 28 ml glass cylindrical bottle (approx 280 mm diameter, 850 mm height), flat bottomed with plastic screw cap | |
Decon 90 | Decon | surface active cleaning agent |