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Microbiology JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/54437-v
What you'll learn
  • ✓ Prepare agar plates, sterile broth, and equipment for deferred growth inhibition assays
  • ✓ Culture inhibitor-producing bacterial isolates and prepare competitor strain inocula
  • ✓ Execute deferred growth inhibition assay and quantify antimicrobial zones of inhibition
  • ✓ Interpret results to assess bacterial competition effects and antimicrobial production
Protocol

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Difficulty
intermediate
Total time
~2–3 days (overnight cultures + incubation periods)
Biosafety
BSL-1

Steps

1
Prepare agar plates, sterile broth, and vaporizer bottles

Prepare all required media and sterile equipment including agar plates, sterile broth, and sterile vaporizer bottles for the assay setup.

▶ 00:45
2
Culture inhibitor-producing isolate overnight

Inoculate sterile broth with the inhibitor-producing bacterial isolate and grow overnight. Spot the culture onto prepared agar plates.

▶ 02:19
3
Prepare spray inoculum of competitor strains

Prepare spray inoculum suspensions of test competitor bacterial strains and apply them by vaporization over the inhibitor-producing isolate colonies on agar plates.

▶ 03:34
4
Quantify zones of inhibition and interpret results

Incubate inoculated plates and measure clearing zones around inhibitor-producing isolates to assess antimicrobial effects on competitor growth.

▶ 04:48
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