Soft Agar Overlay Assay for Detecting Bacteriocin-Mediated Killing in Pseudomonas syringae

Abstract

Source: Hockett, K. L., and Baltrus, D. A. Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds. J. Vis. Exp. (2017)

This video demonstrates the procedure for detecting bacteriocin-mediated killing by spotting supernatant from an antibiotic-induced donor strain onto a soft agar overlay containing a test strain. Clear zones formed after incubation indicate bacterial inhibition.

Protocol

1. Preparation of Overlay and Testing Supernatants for Activity

1. Inoculate a single colony of a P. syringae strain to be tested for sensitivity into 3 ml of KB (King's medium B), incubate overnight with shaking at room temperature.
2. The following morning, back dilute the culture 1/100 into fresh KB. Incubate 3-4 hr with shaking at room temperature.
3. Prepare sterilized water agar by autoclaving a suspension of 0.35-0.7% (w/v) agar in ultrapure water.
   NOTE: A stock of soft water agar can be melted in a microwave and reused repeatedly; a fresh overlay does not need to be prepared for each experiment. If water agar is reuse....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mitomycin C	Santa Cruz Biotechnology	sc-3514	Carcinogenic. Use appropriate PPE (i.e. gloves, eye protection, and face mask), particularly when preparing the stock solution.
Bacteriological grade agar	Genesee Scientific	20-274