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 reused, it is critical to ensure it is completely melted following microwaving. If not, the overlay will have a grainy texture upon solidifying that will make interpretation difficult.
4. Prior to use, maintain the molten soft agar in a 60 °C water bath. Using a sterile serological pipette, transfer 3 ml of soft agar to a sterile culture tube. Return the culture tube to the water bath to maintain the molten state.
5. To pour the overlay, first allow the molten agar to cool (it should feel warm but not hot to touch), but do not allow it to solidify.
6. In a sterile hood, inoculate 100 µl of the tester strain culture into the soft agar and vortex to mix. Rotate culture by hand for 10-15 sec, then pour onto a bottom agar (KB agar). Tilt the plate in all directions to ensure the soft agar evenly covers the bottom agar.
   NOTE: The bottom agar can be any solidified (1.5% agar) medium on which the test strain grows vigorously. For a standard 100 mm diameter Petri dish, use ~20 ml of melted medium. The bottom agar can be prepared several weeks ahead of time (if maintained at 4 °C without drying) or can be prepared the same day.
7. Cover the plate and allow it to solidify for 20- 30 minutes. Be careful not to disturb the plate while it solidifies.
8. Once solidified, spot 2-5 µl of supernatant onto the overlay. Allow the plates to incubate overnight at room temperature. Observe and record results the following morning.
   NOTE: It may be useful to perform and spot from a serial dilution of the supernatant. This will allow researchers to distinguish between bacteriophage and bacteriocin clearing activity. In this case, it is recommended to perform 1:5 or 1:10 dilutions.

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