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

Begin with a molten soft agar maintained at a warm temperature.

Inoculate a Pseudomonas syringae culture, the test strain, into the agar.

Then vortex briefly to distribute the cells evenly.

Pour the mixture onto a solidified agar plate and spread it evenly.

Close the lid and allow it to solidify.

Spot a filtered supernatant onto the surface of the solidified agar.

The supernatant contains bacteriocin, an antibacterial molecule, derived from a stress-induced donor P. syringae strain.

Incubate the plate to allow bacterial growth and diffusion of the bacteriocin through the agar.

The bacteriocin binds to specific surface receptors on the test strain and disrupts its membrane, leading to cell death.

This results in the formation of a clear zone on the agar, indicating the area where the test strain has been killed by the antibacterial molecule bacteriocin.

Dilute the culture 1 to 100 into fresh KB. Incubate the culture for three to four hours with shaking at room temperature. Next, prepare sterilized water agar by autoclaving a suspension of agar in ultra-pure water.

Maintain the molten soft agar in a 60 degree Celsius water bath prior to use. Using a sterile serological pipette, transfer three m