This study investigates the antibacterial properties of bacteriocin derived from Pseudomonas syringae. The methodology involves inoculating soft agar with a bacterial culture and assessing the effects of bacteriocin on a test strain.
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 milliliters of soft agar to a sterile culture tube. Return the culture tube to the water bath to maintain it in a molten state.
Before pouring the overlay, allow the molten agar to cool, but do not allow it to solidify.
It's critical that the agar is maintained in a hot, completely molten state prior to inoculation. If not, the overlay will have a grainy appearance and will be more difficult to interpret.
In a sterile hood, inoculate 100 microliters of the tester strain culture into the soft agar and vortex to mix. Rotate the culture by hand for 10 to 15 seconds, then pour it onto the bottom of an agar plate. Tilt the plate in all directions to ensure the soft agar evenly covers the bottom agar.
Cover the plate and allow it 20 to 30 minutes to solidify. Be careful not to disturb the plate while solidifying. Once solidified, spot two to five microliters of supernatant onto the overlay.
Allow the plates to incubate overnight at room temperature. Observe and record the results the following morning. Using this method, two strains of P. syringae are inhibited by different bacteriocins. Strain A is sensitive to a high molecular weight bacteriocin, tailocin, but not sensitive to an alternative, low-molecular weight bacteriocin.
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