Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The overall goal of this procedure is to screen for agents produced by one bacterial strain, that are inhibitory towards the second bacterial strain. This method can help answer questions in a number of microbiological fields, but is particularly useful in microbial ecology, in understanding antagonistic bacterial interactions. The main advantage of this technique is that it’s inexpensive and easy to perform and interpret.

Demonstrating the procedure will be Aretha, a student in the laboratory. To begin the experiment, inoculate a single colony of P syringae into 3 milliliters of King’s Medium B broth, or KB.Incubate the culture overnight. The following morning, dilute the broth culture at 1 to 100 into 3 milliliters of fresh KB broth.

Incubate the culture for three to four hours with shaking at room temperature. Ensure that the culture has a noticeable increase in turbidity, and then add Mitomycin C.Next, incubate the culture overnight with shaking at room temperature. The next day, pellet one to two milliliters of Mitomycin C induced cultures in a bench-top micro centrifuge.

Remove and sterilize the culture supernatants by passing the supernatant through a 0.22 micrometer pore-size filter. Add sodium chloride to reach a one molar concentration, and add polyethylene glycol, or PEG 8000, to a 10%final concentration, to the sterile supernatant. Repeatedly invert the samples until both the sodium chloride and PEG are completely dissolved.

Incubate the samples in an ice bath for one hour. Next, centrifuge the samples. After spinning, decant the supernatant, and re-suspend the pellet in buffer by pipetting repeatedly.

Remove residual PEG by two sequential extractions with an equal volume of chloroform. Combine with chloroform with the re-suspended pellet and vortex the mixture for 15 seconds. Then centrifuge the mixture at 20, 000 times G for five minutes.

Transfer the upper aqueous phase to a fresh microfuge tube. Repeat this extraction two times, until no white interface between the aqueous and organic phase is visible. Allow residual chloroform to evaporate from extracted supernatants in a fume hood.

Inoculate a single colony of a P syringae strain into three milliliters of KB, and incubate the culture overnight with shaking at room temperature. The following morning, 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. Conversely, Strain B is insensitive to the Tailocin, but is sensitive to the low-molecular weight Bacteriocin. The Tailocin is efficiently recovered following PEG precipitation, while lower molecular weight Bacteriocins are not.

Bacteriophage mediated killing is distinguished from other non-replicative anti-microbials by comparing a dilution series. In the case of bacteriophage mediated clearing, dilution of the supernatant resolved into individual plaques, or clearing zones, while Bacteriocin mediated clearing did not resolve into such plaques. This procedure can be used as an initial screen to identify inhibitory agents, and can be combined with downstream genetic manipulations to identify or confirm the source of this activity.