Assessing the Antimicrobial Potential of Bacteriocin-Producing Murine Fecal Bacterial Isolates

Take mouse fecal suspension comprising lactic acid bacteria, or LAB, that produce bacteriocin, an antimicrobial peptide, and inoculate into a pre-warmed LAB selective soft agar medium.

Pour the medium into an agar plate, forming the first layer.

Overlay a second agar layer and incubate.

The first layer selectively promotes LAB growth, allowing the secreted bacteriocin molecules to diffuse outwards from the colonies into the surrounding medium.

The second layer contains a cell-free medium that allows the diffusion of bacteriocin and prevents the growth of LAB colonies on the surface.

Now, pour a third agar layer containing a bacteriocin-sensitive strain.

The bacteria growing in the third layer encounter the bacteriocin diffusing from the lower layers.

The bacteriocin disrupts the cell membrane of sensitive bacteria, disturbing cellular homeostasis and resulting in cell death, visible as a clear zone on the third layer.

Measure the zone diameter to assess bacteriocin potency.

To count bacteriocin-producing bacterial cells in fecal samples, transfer diluted cells of the bacteriocin-producer to 4 milliliters of pre-warmed MRS soft agar. Mix the agar by vortexing, and pour the cells onto an MRS 1.5% agar plate. This layer is referred to as the first layer.

Transfer another 4 milliliters of cell-free 0.8% MRS soft agar onto the first layer to embed all the cells within the soft agar. This layer is meant to prevent cells from growing as colonies on the surface of the agar plates.

In the sterile hood, dry the plates by taking the lid off for five to 10 minutes before incubating the plates at 30 degrees Celsius for 20 to 24 hours for cell growth and colony formation.

To identify bacteriocin-producing colonies, mix 40 microliters of an overnight culture of an appropriate indicator strain with 4 milliliters of pre-warmed soft agar for each plate. Pour the mixture onto the plates. This layer is referred to as the third layer.

Dry and incubate the plates as before, for cell growth and colony formation. Bacteriocin-producing colonies have clear zones of inhibition around the colonies.