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

1. LAB Counting and Bacteriocin Activity

1. Weigh one fecal pellet of each sample in a 1.5 mL tube and add an adequate volume of ice-cold 0.9% NaCl to achieve a 10% (w/v) solution. Use sterile micro pestles for 1.5 mL tubes to homogenize the fecal suspension and prepare ten-fold serial dilutions in ice-cold 0.9% NaCl.
2. Count the total number of LAB cells.
   1. Transfer diluted cells (100 µL) to 4 mL of prewarmed Man Rogosa Sharpe (MRS) soft agar (50 °C, 0.8% agar). Mix by vortexing before pouring the cells onto a solid MRS agar plate (1.5% agar).
   2. Dry the plate by taking the lid off for 5 – 10 min in the sterile hood before incubating the cells for 20 – 24 h at 30 °C for cell growth and colony formation.
3. Count bacteriocin-producing cells.
   1. Transfer diluted cells (100 µL) of the bacteriocin producer to 4 mL of prewarmed MRS soft agar (50 °C, 0.8% agar), mix by vortexing, and pour the cells onto an MRS agar plate (1.5% agar); this layer is referred to as the first layer.
   2. Transfer another 4 mL of cell-free MRS soft agar (0.8% agar) onto the first layer to embed all cells within the soft agar.
      NOTE: This layer is meant to prevent cells from growing as colonies on the surface of the agar plates. Cells growing on the surface are easily displaced when pouring indicator cells on the top (step 4.3.4) and will, therefore complicate the interpretation of results. This layer is referred to as the second layer.
   3. Dry the plate by taking the lid off for 5 – 10 min in the sterile hood before incubating for 20 – 24 h at 30 °C for cell growth and colony formation.
   4. To identify bacteriocin-producing colonies, mix 40 µL of an overnight culture of an appropriate indicator strain, with 4 mL of prewarmed soft agar. Pour the mixture onto the plate; this layer is referred to as the third layer.
   5. Dry the plate by taking the lid off for 5 – 10 min in the sterile hood before incubating for 20 – 24 h at 30 °C for cell growth and colony formation.
      NOTE: Bacteriocin-producing colonies have clear (inhibition) zones around the colonies (Figure 1).