Example 6
Protocol: Place a 0.22 μm filter on center of the BHI agar plate with phenol red and measure the OD600 of overnight cultures of the different Rothia species or other candidate inhibitors. Dilute the bacterial cultures to OD600 0.5, spread them on filter and grow overnight in their respective growth conditions. This prevented bacteria from physically contacting the agar surface. After overnight growth, remove the filter bearing the bacterial cells and overlay the plate with 4 mL of BHI soft agar (0.5% agar) with phenol sred containing 200 uL of OD600 0.5 Sm Immersing the Sm cells in BHI soft agar and covering the surface of the plate with it allowed to separate Sm from the inhibitory bacteria both physically and temporally. When the soft agar solidifies, it was incubated overnight at 37° C. in aerobic +5% CO2 conditions. The next day pH values were taken at the center of the plate and the side of the plate. The experiments were performed in triplicate. The error bars represent the standard deviation and the statistical significance was determined using Studen't T-test.
Results: The Rothia successfully inhibited the acid production of both Sm UA140 and UA159 in this way (
