Spot-on-Lawn Assay

For the spot-on-lawn assays, 2× AUM was mixed 1:1 (v/v) with sterile, preheated (∼50°C) agar-demi-water solution (30g/L). 1× AUM agar plates were produced using 20 mL of the AUM-agar mixture. A top layer of agar containing the bacteria to be tested was created by mixing 10 mL of the AUM-agar mixture with 0.25 mL of a stationary phase bacterial culture and pouring it on top of a set 1.5× AUM agar plate. Conditioned media was prepared as described above, and 10 μL drops of the conditioned media were dropped on the agar surface in duplicate after the bacterial top layer was poured and let to set. Plates were then allowed to grow overnight at 37°C and the agar plates were inspected for zones of inhibition, caused by the drops of conditioned media. Fresh 1× AUM was dropped in duplicate on the plate as a negative control. Those bacteria that showed a very negative response ε < log(0.1) = −2 to conditioned liquid media were tested to check for inhibition.