To analyze the samples using SERS, the 200 μL output of the sample preparation process was transferred to a sterile 1.5 mL microcentrifuge tube, rinsed with 200 μL of TSB and spun down. The supernatant was aspirated, and 1 mL of fresh TSB was added. The tubes were left open, but covered with Parafilm M (VWR), and incubated for 5 h at 37 °C on an orbital shaker at 160 rpm. Once the incubation time was complete, the Parafilm was removed and the samples were spun down at 4500 × g for 3 min. The supernatant was removed and 1 mL of 1 mM sodium diphosphate buffer (pH 6; Sigma-Aldrich) was used to resuspend the pellets. Sodium diphosphate buffer was found to generate the SERS signal faster for certain species than washing with water.61 (link) The samples were spun down and the rinsing process was repeated 3×. Upon completion of the third centrifugation step, the supernatant was removed to leave approximately 10 μL of sample for SERS analysis. For samples directly from culture, the log-phase microorganisms were pelleted, washed 4× with deionized Millipore water, and then resuspended in 250 μL of deionized Millipore water.