Determination of OD600 and ATP

The relative amount of bacteria in solution was measured by OD600. OD600 is an absorbance measurement at a wavelength of 600 nm. A sample of 150 μL was pipetted from the aliquots and transferred to a clear bottom 96 well plate. The remaining volume of the aliquots was centrifuged at 30,000g for 30 seconds. The supernatant was transferred to a new tube and stored at 4°C until ATP measurements were performed. The ATP concentrations of each aliquot were measured using the luciferin-luciferase assay. This assay is a chemiluminescence measurement where the amount of light produced is dependent on the concentration of ATP in the sample. The luciferin-luciferase solution was made by dissolving 2 mg of potassium luciferin (Gold Biotechnology, St. Louis, MO) and 10 mg of firefly lantern extract (Sigma) into 5 mL of DDW. Extra luciferin-luciferase reagent was stored at -20°C. In a black bottom 96 well plate, 150 μL of sample were added to a well and 20 μL of the luciferin-luciferase solution were added and mixed directly before measuring luminescence. The concentration of ATP (Adenosine 5’-triphosphatte disodium salt hydrate (Grade I, ≥ 99%), Sigma) was determined by comparing the luminescence levels with an ATP standard curve. An ATP working solution was prepared at a concentration of 1,000 nM using standard ATP in DDW. Serial dilution was performed using selective LB to create concentrations for the standard curve. The extracellular ATP concentration is dependent on the number of bacteria cells in culture, therefore the ATP/OD600 ratio was used as an indicator of cell status. The ATP/OD600 ratio can vary due to the age of bacteria, LB, or agar plates. To account for these variables, the ATP/OD600 ratios were normalized against the ATP/OD600 ratio of the control before the drug of interest was added. The ATP/OD600 ratios at a given time point were compared with the control at the same time point to determine if there was a significant difference (α = 0.05) between the ratios. All ATP and OD600 measurements were performed using a Flexstation 3 (Molecular Devices, Sunnyvale, CA). Centrifugation was performed using a Sorvall ST 8R Centrifuge (Thermo Scientific, Waltham, MA).