Measuring Biofilm Cell Lysis by ATP

To measure cell lysis, we used the luminescence assay described in reference 29 (link) to measure increases in extracellular ATP levels using a commercial ATP determination kit (Molecular Probes). Prior to measuring cell lysis, biofilms were grown for 24 h with different concentrations of antibiotics, as described previously, in a 96-well plate. We then washed the plate twice with nuclease-free water and removed excess liquid. After washing, 10 μl of nuclease-free water was added to each well and biofilms were scraped by using inoculation loops or pipette tips. Solutions were transferred to a new 96-well white polystyrene plate (Nunc F96 MicroWell; Thermo Scientific), and 90 μl of ATP standard assay solution from the ATP determination kit (Molecular Probes) was added to each sample. Luminescence was measured with a plate reader.