EdU-Click on E. coli K12 exposed (or not) to ciprofloxacin

Overnight cultures of Escherichia coli K12 (ATCC 25404) were used for the following four treatment groups: (1) treatment with EdU and ciprofloxacin, (2) treatment with EdU and without ciprofloxacin, (3) treatment without EdU and with ciprofloxacin, and (4) treatment without EdU and without ciprofloxacin. Groups (3) and (4) were used as gating controls for groups (1) and (2), respectively. All groups were first grown in a total volume of 2 mL rBHI for 1 hour, then 24 µL of the culture was removed and 20 µL of 10mg.mL-1 ciprofloxacin (or vehicle, 0.1 N HCl) and 4 µL of 10 mM EdU or rPBS was added to each well. After ciprofloxacin (or vehicle) treatment, bacteria were incubated anaerobically at 37°C for 3 hours. The bacterial cells were fixed and then underwent a click reaction with Alexa Fluor 647 azide (APC channel) to make newly replicated bacteria fluoresece. They were also stained with Sybr Green I nucleic acid stain (FITC channel) to capture all bacteria. File name key: wEwC = Group (1) treatment with EdU and with ciprofloxacin wEwoC = Group (2) treatment with EdU and without ciprofloxacin woEwC = Group (3) treatment without EdU and with ciprofloxacin woEwC = Group (4) treatment without EdU and without ciprofloxacin