An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells

For probe accumulation analysis, streak glycerol stocks of the bacterial strains onto LB agar plates for an overnight incubation at 37 degrees Celsius. The next morning, pick a single colony from the plate for overnight culture in lysogeny broth at 37 degrees Celsius. The next morning, dilute the overnight culture approximately 50-fold in fresh medium.

When the culture reaches mid-log phase, pellet the bacteria by centrifugation, and decant the medium. Resuspend the bacteria in 1 milliliter of PBS, and centrifuge the bacteria again. Decant the supernatant, and resuspend the washed pellet in PBS to an optical density at 600 nanometers of two.

Add 10.1 microliters of 10 millimolar CCCP in PBS to 1 milliliter of bacteria, and incubate the bacteria at 37 degrees Celsius for 10 minutes. At the end of the incubation, collect the bacteria by centrifugation, and resuspend the pellet in 1 milliliter of 10-to-100 micromolar fluorescent antibiotic solution in PBS.

After a 30-minute incubation at 37 degrees Celsius, wash the cells by centrifugation four times in 1 milliliter of cold PBS per wash. After the wash, lyse the bacteria with 180 microliters of lysis buffer, and 70 microliters of lysozyme.

After 30 minutes at 37 degrees Celsius, freeze-thaw the bacteria three times at minus 78 degrees Celsius for 5 minutes and 34 degrees Celsius for 15 minutes respectively. After the last round of freeze-thawing, sonicate the sample for 20 minutes, followed by a 30-minute incubation at 65 degrees Celsius.

At the end of the incubation, collect the lysed sample by centrifugation, and strain the tube contents through a 10-kilodalton filter membrane. Wash the filter four times with 100 microliters of water per wash, and aliquot each wash into individual wells of a black flat-bottom 96-well plate. Then measure the fluorescence intensity on a plate reader with excitation and emission wavelengths appropriate to the fluorophore.