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

Take assay tubes containing bacterial samples.

In the test sample, add inhibitor molecules to block the antibiotic efflux pump of the bacteria.

Centrifuge and discard the supernatant.

Add fluorophore-conjugated antibiotic probes that enter the bacterial cells.

In the control sample, the efflux pump extrudes the probes into the extracellular medium.

In the inhibitor-treated sample, the blocked efflux pump cannot expel the probes, causing their accumulation.

Centrifuge the samples. Discard the supernatant containing extracellular probes.

Add lysis buffer followed by lysozyme to weaken the bacterial cell wall.

Perform freeze-thaw cycles, sonication, and high-temperature exposure to lyse the cells.

Centrifuge. Transfer the supernatant to a filter membrane.

Wash and collect the filtrate containing the probes.

Measure the fluorescence intensity of the filtrate.

A higher fluorescence intensity in inhibitor-treated bacterial samples suggests loss of efflux function, leading to intracellular probe accumulation.

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 singl