Microfluidic Time-Lapse Microscopy to Study Antibiotic Persistence in Bacteria

Begin with exponentially growing bacterial cells expressing a fluorescently labeled DNA-binding fusion protein to visualize the nucleoid.

Add the cells to the cell inlets of a pre-prepared microfluidic plate.

Next, introduce antibiotic-containing media and antibiotic-free media into separate solution inlets.

Seal the plate, mount it onto the microscope stage, and initiate time-lapse imaging.

First, load the cells into the culture chamber.

Then, perfuse antibiotic-free media into the chamber to support exponential cell growth.

Finally, introduce the antibiotic-containing media.

The antibiotic induces double-strand DNA breaks, killing susceptible cells. These cells exhibit compacted, non-replicating nucleoids with stable fluorescence.

Switch back to antibiotic-free media to promote recovery.

Persister cells-a small subpopulation that survives antibiotic treatment-resume DNA replication, elongate, and divide.

This recovery leads to increased fluorescence in persister cells, allowing them to be visually distinguished from susceptible cells.

Remove the conservation solution from every well of the microfluidic plate, and replace it with fresh culture medium. Seal the microfluidic plate with the man