This study investigates the effects of antibiotics on bacterial cells using a microfluidic platform. By visualizing nucleoid structures, the research highlights the behavior of persister cells under antibiotic stress.
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 manifold system by clicking on the Seal button or through the microfluidic software.
Next, on the microfluidic software interface, perform a first priming sequence by clicking on Run Liquid Priming Sequence. Incubate the plate in a thermostatically controlled cabinet of the microscope at 37 degrees Celsius for a minimum of two hours before starting the microscopy imaging. Then start a second Run Liquid Priming Sequence before beginning the experiment.
Seal off the microfluidic plate by clicking on Unseal Plate on the microfluidic software interface. Replace the medium in the wells with 200 microliters of fresh medium, antibiotic containing fresh medium, and 0.01 OD diluted culture sample in the fresh medium. After sealing the microfluidic plate as demonstrated before, place the plate onto the microscope objective inside the microscope cabinet.
In the microfluidic software, click on the Run Cell Loading Sequence to allow the loading of the cells into the microfluidic plate. Set an optimal focus using the transmitted light mode, and select several regions of interest where an appropriate cell number of up to 300 cells per field can be observed. On the microfluidic software, edit the Run a Custom Sequence to program the injection of fresh medium at 6.9 kilopascals for six hours to wells one and two, followed by the medium containing antibiotic at 6.9 kilopascals for six hours to well three, and finally the fresh medium at 6.9 kilopascals for 24 hours to wells four and five.
Perform microscopy imaging in time-lapse mode with one frame every 15 minutes using transmitted light and the excitation light source for the fluorescent reporter, and start the microfluidic program. Open the ImageJ or Fiji software on the computer, and drag the hyperstack time-lapse microscopy images into the Fiji loading bar. Use Image, followed by Color, and then Make Composite to fuse the different channels of the hyperstack.
Use Image, Color, and then Arrange Channels if the channels do not correspond to the desired color. Open the MicrobeJ plugin, and detect the bacterial cells using the manual editing interface. Delete the automatically detected cells, and manually outline the persister cells of interest frame by frame.
After detection, use the Result icon in the MicrobeJ manual editing interface to generate a ResultJ table. Use the ResultJ table to gain insights into different parameters of interest of the single-cell analysis, and save the ResultJ file.
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