Microscopic Analysis of Bacterial Growth Under Stress Using Microfluidic Chambers

Begin with a microfluidic plate filled with a pre-warmed nutrient medium.

Attach the plate to the manifold system and seal the setup. Drive the medium through the channels to remove any air bubbles.

Place the setup on a microscope stage and preheat it to prevent plate expansion during imaging.

Next, remove the medium, introduce a bacterial culture into the cell inlets, and a stress-inducing medium into the solution inlets.

The stress-inducing medium contains an antibiotic that halts cell division without inhibiting an increase in cellular mass.

Seal the system, then initiate cell loading to transfer the bacteria into the culture chamber of the plate.

Using transmitted light, locate a field of view containing evenly distributed single cells.

Inject the chamber with the stress-inducing medium to expose the cells to the antibiotic.

Using phase-contrast mode, capture time-lapse images to observe the formation of elongated filamentous cells with increased DNA content caused by antibiotic-mediated inhibition of cell division.

First, remove the conservation solution from the microfluidic plate and replace it with fresh medium preheated to 37 degrees Celsius as described in the Microfluidic Software User Guide. Attach the microfluidic plate to the manifold system.

And in the microfluidic software, click on the Seal button to seal on the plate to the manifold system. After that, click on the Priming button. Position the microfluidic plate with the manifold system on the microscope stage and preheat at 37 degrees Celsius for two hours to avoid the dilation of the microfluidic chamber.

Seal off the microfluidic plate on the microfluidic system by clicking on the Seal Off button. Replace the medium from well eight with 150 microliters of culture sample and replace the medium from well one to five with the desired medium, with or without the stress-inducing reagent. Seal the microfluidic plate and place it on the microscope stage.

In the microfluidic software, run the cell loading procedure. Check that the loading of the cells is satisfactory by looking under the microscope in transmitted light. Carefully focus in transmitted-light mode and select several fields of view that show isolated bacteria and are not overcrowded, around 10 to 20 cells per 100 square microliters.

In the microfluidic software, click on the Run a Custom Sequence button, then program the injection of the stress-inducing medium during 10 minutes at two PSI, followed by injection at one PSI for the wanted duration of the stress treatment. Perform microscopy imaging in the time-lapse mode with one frame every 10 minutes, using phase contrast in transmitted light and a 560 nanometer excitation light source for the mCherry signal if required. Start the microscopic image acquisition and the microfluidic injection protocol at the same time.