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 Softwar