Microfluidic Patterning and Fluorescence-Based Tracking of Single-Cell Bacterial Growth

Begin with a fluorescently tagged bacterial culture added to a carbon-free buffer.

Centrifuge to pellet the bacteria and discard the supernatant.

Resuspend the pellet in a fresh carbon-free buffer containing a mild detergent.

The lack of a carbon source halts growth, while the detergent prevents clumping and keeps cells suspended.

Load the bacterial suspension into a syringe and connect it to a pump.

Inject the suspension into the inlet of a microfluidic chip containing a channel with floor-embedded traps.

Slowly withdraw the liquid from the channel outlet.

As the liquid recedes, capillary forces guide the bacteria toward the outlet.

In this process, single bacterial cells settle into the traps, resulting in bacterial patterning.

Flush the channel with a prewarmed, nutrient-rich broth continuously.

The bacteria resume growth and form microcolonies inside the traps.

Use fluorescence microscopy to capture time-lapse images and analyze bacterial growth in a controlled environment.

Pipette one milliliter of MOPS medium into a centrifuge vial and add 10 microliters of 0.132 molar potassium phosphate.

Aliquot 100 microliters of the overnight culture into the centrifuge vial and centrifuge the culture