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 at 2,300 times g for two minutes. Gently discard the supernatant to resuspend the pellet in one milliliter of fresh MOPS medium with 0.015% of Tween 20 and 0.01% of potassium phosphate and load the bacterial suspension in a one milliliter syringe. To secure the syringe and the tubing connection, directly insert a needle into the tubing.

Now mount the syringe on the syringe pump and inject the suspension into the microfluidic chip through the inlet located at the upstream part of the channel until the suspension covers the template region with traps. Set the syringe pump at a flow rate of 0.07 to 0.2 microliters per minute to withdraw the bacterial suspension and monitor the patterning process via microscope software. Once the template has been patterned with cells, increase the withdrawal flow rate to quickly empty the microfluidic channel and flush it with fresh LB that was previously degassed for at least 30 minutes and prewarmed at 30 degrees Celsius.

Now set the syringe pump at a flow rate of two microliters per minute to gently flush the channel. Once the channel has been filled, again increase the flow rate. Acquire images of growing bacteria at the desired magnification and time interval.