A Microfluidic Device for Real-Time Imaging and Pressure Tracking During Biofilm Formation

Begin with a microfluidic device mounted on a microscope stage.

The device comprises a polymer-glass assembly enclosing a microchannel with cylindrical pillars that mimic a porous medium.

A syringe pump delivers culture medium through a filter-fitted syringe into the channel, which drains via an outlet tubing into a waste container.

Monitor the baseline fluid pressure using a sensor connected to the channel.

Place the outlet tubing into a vial containing a bacterial suspension.

Reverse the flow until the suspension fills the channel, while the filter prevents bacteria from entering the syringe.

Incubate to allow the bacterial cells to adhere to surfaces, proliferate, and initiate biofilm formation.

Then, remove the filter and return the outlet to the waste container.

Resume medium flow and monitor the pressure as the biofilm grows and blocks the channel.

Capture time-lapse microscopic images to visualize biofilm growth over time. Correlate biofilm growth with pressure shifts to study bioclogging in porous media.

Begin by turning on the box incubator of the microscope three hours before the experiment to ensure a stable temperature of 25 degrees Celsius.

Connect the inlet and outlet tubing to the microfluidic device. Secure the connection between the tubing and the syringe by inserting a needle having an outer diameter of 0.6 millimeters into the inlet tubing. Place the microfluidic device, 30 milliliters of deionized water, and 30 milliliters of culture medium in a vacuum desiccator and degas them for at least one hour.

Once done, slowly pull the culture medium and the deionized water into two separate 30 milliliter syringes. Then, mount the microfluidic device on the microscope and place the outlet tubing in a waste container. Connect the syringe filled with deionized water to the microfluidic channel through the microfluidic tubing and slowly inject the water until it exits from the pressure sensor outlet.

Fill the pressure sensor with water and flush all the bubbles from the tubing connecting the microfluidic channel and the pressure sensor. Close the outlet of the pressure sensor with the screws dedicated to the pressure sensor. Fill the rest of the microfluidic channel with deionized water.

Next, place a 1.2 micrometer filter on the culture media syringe. Remove the water syringe and carefully connect the syringe to the inlet microfluidic tubing. After mounting the syringe on the syringe pump, flush the channel with the culture medium at a flow rate of two milliliters per hour for one hour.

Thereafter, set the syringe pump to the desired flow rate during the experiment and set the pressure reading of the pressure sensors to zero. Next, pipette 1 milliliter of the Bacillus subtilis NCIB 3610 culture of an optical density of 0.1 at a 600 nanometer wavelength in a 1.5 milliliter centrifuge vial. To load the bacterial culture into the microfluidic channel, place the outlet tube into the centrifuge vial and wait for five minutes to remove any potential air bubbles from the tube's outlet.

Once done, withdraw 150 microliters of bacterial solution at a flow rate of 1 milliliter per hour until the microfluidic channel is filled with the bacterial culture. Then, carefully remove the culture media syringe filter, and place the outlet into the waste container. Leave the bacterial cells at zero-flow conditions in the microfluidic channel for three hours to allow their surface attachment in the porous medium.

To start the experiment, start the flow by setting the syringe pump to the desired flow rate and start the pressure reading at 1 hertz before acquiring the images of the growing biofilms at the desired time interval, optical configuration, and magnification.