This article describes a method for visualizing bacterial movement through a microfluidic device. The setup allows for the observation of individual bacterial cells as they navigate through porous channels.
Take a microfluidic device consisting of an inlet tube connected to a microfluidic chamber made of porous channels for bacterial movement.
The chamber is connected to an observation chamber that is attached to a syringe pump through an outlet tube.
Saturate the chambers with a buffer to remove any air bubbles.
Place the device on the microscope stage and position the objective lens above the observation chamber.
Adjust the microscope settings to clearly visualize individual bacterial cells.
Place the inlet tubing into a suspension of fluorescently labeled bacteria.
Start the flow to draw bacteria into the microfluidic chamber, where they pass through the porous channels to reach the observation chamber.
Image the observation chamber at regular intervals.
Generate a breakthrough curve, which represents the fraction of bacteria that pass through the porous matrix over time.
Remove the microfluidic from vacuum and place it on the microscope stage. Use the syringe pump to saturate it with motility buffer.
Using Brightfield microscopy or phase contrast, adjust the magnification to visualize individual bacterial cells and focus on the center of the observation channel. Switch the light path settings to fluorescence microscopy. Adjust focus through an offset and the camera exposure time to resolve individual bacterial cells, in this case, 100 milliseconds.
Next, insert the inlet tubing into a two milliliter tube containing the bacterial suspension. Reverse the pump direction and start withdrawing the suspension at a flow rate of one microliter per minute.
Scan the cross-section of the entire observation channel, recording an image every minute.
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