This article describes a protocol for using a microfluidic plate to culture bacteria for experimental purposes. The method involves priming the channels, seeding the bacteria, and facilitating their attachment under controlled conditions.
Take a microfluidic plate containing pairs of input and output wells connected by microfluidic channels, which include serpentine channels and an experimental channel.
To prime the channels, add media to the output well.
Place the plate into a plate stage and seal it with a sterilized cover.
Activate the media flow from the output well to the input well to flush out any trapped air pockets within the channels.
Remove the plate. Discard the residual media from the output well and add a bacterial culture.
To the input well, add fresh media to prevent air backflow. Place the plate back into the stage.
While observing under a microscope, resume the flow to draw cells into the experimental channel.
Adjust the flow rate to avoid cell entry into the serpentine channels to prevent clogging.
Stop the flow and incubate to facilitate bacterial attachment.
Remove the plate and discard the residual bacterial culture and media.
The bacteria within the microfluidic plate are now ready for further experiments.
For priming, first remove the 48-well microfluidic plate from the packaging, without touching the glass surface at the bottom of the plate, and clean the glass slide at the plate bottom with a lens tissue. To prime the microfluidic channels, pipette 200 microliters of 37 degrees Celsius minimal medium into the output well, taking care to avoid bubbles. Then place the plate onto the plate stage.
Wipe the interface with ethanol, sealing it onto the plate stage once the ethanol has dried. In manual mode on the control module, set the fluid as Luria-Bertani broth at 37 degrees Celsius, and max sheer as five dyne per square centimeter. Click the output wells to activate the flow from the output to the input well, to prime the channels.
After five minutes, pause the flow to prepare for seeding, and carefully remove the plate from the stage. Then, aspirate any residual medium from the output well without removing any medium from the inner circle that leads to the microfluidic channels. To seed the experimental channels, add 300 microliters of minimal medium into the input well, followed by the addition of 300 microliters of bacterial culture into the output well.
Return the plate to the imaging stage making sure to wipe the interface with ethanol before placing it onto the plate, and use the live camera feed to focus on a single channel. While visually monitoring the live feed, resume the flow at 1 to 2 dyne per square centimeter for approximately 2 to 4 seconds, to allow the cells to enter the experimental channel but not the serpentine channels, and leave the plate on the temperature controlled stage for one hour, to allow the cells to attach. At the end of the attachment period, carefully remove the plate from the stage and aspirate the bacteria from the output well without disturbing the channel.
Then, use a new pipette tip to remove the medium from the input wells.
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