Single-cell Microfluidic Analysis of Bacillus subtilis

The overall goal of this microfluidic experimental set-up is to enable the longterm observation of individual bacterial cells under constant, highly controlled environmental conditions. This method can help answer key questions in the bacterial physiology field, such as what are the longterm response patterns of individual cells to stress or how does a heterogeneous phenotype across a population vary with time. The main advantage of this technique is that we can observe individual cell lineages for tens or even hundreds of generations, under constant, uniform and highly controlled environmental conditions.

After preparing PDMS polymer according to the text protocol, place a silicone master on a piece of unbroken aluminum foil in a polystyrene Petri dish. Pour the degassed PDMS mixture over the master to a depth of approximately five millimeters. Then degas the dish for at least 10 minutes.

Cure the PDMS in an oven at 60 degrees Celsius for at least one hour and cool it to room temperature. Then remove the aluminum foil containing the master and cured PDMS from the plate. Carefully score it and peel off the aluminum foil from the back of the master.

Then carefully peel the PDMS from the master. As a wafer, typically include several microfluidic devices with slightly different dimensions. Use a clean, sharp scalpel to cut a single device to the appropriate size.

Place a piece of translucent office tape over the patterned side of the device to enhance the visibility of the patterned features. The inlet and outlet ports are identified as circular areas at opposite ends of the visible fluidic channels. Flip the PDMS device with the pattern side down and use a 0.75 millimeter biopsy punch to punch through the device to the marked dots.

Then discard the punchings. A tweezer may be required to remove the punchings from the device. Insert the cleaned and dried cover glass and PDMS device into an oxygen plasma cleaner and plasma treat the device with the settings shown here.

Immediately after the plasma treatment finishes, remove the device and cover glass from the plasma cleaner. Invert the PDMS and place it on the center of the cover glass so that the pattern side contacts the glass. Ensure that the feeding channels are aligned with the long access of the cover glass.

Press down very gently to ensure that the PDMS seals against the glass. Bake the assembled device in an oven at 60 degrees Celsius for at least one hour. After baking, manually verify that the PDMS is bonded to the glass by gently pushing on one corner of the device.

After cutting polymer tubing according to the text protocol, assemble Y-junctions for pinch valves, if using them, by cutting and attaching two-centimeter lengths of flexible silicone tubing to the top barbs of the Y and one-centimeter lengths of silicone tubing to the bottom barb of the Y.Next, cut 10-centimeter lengths of polymer tubing then connect them at one end to the switch junction by inserting them into the one-centimeter silicone tubing segments on the bottom barb of the Y.Connect the lengths of tubing at the other end to 21-gauge needles that have been removed from their plastic syringe adapters and bent approximately 90 degrees about nine millimeters from the needle end. Connect 21-gauge blunt needles to one end of the tubing segments that will run from the syringes to the switch apparatus by inserting the needles into the tubing. Insert the other ends of each length of tubing into the silicone tubing segments on the inlet barbs of the Y-junctions.

After filling syringes with bacterial growth medium, supplemented with BSA, according to the text protocol, connect the loaded syringes to the prepared 21-gauge needles, attach to the inlet tubing and load the syringes into the syringe pumps. Purge air from the inlet plumbing by running the syringe pumps at a high flow rate and orienting the syringe pumps vertically, tapping the syringes to bring air bubbles to the top. Once air has been purged from the syringe, position the syringe pump horizontally and progressively tap or flick the polymer lines from the syringes, up through the switch apparatus to dislodge and purge any air bubbles.

Repeat this process for the second bank of syringes if switching media. After the air bubbles are purged, set the phase-two syringe pump to 1.5 microliters per minute then pause the flow. Place small binder clips onto the segments of flexible tubing on the branch of the Y-junctions corresponding to the second medium phase and continue to run the phase-one pump to purge the second medium downstream of the Y-junction.

To load the PDMS device, begin by gently placing empty, thin gel-loading micropipette tips into the outlet holes of the microfluidic device. Using thin gel-loading tips with the P200 micropipette, passivate the device by injecting medium containing one milligram per milliliter BSA into the inlet holes, observing the tips and the outlet holes to monitor filling of the microfluidic channels. Incubate the device at room temperature for approximately five minutes.

Next, using similar tips, load each channel with the re-suspended cells using the tips in the outlet channel to monitor the progress of the cells through the device. Gently remove the gel-loading tips from the outlet holes, working one at a time to prevent damage to the device. Centrifuge the device in a benchtop microcentrifuge in an appropriate rotor adapter at approximately 6, 000 times g for 10 minutes.

Verify successful loading under the microscope but without affixing the device to the slide holder stage insert. Carefully mount the loaded device onto a stage insert by taping the cover glass on either side of the PDMS to the bottom of the stage insert. Invert the stage insert device assembly so that the PDMS is facing up and place it on a soft surface such as a dust-free wipe.

Adjust the flow rate of the phase-one syringe pump to 35 microliters per minute. Then working with one lane at a time, insert the inlet needle and then the outlet needle that runs to the waste beaker. After visually inspecting the device for leaks, examine the outlet tubing for the appearance of excess cells that appear as a turbid stripe in the medium meniscus, which will slowly move towards the waste beaker.

Once the device has run for approximately 15 minutes, set the phase-one syringe pump to 1.5 microliters per minute and pause the flow. Bring the entire pump and device apparatus to an inverted fluorescent microscope and restart the phase-one medium flow at 1.5 microliters per minute. Carefully mount the stage insert with the device on to the microscope, using tape as necessary to route the inlet and outlet tubing.

Locate the appropriate positions on the device for imaging and begin imaging as desired. Note that the cells typically require a few hours to read steady-state, exponential phase growth, and the onset of image acquisition may be delayed as desired so that imaging begins after the equilibration period. To switch medium between successive rounds of imaging, pause the flow of the phase-one syringe pump, carefully unclip the binder clips from the phase-two silicone tubing, moving each one to the silicone tubing on the phase-one branch of the Y-junction, then unpause the flow of the phase-two syringe pump and continue imaging.

As assessed by phase contrast microscopy, before attaching the microfluidic plumbing to the device, initial cell loading would be considered successful if all or nearly all of the microfluidic side channels contained one or more bacterial cells. Once the device is initially loaded, it is equilibrated at a relatively high flow rate. As shown here, microscopic inspection of an equilibrated device should reveal a few cells confined in single file in most of the side channels.

Once an equilibrated device is placed on the microscope under flow at 1.5 microliters per minute at 37 degrees Celsius, the cells will resume uniform and constant exponential phase growth over the course of approximately two hours. Cells that have resumed exponential phase growth can be reliably imaged using bright-field or fluorescence microscopy for at least 24 hours. Introducing a medium switch expands the range of experiments that can be conducted but also increases the frequency of catastrophic cell death events through the presence of air bubbles or cell clusters at the inlet that become dislodged and pass through the feeding channel.

Once mastered, this technique can be done in approximately five hours, if it is performed properly. By using this procedure, with different fluorescent reporters, strains and environmental conditions, a wide variety of physiological phenomena can be examined, including very rare or transient events.