11. To generate the two inlet channels, insert the steel catheter
coupler half into the hole punched into the device and half into
a 60-cm-long silicone tubing (see Note 8).
12. To generate the two outlet channels, insert the steel catheter
coupler half into the hole punched into the device and half into
a 40-cm-long silicone tubing.
13. Seal the four connections between the device and the silicone
tubing using a drop of PDMS-mix and incubate the device
again at 80
C for 2 h.
14. Perfuse ultrapure water both through the inlet and the outlet
channels, to make sure that there are no leakages or PDMS
obstructions.
15. Transfer the microfluidic device into a beaker, cover it with
aluminum, and autoclave it. The device is now ready to use
(Fig. 1d).
3.7 Microfluidic
System Assembly
and Disassembly
for Time-Lapse
Experiment
1. Carry out the handling of bacterial cultures and the assembly of
the microfluidic device inside a biosafety cabinet.
2. Clean two broad-tip tweezers with 70% ethanol.
3. Place a Whatman filter paper into the lid of a petri dish and wet
it with 5 mL of prewarmed complete 7H9 medium. Pour out
the excess medium (see Note 23).
4. Take out two the semipermeable membrane rectangles from
the stack of membranes prepared before (from Subheading
3.3) and place it on the wet Whatman filter paper.
5. Wait for a few minutes for the membranes to get wet and then
flatten them carefully with the clean ends of the tweezers.
6. Arrange the membranes parallel to each other and distance
them at least 1 cm apart.
7. Close the petri dish with the bottom to prevent the membranes
from drying out while performing the other operations.
8. Place the sterilized microfluidic device in the middle of the
transparent acrylic frame (Fig. 1c), centering the device also
with respect to the center of the aluminum frame, and keeping
the channels side up.
9. Apply moderate pressure on the microfluidic device, using the
side of a 1-mL pipette tip, to make the flat bottom of the device
adhering to the acrylic surface.
10. Fill either one or two 60-mL syringes with complete 7H9
medium and connect each syringe to the inlet tubing through
a female Luer connector (see Note 8). This procedure serves to
prime the tubing until the medium reaches the inlet ports but
without coming out of the holes.
216
Giulia Manina and Neeraj Dhar
coupler half into the hole punched into the device and half into
a 60-cm-long silicone tubing (see Note 8).
12. To generate the two outlet channels, insert the steel catheter
coupler half into the hole punched into the device and half into
a 40-cm-long silicone tubing.
13. Seal the four connections between the device and the silicone
tubing using a drop of PDMS-mix and incubate the device
again at 80
C for 2 h.
14. Perfuse ultrapure water both through the inlet and the outlet
channels, to make sure that there are no leakages or PDMS
obstructions.
15. Transfer the microfluidic device into a beaker, cover it with
aluminum, and autoclave it. The device is now ready to use
(Fig. 1d).
3.7 Microfluidic
System Assembly
and Disassembly
for Time-Lapse
Experiment
1. Carry out the handling of bacterial cultures and the assembly of
the microfluidic device inside a biosafety cabinet.
2. Clean two broad-tip tweezers with 70% ethanol.
3. Place a Whatman filter paper into the lid of a petri dish and wet
it with 5 mL of prewarmed complete 7H9 medium. Pour out
the excess medium (see Note 23).
4. Take out two the semipermeable membrane rectangles from
the stack of membranes prepared before (from Subheading
3.3) and place it on the wet Whatman filter paper.
5. Wait for a few minutes for the membranes to get wet and then
flatten them carefully with the clean ends of the tweezers.
6. Arrange the membranes parallel to each other and distance
them at least 1 cm apart.
7. Close the petri dish with the bottom to prevent the membranes
from drying out while performing the other operations.
8. Place the sterilized microfluidic device in the middle of the
transparent acrylic frame (Fig. 1c), centering the device also
with respect to the center of the aluminum frame, and keeping
the channels side up.
9. Apply moderate pressure on the microfluidic device, using the
side of a 1-mL pipette tip, to make the flat bottom of the device
adhering to the acrylic surface.
10. Fill either one or two 60-mL syringes with complete 7H9
medium and connect each syringe to the inlet tubing through
a female Luer connector (see Note 8). This procedure serves to
prime the tubing until the medium reaches the inlet ports but
without coming out of the holes.
216
Giulia Manina and Neeraj Dhar