11. Take the aluminum frame and spread a very small amount of
immersion oil around the opening (Fig. 1c) (see Note 24).
12. Pick up a 24 Â 50 mm coverslip using the broad-tip tweezers
and place it in the center of the opening of the aluminum frame
(Fig. 1c) (see Note 25).
13. Anticipate the position of the 6 bacterial strains on the two
membranes. Draw 3 imaginary lines perpendicular to the
length of each membrane, to generate an area on the left, one
in the center, and one on the right that will correspond to the
3 widest zones of each microfluidic serpentine (Fig. 1b, c).
14. Additionally, mark on the edge of the acrylic layer the bacterial
strains that will be positioned in the corresponding area.
15. Dispense 1 μL of filtered bacteria (from Subheading 3.1),
either independent cultures of the same bacterial strain or
different bacterial strains, onto the center of each subsection
of the membranes, and spread the inoculum gently using the
side of a 20-μL pipette tip (see Note 26).
16. Leave the petri dish open in a biosafety cabinet under the
laminar flow for about 5 min, until the medium is absorbed
on the membrane. Do not let the membrane dry out.
17. Gently lift one semipermeable membrane at a time from the
Whatman filter and place it over each microfluidic serpentine,
with the bacteria side face up. Make sure there are no air
bubbles trapped between the membrane and that the serpentine is completely covered by the membrane, to prevent
leakages.
18. In one movement, flip the upper acrylic layer and place it on
the lower aluminum frame, by centering the 8 screws in the
holes of the bottom frame, and aligning the microfluidic device
with the coverslip on the base (Fig. 1c).
19. Apply moderate pressure with three fingertips on the acrylic
layer in correspondence with the device and start rotating the
screws. Once all the screws have been positioned, start tightening them, making sure to apply equal pressure and to tighten
each screw to the same extent (see Note 27).
20. Place the outlet tubings into a waste receptacle that contains
concentrated tuberculocidal disinfectant and cover it with parafilm, without squashing the tubings and keeping them visible.
21. Transfer the assembled device to the microscope. Spread a
generous amount of immersion oil on the bottom of the
coverslip as well as on the 100Â objective lens and clamp the
device frame to the microscope stage (see Note 28) (Fig. 1c, d).
22. Place the syringe/s on the infusion pump (see Note 8).
Microfluidic Microscopy of Mycobacteria
217
immersion oil around the opening (Fig. 1c) (see Note 24).
12. Pick up a 24 Â 50 mm coverslip using the broad-tip tweezers
and place it in the center of the opening of the aluminum frame
(Fig. 1c) (see Note 25).
13. Anticipate the position of the 6 bacterial strains on the two
membranes. Draw 3 imaginary lines perpendicular to the
length of each membrane, to generate an area on the left, one
in the center, and one on the right that will correspond to the
3 widest zones of each microfluidic serpentine (Fig. 1b, c).
14. Additionally, mark on the edge of the acrylic layer the bacterial
strains that will be positioned in the corresponding area.
15. Dispense 1 μL of filtered bacteria (from Subheading 3.1),
either independent cultures of the same bacterial strain or
different bacterial strains, onto the center of each subsection
of the membranes, and spread the inoculum gently using the
side of a 20-μL pipette tip (see Note 26).
16. Leave the petri dish open in a biosafety cabinet under the
laminar flow for about 5 min, until the medium is absorbed
on the membrane. Do not let the membrane dry out.
17. Gently lift one semipermeable membrane at a time from the
Whatman filter and place it over each microfluidic serpentine,
with the bacteria side face up. Make sure there are no air
bubbles trapped between the membrane and that the serpentine is completely covered by the membrane, to prevent
leakages.
18. In one movement, flip the upper acrylic layer and place it on
the lower aluminum frame, by centering the 8 screws in the
holes of the bottom frame, and aligning the microfluidic device
with the coverslip on the base (Fig. 1c).
19. Apply moderate pressure with three fingertips on the acrylic
layer in correspondence with the device and start rotating the
screws. Once all the screws have been positioned, start tightening them, making sure to apply equal pressure and to tighten
each screw to the same extent (see Note 27).
20. Place the outlet tubings into a waste receptacle that contains
concentrated tuberculocidal disinfectant and cover it with parafilm, without squashing the tubings and keeping them visible.
21. Transfer the assembled device to the microscope. Spread a
generous amount of immersion oil on the bottom of the
coverslip as well as on the 100Â objective lens and clamp the
device frame to the microscope stage (see Note 28) (Fig. 1c, d).
22. Place the syringe/s on the infusion pump (see Note 8).
Microfluidic Microscopy of Mycobacteria
217