17. Use of agarose pads in μDish allows imaging of multiple strains
or different conditions in parallel. These dishes can also be
sealed with grease to avoid evaporation or when working
with M. tuberculosis. When conducting short-term time-lapse
experiments, medium can be dispensed inside the dish at the
edge, to delay evaporation.
18. These washing steps are required to get rid of sodium azide
from the membranes.
19. To cut the membranes uniformly, draw two adjacent rectangles
of 11 Â 50 mm size on one side of the glass sheet, using a dark
fine-pointed marker. Cut the membranes on the opposite side
of the glass sheet, previously cleaned with 70% ethanol. Place
the dialysis membrane above the drawing, align the ruler to the
drawn outline, and, by applying strong pressure on the ruler,
cut the membrane along the edges using a sharp scalpel. Make
sure to first cut away the two long sides where the membrane
folds, and then, the remaining sides of the two rectangles by
aligning the ruler on the drawn contours. If the membrane
dries, soak it into the water, while if it gets damaged, discard it.
20. The 24 Â 50 mm rectangles represent the outline of the glass
coverslips on which the device will be superimposed and will
aid in cutting along the contour of the device accurately. It is
also important to leave the rectangles sufficiently interspaced
and far from the edge of the wafer, about 10 mm.
21. When preparing the microfluidic device, work in a dust-free
environment and clean all surfaces that come in contact with
the PDMS. When preparing the PDMS mold, wear a lab coat,
clean pair of gloves, hair-cover, and face-mask.
22. To keep the cutting mat clean, use isopropyl alcohol and
Kimtech wipes to remove PDMS residues produced during
cutting and punching. In addition, use Scotch Magic tape to
remove PDMS residue and dust from the device.
23. The filter paper should be wet but not soaking in medium, as
this would cause the bacteria to float away.
24. Avoid using excess of immersion oil because it can overflow
onto the top of the coverslip and come into contact with the
bacteria, affecting their growth and imaging.
25. The immersion oil helps the coverslip adhere to the surface and
generates a cushioning bearing between the glass and the
aluminum frame.
26. This operation is very delicate and attention must be paid that
the halos of the three bacterial cultures, spread on the same
membrane, do not overlap, to avoid cross-contamination
among different strains. Alternatively, only two strains per
membrane can be used, spreading them at the two
opposite ends.
Microfluidic Microscopy of Mycobacteria
225
or different conditions in parallel. These dishes can also be
sealed with grease to avoid evaporation or when working
with M. tuberculosis. When conducting short-term time-lapse
experiments, medium can be dispensed inside the dish at the
edge, to delay evaporation.
18. These washing steps are required to get rid of sodium azide
from the membranes.
19. To cut the membranes uniformly, draw two adjacent rectangles
of 11 Â 50 mm size on one side of the glass sheet, using a dark
fine-pointed marker. Cut the membranes on the opposite side
of the glass sheet, previously cleaned with 70% ethanol. Place
the dialysis membrane above the drawing, align the ruler to the
drawn outline, and, by applying strong pressure on the ruler,
cut the membrane along the edges using a sharp scalpel. Make
sure to first cut away the two long sides where the membrane
folds, and then, the remaining sides of the two rectangles by
aligning the ruler on the drawn contours. If the membrane
dries, soak it into the water, while if it gets damaged, discard it.
20. The 24 Â 50 mm rectangles represent the outline of the glass
coverslips on which the device will be superimposed and will
aid in cutting along the contour of the device accurately. It is
also important to leave the rectangles sufficiently interspaced
and far from the edge of the wafer, about 10 mm.
21. When preparing the microfluidic device, work in a dust-free
environment and clean all surfaces that come in contact with
the PDMS. When preparing the PDMS mold, wear a lab coat,
clean pair of gloves, hair-cover, and face-mask.
22. To keep the cutting mat clean, use isopropyl alcohol and
Kimtech wipes to remove PDMS residues produced during
cutting and punching. In addition, use Scotch Magic tape to
remove PDMS residue and dust from the device.
23. The filter paper should be wet but not soaking in medium, as
this would cause the bacteria to float away.
24. Avoid using excess of immersion oil because it can overflow
onto the top of the coverslip and come into contact with the
bacteria, affecting their growth and imaging.
25. The immersion oil helps the coverslip adhere to the surface and
generates a cushioning bearing between the glass and the
aluminum frame.
26. This operation is very delicate and attention must be paid that
the halos of the three bacterial cultures, spread on the same
membrane, do not overlap, to avoid cross-contamination
among different strains. Alternatively, only two strains per
membrane can be used, spreading them at the two
opposite ends.
Microfluidic Microscopy of Mycobacteria
225