18. Add 10μL of Alamar Blue reagent to each well (see Note 31).
19. Place plastic lid on the 96-well plate.
20. Turn the microplate spectrophotometer on and start the
Microplate program (see Note 32).
21. Remove the lid of the 96-well plate and insert the plate into the
microplate spectrophotometer.
22. Read the 96-well plate using dual wavelengths of 600 and
570 nm (see Note 33).
23. Replace the lid then wrap the plate in foil to protect from light
and place in a 37
C benchtop incubator.
24. Repeat steps 20–23 after 24, 48, and 72 h (see Note 34).
25. Dispose of the 96-well plate and lid following biosafetyapproved procedures.
3.2 Concentration
of Spent Culture
Filtrates from
Mycobacterium spp.
1. Obtain a clean stainless-steel dispensing pressure vessel; cover
all openings with aluminum foil, and autoclave.
2. Obtain a clean Amicon-stirred ultrafiltration cell and tubing for
connection to the stainless-steel dispensing pressure vessel.
Rinse all tubing, O-rings, and components of the ultrafiltration
unit with 70% ethanol and allow to air-dry (see Note 35).
3. Place the 5 kDa MWCO ultrafiltration membrane, with the
shiny side down, in 90% isopropanol. Cover and allow to
equilibrate for 10 min.
4. Discard the isopropanol and equilibrate the membrane in
pyrogen-free, 0.2μm filtered water for 30 min.
5. Assemble the stirred ultrafiltration cell with the shiny side of
the 5 kDa MWCO ultrafiltration membrane facing up. It is
important that all O-rings are properly seated to prevent leakage (see Note 36).
6. Transfer the assembled stirred ultrafiltration cell and stainlesssteel dispensing pressure vessel to a 4
C chromatography
cabinet or cold room.
7. Remove the aluminum foil and fill the stainless-steel dispensing
pressure vessel with CFP. Close the stainless-steel dispensing
pressure vessel per assembly instructions and connect tubing
from compressed nitrogen to the input port of the reservoir (see
Note 37).
8. Connect tubing to the output port of the stainless-steel dispensing pressure vessel and connect the other end to the input
port on the top of the stirred ultrafiltration cell (see Fig. 2, for
assembly). It is important that all connections and fittings are
properly engaged and sealed to prevent leakage.
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Megan Lucas et al.
19. Place plastic lid on the 96-well plate.
20. Turn the microplate spectrophotometer on and start the
Microplate program (see Note 32).
21. Remove the lid of the 96-well plate and insert the plate into the
microplate spectrophotometer.
22. Read the 96-well plate using dual wavelengths of 600 and
570 nm (see Note 33).
23. Replace the lid then wrap the plate in foil to protect from light
and place in a 37
C benchtop incubator.
24. Repeat steps 20–23 after 24, 48, and 72 h (see Note 34).
25. Dispose of the 96-well plate and lid following biosafetyapproved procedures.
3.2 Concentration
of Spent Culture
Filtrates from
Mycobacterium spp.
1. Obtain a clean stainless-steel dispensing pressure vessel; cover
all openings with aluminum foil, and autoclave.
2. Obtain a clean Amicon-stirred ultrafiltration cell and tubing for
connection to the stainless-steel dispensing pressure vessel.
Rinse all tubing, O-rings, and components of the ultrafiltration
unit with 70% ethanol and allow to air-dry (see Note 35).
3. Place the 5 kDa MWCO ultrafiltration membrane, with the
shiny side down, in 90% isopropanol. Cover and allow to
equilibrate for 10 min.
4. Discard the isopropanol and equilibrate the membrane in
pyrogen-free, 0.2μm filtered water for 30 min.
5. Assemble the stirred ultrafiltration cell with the shiny side of
the 5 kDa MWCO ultrafiltration membrane facing up. It is
important that all O-rings are properly seated to prevent leakage (see Note 36).
6. Transfer the assembled stirred ultrafiltration cell and stainlesssteel dispensing pressure vessel to a 4
C chromatography
cabinet or cold room.
7. Remove the aluminum foil and fill the stainless-steel dispensing
pressure vessel with CFP. Close the stainless-steel dispensing
pressure vessel per assembly instructions and connect tubing
from compressed nitrogen to the input port of the reservoir (see
Note 37).
8. Connect tubing to the output port of the stainless-steel dispensing pressure vessel and connect the other end to the input
port on the top of the stirred ultrafiltration cell (see Fig. 2, for
assembly). It is important that all connections and fittings are
properly engaged and sealed to prevent leakage.
86
Megan Lucas et al.