5. Seal hypoxic chamber door with autoclave tape to ensure a
tight seal and no gas leakage.
6. Incubate for 3 days without opening hypoxic chamber (see
Note 15).
7. Remove microplates from hypoxic chamber. Using a plate
dispenser, add 60 μL of complete replicating medium by two
rounds of 30 μL to initiate the outgrowth phase (see Note 16).
8. Dip the cotton portion of an individually wrapped wooden
cotton swab (Puritan, sterile) into 70% ethanol (approximately
10 mL in a 15 mL Falcon tube), remove excess ethanol out of
the cotton by pushing the cotton against the sterile interior of
the Falcon tube, and then use the tip to quickly wipe the entire
surface of the 384-well plate. This step will remove any small
drops of culture medium that may have sprayed onto the
surface.
9. Stack the microplates in groups of three or four in a plastic
container and prop open lid as described in Subheading 3.2,
step 4.
10. Incubate the plates for 7 days at 20% O 2 and 5% CO 2 (see
Note 17).
11. Remove microplate lids and allow plates to sit uncovered for
approximately 20 min (see Notes 18 and 19).
12. Read the OD 580 of the entire plate (see Notes 20 and 21).
3.3 Nonreplicating
Protocol for 96-Well
Format
1. Using a 12-channel multichannel pipette, add 200 μL of nonreplicating M. tuberculosis at an OD 580 of 0.1 to a 96-well clearbottomed, tissue culture-treated microplate.
2. Add 2 μL of test agents in DMSO (see Note 13). Mix up/down
at least five to ten times while stirring in a circular motion at the
same time to ensure DMSO is adequately dissolved.
3. Add 2 μL of control compounds oxyphenbutazone and rifampicin at 100 μM and 10 μg/mL, respectively, and 2 μL of
DMSO vehicle control (see Note 14).
4. Replace lid and incubate for 7 days at 1% O 2 and 5% CO 2 (see
Note 22).
5. Remove plate(s) from hypoxic chamber.
6. Set up a standard outgrowth plate by adding 200 μL of
medium to every well in a new 96-well microplate.
7. Mix assay plate containing M. tuberculosis and test agents by
pipetting 150 μL up and down ten times with a 200 μL pipette
while simultaneously using the tips to stir the well bottoms (see
Note 23).
Multi-stress Model for Nonreplicating M. tuberculosis
621
tight seal and no gas leakage.
6. Incubate for 3 days without opening hypoxic chamber (see
Note 15).
7. Remove microplates from hypoxic chamber. Using a plate
dispenser, add 60 μL of complete replicating medium by two
rounds of 30 μL to initiate the outgrowth phase (see Note 16).
8. Dip the cotton portion of an individually wrapped wooden
cotton swab (Puritan, sterile) into 70% ethanol (approximately
10 mL in a 15 mL Falcon tube), remove excess ethanol out of
the cotton by pushing the cotton against the sterile interior of
the Falcon tube, and then use the tip to quickly wipe the entire
surface of the 384-well plate. This step will remove any small
drops of culture medium that may have sprayed onto the
surface.
9. Stack the microplates in groups of three or four in a plastic
container and prop open lid as described in Subheading 3.2,
step 4.
10. Incubate the plates for 7 days at 20% O 2 and 5% CO 2 (see
Note 17).
11. Remove microplate lids and allow plates to sit uncovered for
approximately 20 min (see Notes 18 and 19).
12. Read the OD 580 of the entire plate (see Notes 20 and 21).
3.3 Nonreplicating
Protocol for 96-Well
Format
1. Using a 12-channel multichannel pipette, add 200 μL of nonreplicating M. tuberculosis at an OD 580 of 0.1 to a 96-well clearbottomed, tissue culture-treated microplate.
2. Add 2 μL of test agents in DMSO (see Note 13). Mix up/down
at least five to ten times while stirring in a circular motion at the
same time to ensure DMSO is adequately dissolved.
3. Add 2 μL of control compounds oxyphenbutazone and rifampicin at 100 μM and 10 μg/mL, respectively, and 2 μL of
DMSO vehicle control (see Note 14).
4. Replace lid and incubate for 7 days at 1% O 2 and 5% CO 2 (see
Note 22).
5. Remove plate(s) from hypoxic chamber.
6. Set up a standard outgrowth plate by adding 200 μL of
medium to every well in a new 96-well microplate.
7. Mix assay plate containing M. tuberculosis and test agents by
pipetting 150 μL up and down ten times with a 200 μL pipette
while simultaneously using the tips to stir the well bottoms (see
Note 23).
Multi-stress Model for Nonreplicating M. tuberculosis
621