3.7 Preparation
of M. tuberculosis
for Replicating Assays
1. Grow M. tuberculosis wild type or ΔpanCDΔlysA (see Note 2) to
an OD 580 of 0.4–0.7 in strain-appropriate replicating complete
medium.
2. Dilute the exponentially growing culture to an OD 580 of 0.01
in the same medium.
3.8 Replicating
Assay Protocol
for 384-Well Format
1. Using a MultiDrop Combi plate dispenser and a low volume
cassette, add 50 μL of replicating cells in 7H9-ADN or
7H9-OADC (containing appropriate supplements for the
M. tuberculosis ΔpanCDΔlysA strain) into a 384-well assay
plate.
2. Robotically dispense 500 nL of compounds or DMSO controls
directly into the wells containing M. tuberculosis (see Note 31).
3. Appropriate controls include those listed for the nonreplicating
assay in 96-well plates (see Subheading 3.3, step 3).
4. Place plates into plastic containers as described in Subheading
3.2, step 8.
5. Incubate plates for 7 days at 37
C with 20% O 2 and 5% CO 2 .
6. At day 7, remove plates from incubator. Remove lids and allow
plates to equilibrate to room temperature for 20 min.
7. Replace lids and read OD 580 (see Note 32).
3.9 Replicating
Assay Protocol
for 96-Well Format
1. Using a multichannel pipettor, dispense 200 μL of replicating
cells in 7H9-ADN or 7H9-OADC (containing appropriate
supplements for the M. tuberculosis ΔpanCDΔlysA strain)
from a reagent trough into a 96-well plate.
2. Fill well A12 with cell-free R medium for blanking the plate
when reading OD 580 and fill the remaining 95 wells with R
M. tuberculosis.
3. Keep columns 1 and 12 compound and DMSO-free. Add 2 μL
of compound or DMSO vehicle control to each remaining well
and mix up and down five times while also stirring the tip along
the well bottom in a circular motion. Column 11 will contain
the DMSO vehicle controls (rows A–H).
4. Use one or more of the following positive controls that possess
activity on replicating M. tuberculosis such as rifampicin, isoniazid, ethionamide, ethambutol, streptomycin, kanamycin, and
PAS (see Note 33).
5. Replace the lid and incubate plates for 7 days at 37
C with 20%
O 2 and 5% CO 2 . Place plates into plastic containers as
described in Subheading 3.2, step 8.
6. On day 7, remove plates from incubator. Remove lids and allow
plates to equilibrate to room temperature for 20 min.
Multi-stress Model for Nonreplicating M. tuberculosis
625
of M. tuberculosis
for Replicating Assays
1. Grow M. tuberculosis wild type or ΔpanCDΔlysA (see Note 2) to
an OD 580 of 0.4–0.7 in strain-appropriate replicating complete
medium.
2. Dilute the exponentially growing culture to an OD 580 of 0.01
in the same medium.
3.8 Replicating
Assay Protocol
for 384-Well Format
1. Using a MultiDrop Combi plate dispenser and a low volume
cassette, add 50 μL of replicating cells in 7H9-ADN or
7H9-OADC (containing appropriate supplements for the
M. tuberculosis ΔpanCDΔlysA strain) into a 384-well assay
plate.
2. Robotically dispense 500 nL of compounds or DMSO controls
directly into the wells containing M. tuberculosis (see Note 31).
3. Appropriate controls include those listed for the nonreplicating
assay in 96-well plates (see Subheading 3.3, step 3).
4. Place plates into plastic containers as described in Subheading
3.2, step 8.
5. Incubate plates for 7 days at 37
C with 20% O 2 and 5% CO 2 .
6. At day 7, remove plates from incubator. Remove lids and allow
plates to equilibrate to room temperature for 20 min.
7. Replace lids and read OD 580 (see Note 32).
3.9 Replicating
Assay Protocol
for 96-Well Format
1. Using a multichannel pipettor, dispense 200 μL of replicating
cells in 7H9-ADN or 7H9-OADC (containing appropriate
supplements for the M. tuberculosis ΔpanCDΔlysA strain)
from a reagent trough into a 96-well plate.
2. Fill well A12 with cell-free R medium for blanking the plate
when reading OD 580 and fill the remaining 95 wells with R
M. tuberculosis.
3. Keep columns 1 and 12 compound and DMSO-free. Add 2 μL
of compound or DMSO vehicle control to each remaining well
and mix up and down five times while also stirring the tip along
the well bottom in a circular motion. Column 11 will contain
the DMSO vehicle controls (rows A–H).
4. Use one or more of the following positive controls that possess
activity on replicating M. tuberculosis such as rifampicin, isoniazid, ethionamide, ethambutol, streptomycin, kanamycin, and
PAS (see Note 33).
5. Replace the lid and incubate plates for 7 days at 37
C with 20%
O 2 and 5% CO 2 . Place plates into plastic containers as
described in Subheading 3.2, step 8.
6. On day 7, remove plates from incubator. Remove lids and allow
plates to equilibrate to room temperature for 20 min.
Multi-stress Model for Nonreplicating M. tuberculosis
625