7. 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).
8. Replace lid and incubate for 3–7 days at 1% O 2 and 5% CO 2 (see
Note 22).
9. Remove plate(s) from hypoxic chamber.
10. 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).
11. Transfer 25–30 μL of triplicates from assay plate to new 96-well
plates using wells A1–A12.
12. Make tenfold serial dilutions (10
0 , 10
À1
, 10
À2
, 10
À3 , 10
À4 ,
and 10
À5
) from rows A to F by serial diluting 10 μL into 90 μL
of PBS-Tyl or 7H9-ADN and mixing five times with a pipette
set at 70 μL per dilution. Change tips with each dilution.
13. Spread 10 μL of each dilution on each section of two tristyle
7H11-OADC agar plates as groups of dilutions (10
0 , 10
À1 ,
and 10
À2 , and 10
À3 , 10
À4 , and 10
À5 ).
14. Incubate agar plates for 2.5–4 weeks at 37
C at 20% O 2 and 5%
CO 2 and enumerate colonies (see Notes 25–27).
3.5 Comprehensive
Nonreplicating Stress
Deconvolution
Deconvolution assays are used to determine which of the stresses in
the nonreplicating-assay conspired with the test agent to kill nonreplicating M. tuberculosis. This entails removing stresses one-byone and inquiring if the test agent retains the ability to kill nonreplicating M. tuberculosis (Table 2). A complete deconvolution
queries 16 variations of the 4 stresses: pH, butyrate, nitrite, and
oxygen levels.
1. Prepare eight different deconvolution media (A–H) in 40 mL
volumes in 50 mL Falcon tubes as shown in Table 2.
2. Grow M. tuberculosis (see Notes 1 and 7) to mid-log phase
(OD 580 ¼ 0.4–0.7) in complete replicating medium.
3. Centrifuge cells at 3098 Â g for 8 min at RT at maximum
deceleration and acceleration.
4. Resuspend the bacterial pellet in 1 mL of PBS–Tyl. Add the
original culture volume of PBS–Tyl. For example, if 45 mL of
culture volume was washed, the pellet should be resuspended
to homogeneity first with 1 mL of PBS-Tyl, followed by an
additional 44 mL of PBS-Tyl.
5. Centrifuge cells one more time as in step 3 and resuspend cells
in PBS-Tyl at an OD 580 of 1.0.
6. Dilute cells one to ten in each medium (A–H) in 50 mL Falcon
tubes to an OD 580 ¼ 0.1.
Multi-stress Model for Nonreplicating M. tuberculosis
623
DMSO vehicle control (see Note 14).
8. Replace lid and incubate for 3–7 days at 1% O 2 and 5% CO 2 (see
Note 22).
9. Remove plate(s) from hypoxic chamber.
10. 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).
11. Transfer 25–30 μL of triplicates from assay plate to new 96-well
plates using wells A1–A12.
12. Make tenfold serial dilutions (10
0 , 10
À1
, 10
À2
, 10
À3 , 10
À4 ,
and 10
À5
) from rows A to F by serial diluting 10 μL into 90 μL
of PBS-Tyl or 7H9-ADN and mixing five times with a pipette
set at 70 μL per dilution. Change tips with each dilution.
13. Spread 10 μL of each dilution on each section of two tristyle
7H11-OADC agar plates as groups of dilutions (10
0 , 10
À1 ,
and 10
À2 , and 10
À3 , 10
À4 , and 10
À5 ).
14. Incubate agar plates for 2.5–4 weeks at 37
C at 20% O 2 and 5%
CO 2 and enumerate colonies (see Notes 25–27).
3.5 Comprehensive
Nonreplicating Stress
Deconvolution
Deconvolution assays are used to determine which of the stresses in
the nonreplicating-assay conspired with the test agent to kill nonreplicating M. tuberculosis. This entails removing stresses one-byone and inquiring if the test agent retains the ability to kill nonreplicating M. tuberculosis (Table 2). A complete deconvolution
queries 16 variations of the 4 stresses: pH, butyrate, nitrite, and
oxygen levels.
1. Prepare eight different deconvolution media (A–H) in 40 mL
volumes in 50 mL Falcon tubes as shown in Table 2.
2. Grow M. tuberculosis (see Notes 1 and 7) to mid-log phase
(OD 580 ¼ 0.4–0.7) in complete replicating medium.
3. Centrifuge cells at 3098 Â g for 8 min at RT at maximum
deceleration and acceleration.
4. Resuspend the bacterial pellet in 1 mL of PBS–Tyl. Add the
original culture volume of PBS–Tyl. For example, if 45 mL of
culture volume was washed, the pellet should be resuspended
to homogeneity first with 1 mL of PBS-Tyl, followed by an
additional 44 mL of PBS-Tyl.
5. Centrifuge cells one more time as in step 3 and resuspend cells
in PBS-Tyl at an OD 580 of 1.0.
6. Dilute cells one to ten in each medium (A–H) in 50 mL Falcon
tubes to an OD 580 ¼ 0.1.
Multi-stress Model for Nonreplicating M. tuberculosis
623