3 Methods
3.1 Preparation
of Nonreplicating
M. tuberculosis
1. Grow M. tuberculosis (see Notes 1 and 7) to mid-log phase
(OD 580 ¼ 0.4–0.7) in complete replicating medium in roller
bottles. Media should be modified as appropriate for the strain
being used (see items 3 and 4 in Subheading 2.1).
2. Centrifuge cells at 3098 Â g for 8 min at RT.
3. Resuspend the bacterial pellet in 1 mL of PBS–Tyloxapol
(PBS-Tyl). Add the original culture volume of PBS–Tyloxapol.
For example, if 45 mL culture volume was centrifuged, the
pellet should be resuspended to homogeneity first with 1 mL of
PBS-Tyl, followed by an additional 44 mL of PBS-Tyl.
4. Wash cells one more time.
5. Resuspend pellet in 1 mL of nonreplicating medium. Add
nonreplicating medium to ~3/4 of the original culture volume.
For a 45 mL original culture, this is approximately 33 mL (see
Note 8).
6. Remove 1 mL aliquot to measure OD 580 .
7. If cells will be used for CFU experiments, skip step 8 and
proceed to optional step 9.
8. Adjust optical density to OD 580 ¼ 0.1 using nonreplicating
medium.
9. Optional step: If these cells will be used to prepare a single-cell
suspension (Subheading 3.4, step 1), do not dilute the cells
until after the slow-speed centrifugation to obtain single cells
(see Note 9).
10. Add NaNO 2 to a final concentration of 0.5 mM from freshly
prepared 1 M stock solution.
11. Allow cells to sit in nonreplicating medium containing NaNO 2
for approximately 30 min at RT (see Note 10).
3.2 Nonreplicating
Protocol for 384-Well
Format
1. Add 15 μL of nonreplicating M. tuberculosis to every well in
384-well assay plates using a MultiDrop Combi plate dispenser
and a low volume cassette (see Notes 11).
2. Robotically dispense 150 nL of test compound in 100% DMSO
to achieve 1% DMSO final (see Notes 12 and 13).
3. Add 150 nL of control compounds oxyphenbutazone (final
100 μM) and/or rifampicin (final 10 μg/mL) or DMSO vehicle control (see Note 14).
4. Stack up to three to four microplates in plastic boxes with snap
lids (a total of six to eight plates) and move to a 37
C hypoxic
chamber set at 1% O 2 and 5% CO 2 . Put a sterile cotton swab
under the lid of the plastic box to prop open one of the four
corners.
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Ben Gold et al.
3.1 Preparation
of Nonreplicating
M. tuberculosis
1. Grow M. tuberculosis (see Notes 1 and 7) to mid-log phase
(OD 580 ¼ 0.4–0.7) in complete replicating medium in roller
bottles. Media should be modified as appropriate for the strain
being used (see items 3 and 4 in Subheading 2.1).
2. Centrifuge cells at 3098 Â g for 8 min at RT.
3. Resuspend the bacterial pellet in 1 mL of PBS–Tyloxapol
(PBS-Tyl). Add the original culture volume of PBS–Tyloxapol.
For example, if 45 mL culture volume was centrifuged, the
pellet should be resuspended to homogeneity first with 1 mL of
PBS-Tyl, followed by an additional 44 mL of PBS-Tyl.
4. Wash cells one more time.
5. Resuspend pellet in 1 mL of nonreplicating medium. Add
nonreplicating medium to ~3/4 of the original culture volume.
For a 45 mL original culture, this is approximately 33 mL (see
Note 8).
6. Remove 1 mL aliquot to measure OD 580 .
7. If cells will be used for CFU experiments, skip step 8 and
proceed to optional step 9.
8. Adjust optical density to OD 580 ¼ 0.1 using nonreplicating
medium.
9. Optional step: If these cells will be used to prepare a single-cell
suspension (Subheading 3.4, step 1), do not dilute the cells
until after the slow-speed centrifugation to obtain single cells
(see Note 9).
10. Add NaNO 2 to a final concentration of 0.5 mM from freshly
prepared 1 M stock solution.
11. Allow cells to sit in nonreplicating medium containing NaNO 2
for approximately 30 min at RT (see Note 10).
3.2 Nonreplicating
Protocol for 384-Well
Format
1. Add 15 μL of nonreplicating M. tuberculosis to every well in
384-well assay plates using a MultiDrop Combi plate dispenser
and a low volume cassette (see Notes 11).
2. Robotically dispense 150 nL of test compound in 100% DMSO
to achieve 1% DMSO final (see Notes 12 and 13).
3. Add 150 nL of control compounds oxyphenbutazone (final
100 μM) and/or rifampicin (final 10 μg/mL) or DMSO vehicle control (see Note 14).
4. Stack up to three to four microplates in plastic boxes with snap
lids (a total of six to eight plates) and move to a 37
C hypoxic
chamber set at 1% O 2 and 5% CO 2 . Put a sterile cotton swab
under the lid of the plastic box to prop open one of the four
corners.
620
Ben Gold et al.