4. Obtain a “day 0” sample: transfer a 500 μL aliquot from the
starting log-phase culture to a 2 mL screw cap tube, and pellet
via centrifugation at 10,000 rpm (9500 Â g) for 5 min.
5. Resuspend the pellet in 1 mL 4% PFA and store at 4
C
overnight.
6. The next day, surface-decontaminate the screw cap tube(s) and
remove from the BSL-3 facility.
7. Pellet the fixed bacteria via centrifugation at 12,000 rpm
(13,500 Â g) for 5 min.
8. Resuspend the pelleted bacteria in 200 μL PBS-Tw (0.1%) and
transfer to a 96-well plate.
9. Store at 4
C, protected from light, until flow cytometry analysis at the end of the assay.
10. Follow steps 4–9 to collect and process 500 μL aliquots from
the cultures set up in step 3 every 3 days for 12 days.
11. For tests of reporter response to NO stress, grow the reporter
M. tuberculosis strain in stirred, aerated 125 mL Erlenmeyer
flasks to log-phase (OD 600 ~ 0.6) in 35 mL 7H9, pH 7.0
medium containing 50 μg/mL hygromycin for maintenance
of the reporter plasmid.
12. Subculture the log phase culture to OD 600 ¼ 0.05 in stirred,
aerated 125 mL Erlenmeyer flasks containing 35 mL of the
various test media: (1) 7H9, pH 7.0, and (2) 7H9, pH 7.0,
100 μM DETA NONOate, all containing 50 μg/mL
hygromycin.
13. Collect day 0 samples as in steps 4–9, and repeat to collect and
process 500 μL aliquots from the cultures set up in step 12 for
day 1 and day 2 samples.
14. For tests of reporter response to hypoxia, grow the reporter
M. tuberculosis strain in stirred, aerated 125 mL Erlenmeyer
flasks to log-phase (OD 600 ~ 0.6) in 35 mL 7H9, pH 7.0
medium containing 50 μg/mL hygromycin for maintenance
of the reporter plasmid.
15. Subculture the log phase bacteria to OD 600 ¼ 0.05 in two
separate stirred, aerated 125 mL Erlenmeyer flasks containing
35 mL of 7H9, pH 7.0, 50 μg/mL hygromycin.
16. Maintain one flask at 37
C with 5% CO 2 and atmospheric O 2 ,
and the second flask at 37
C with 5% CO 2 and 1% O 2
(we utilize a BioSpherix subchamber system for O 2 control).
17. Collect day 0 samples as in steps 4–9, and repeat to collect and
process 500 μL aliquots from the cultures set up in step 15 for
day 6 samples.
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David Giacalone et al.
starting log-phase culture to a 2 mL screw cap tube, and pellet
via centrifugation at 10,000 rpm (9500 Â g) for 5 min.
5. Resuspend the pellet in 1 mL 4% PFA and store at 4
C
overnight.
6. The next day, surface-decontaminate the screw cap tube(s) and
remove from the BSL-3 facility.
7. Pellet the fixed bacteria via centrifugation at 12,000 rpm
(13,500 Â g) for 5 min.
8. Resuspend the pelleted bacteria in 200 μL PBS-Tw (0.1%) and
transfer to a 96-well plate.
9. Store at 4
C, protected from light, until flow cytometry analysis at the end of the assay.
10. Follow steps 4–9 to collect and process 500 μL aliquots from
the cultures set up in step 3 every 3 days for 12 days.
11. For tests of reporter response to NO stress, grow the reporter
M. tuberculosis strain in stirred, aerated 125 mL Erlenmeyer
flasks to log-phase (OD 600 ~ 0.6) in 35 mL 7H9, pH 7.0
medium containing 50 μg/mL hygromycin for maintenance
of the reporter plasmid.
12. Subculture the log phase culture to OD 600 ¼ 0.05 in stirred,
aerated 125 mL Erlenmeyer flasks containing 35 mL of the
various test media: (1) 7H9, pH 7.0, and (2) 7H9, pH 7.0,
100 μM DETA NONOate, all containing 50 μg/mL
hygromycin.
13. Collect day 0 samples as in steps 4–9, and repeat to collect and
process 500 μL aliquots from the cultures set up in step 12 for
day 1 and day 2 samples.
14. For tests of reporter response to hypoxia, grow the reporter
M. tuberculosis strain in stirred, aerated 125 mL Erlenmeyer
flasks to log-phase (OD 600 ~ 0.6) in 35 mL 7H9, pH 7.0
medium containing 50 μg/mL hygromycin for maintenance
of the reporter plasmid.
15. Subculture the log phase bacteria to OD 600 ¼ 0.05 in two
separate stirred, aerated 125 mL Erlenmeyer flasks containing
35 mL of 7H9, pH 7.0, 50 μg/mL hygromycin.
16. Maintain one flask at 37
C with 5% CO 2 and atmospheric O 2 ,
and the second flask at 37
C with 5% CO 2 and 1% O 2
(we utilize a BioSpherix subchamber system for O 2 control).
17. Collect day 0 samples as in steps 4–9, and repeat to collect and
process 500 μL aliquots from the cultures set up in step 15 for
day 6 samples.
372
David Giacalone et al.