2.5 Comprehensive
Nonreplicating Stress
Deconvolution
1. See Subheading 2.1, items 1–15.
2. NR deconvolution base medium: Prepare 500 mL of NR complete medium as described in Subheading 2.1, item 12 (for
M. tuberculosis wild type) or Subheading 2.1, item 13 (for
M. tuberculosis ΔpanCDΔlysA) with the following modifications: do not adjust pH and omit adding butyrate and NaNO 2 .
3. NR deconvolution media A–H: Aliquot 40 mL of NR deconvolution base medium into 8 Â 50 mL sterile tubes labeled A–
H. Add 20 μL of 99% w/v butyrate to appropriate tubes
according to Table 2. Adjust pH of all tubes to 5.0 or 7.0
according to Table 2. Filter sterilize all eight deconvolution
media. NaNO 2 is added to appropriate deconvolution media at
0.5 mM immediately prior to initiating experiment.
2.6 Identification
of NaNO 2 -Dependent
Compounds
1. See Subheading 2.4.
noethyl)-N-(2-ammonioethyl)amino)diazen-1-ium-1,2-diolate in 1 mL of 0.01 N NaOH and store at À80
C. Dilute
DETA-NO stock in 7H9 complete medium to prepare 100Â
stock solutions (10, 20, and 40 mM) prior to addition to
M. tuberculosis.
2.7 Preparation
of M. tuberculosis
for Replicating Assays
1. See Subheading 2.1, items 1–4.
2.8 Replicating
Assay Protocol
for 384-Well Format
1. See Subheading 2.1, items 1–4.
2. 384-well black, clear flat bottom, tissue culture-treated microplates with lids (see Note 4).
3. Multidrop Combi Reagent Dispenser with small tube cassette.
4. Plastic containers with clip-lids that hold up to twelve 96- or
384-well lidded microplates in two stacks (approximately
10
00
 7
00
 5
00 ; length  width  height) (see Note 5).
2.9 Replicating
Assay Protocol
for 96-Well Format
1. See Subheading 2.1, items 1–4.
2. 96-well, sterile tissue culture-treated microplates with lids.
3. One or more of the following control compounds: rifampicin,
isoniazid, ethionamide, ethambutol, streptomycin, kanamycin,
and/or para-aminosalicylic acid at 10 mM in DMSO.
2.10 Data Analysis
1. Data analysis software such as Microsoft Excel, Prism 6.0
(GraphPad), or Collaborative Drug Discovery (CDD).
2. See Subheading 2.4 for CFU assays.
Multi-stress Model for Nonreplicating M. tuberculosis
619
Nonreplicating Stress
Deconvolution
1. See Subheading 2.1, items 1–15.
2. NR deconvolution base medium: Prepare 500 mL of NR complete medium as described in Subheading 2.1, item 12 (for
M. tuberculosis wild type) or Subheading 2.1, item 13 (for
M. tuberculosis ΔpanCDΔlysA) with the following modifications: do not adjust pH and omit adding butyrate and NaNO 2 .
3. NR deconvolution media A–H: Aliquot 40 mL of NR deconvolution base medium into 8 Â 50 mL sterile tubes labeled A–
H. Add 20 μL of 99% w/v butyrate to appropriate tubes
according to Table 2. Adjust pH of all tubes to 5.0 or 7.0
according to Table 2. Filter sterilize all eight deconvolution
media. NaNO 2 is added to appropriate deconvolution media at
0.5 mM immediately prior to initiating experiment.
2.6 Identification
of NaNO 2 -Dependent
Compounds
1. See Subheading 2.4.
noethyl)-N-(2-ammonioethyl)amino)diazen-1-ium-1,2-diolate in 1 mL of 0.01 N NaOH and store at À80
C. Dilute
DETA-NO stock in 7H9 complete medium to prepare 100Â
stock solutions (10, 20, and 40 mM) prior to addition to
M. tuberculosis.
2.7 Preparation
of M. tuberculosis
for Replicating Assays
1. See Subheading 2.1, items 1–4.
2.8 Replicating
Assay Protocol
for 384-Well Format
1. See Subheading 2.1, items 1–4.
2. 384-well black, clear flat bottom, tissue culture-treated microplates with lids (see Note 4).
3. Multidrop Combi Reagent Dispenser with small tube cassette.
4. Plastic containers with clip-lids that hold up to twelve 96- or
384-well lidded microplates in two stacks (approximately
10
00
 7
00
 5
00 ; length  width  height) (see Note 5).
2.9 Replicating
Assay Protocol
for 96-Well Format
1. See Subheading 2.1, items 1–4.
2. 96-well, sterile tissue culture-treated microplates with lids.
3. One or more of the following control compounds: rifampicin,
isoniazid, ethionamide, ethambutol, streptomycin, kanamycin,
and/or para-aminosalicylic acid at 10 mM in DMSO.
2.10 Data Analysis
1. Data analysis software such as Microsoft Excel, Prism 6.0
(GraphPad), or Collaborative Drug Discovery (CDD).
2. See Subheading 2.4 for CFU assays.
Multi-stress Model for Nonreplicating M. tuberculosis
619