2 Materials
2.1 Preparation
of Nonreplicating
(NR) M. tuberculosis
1. M. tuberculosis wild-type H37Rv or ΔpanCDΔlysA (see
Note 1).
2. Albumin-Dextrose-NaCl (ADN) 10Â stock: mix 5 g of BSA
(see Note 2), 2 g of D-glucose, and 0.85 g of NaCl and add
double-distilled water (ddH 2 O) to 100 mL. Filter sterilize and
wrap in foil at 4
C.
3. Replicating (R) medium for M. tuberculosis wild-type: dissolve
4.7 g of 7H9 powder and 2 mL of 100% glycerol in 900 mL of
ddH 2 O (7H9 medium). Autoclave and add 100 mL of ADN or
oleic acid–albumin–dextrose–catalase (OADC) and 10 mL of
2% Tyloxapol. These media are called 7H9-ADN or
7H9-OADC, respectively. M. tuberculosis wild type can use
either ADN or OADC, and M. tuberculosis ΔpanCDΔlysA
requires OADC.
4. Replicating medium for M. tuberculosis ΔpanCDΔlysA: To
7H9-OADC, add 6 mL of 50% glycerol in water, 10 mL of
20% w/v casamino acids, 1 mL of 24 mg/mL pantothenate,
and 3.13 mL of 80 mg/mL lysine.
5. Albumin-NaCl (AN) 10Â stock: mix 5 g of BSA (see Note 2),
0.85 g of NaCl and add ddH 2 O to 100 mL. Filter sterilize and
wrap in foil at 4
C.
6. NH 4 Cl 1000Â stock: dissolve 10 g of NH 4 Cl in 100 mL of
ddH 2 O, and filter sterilize.
7. ZnSO 4 10,000Â stock: dissolve 1 g of ZnSO 4 in 100 mL of
ddH 2 O, followed by filter sterilization.
8. 1 M NaNO 2 : dissolve 69 mg of NaNO 2 in 1 mL of ddH 2 O.Prepare a fresh stock of NaNO 2 the day of the experiment.
9. 2% v/v Tyloxapol: dissolve 2 mL of 100% Tyloxapol in 98 mL
of ddH 2 O and filter sterilize. Wrap in foil at 4
C.
10. PBS–Tyloxapol (PBS-Tyl): dissolve 10 mL of 2% v/v Tyloxapol in 1 L of Dulbecco’s PBS without Ca
2+ and Mg
2+ . Filter
sterilize and wrap in foil at 4
C.
11. Modified Sauton’s medium: dissolve 0.5 g of KH 2 PO 4 , 0.5 g
of MgSO 4 , 0.05 g of ferric ammonium citrate in 900 mL of
ddH 2 O. Prepare fresh and use immediately without filter
sterilization.
12. Nonreplicating complete medium (without NaNO 2 ) for
M. tuberculosis wild type: mix 900 mL of modified Sauton’s
medium, 100 mL of AN, 10 mL of 2% Tyloxapol, 100 μL of
10% w/v ZnSO 4 , 1 mL of 10% w/v NH 4 Cl, and 500 μL of
99% w/v butyrate. Adjust pH to 5.0 with 2 N NaOH. Filter
sterilize (see Note 3).
Multi-stress Model for Nonreplicating M. tuberculosis
617
2.1 Preparation
of Nonreplicating
(NR) M. tuberculosis
1. M. tuberculosis wild-type H37Rv or ΔpanCDΔlysA (see
Note 1).
2. Albumin-Dextrose-NaCl (ADN) 10Â stock: mix 5 g of BSA
(see Note 2), 2 g of D-glucose, and 0.85 g of NaCl and add
double-distilled water (ddH 2 O) to 100 mL. Filter sterilize and
wrap in foil at 4
C.
3. Replicating (R) medium for M. tuberculosis wild-type: dissolve
4.7 g of 7H9 powder and 2 mL of 100% glycerol in 900 mL of
ddH 2 O (7H9 medium). Autoclave and add 100 mL of ADN or
oleic acid–albumin–dextrose–catalase (OADC) and 10 mL of
2% Tyloxapol. These media are called 7H9-ADN or
7H9-OADC, respectively. M. tuberculosis wild type can use
either ADN or OADC, and M. tuberculosis ΔpanCDΔlysA
requires OADC.
4. Replicating medium for M. tuberculosis ΔpanCDΔlysA: To
7H9-OADC, add 6 mL of 50% glycerol in water, 10 mL of
20% w/v casamino acids, 1 mL of 24 mg/mL pantothenate,
and 3.13 mL of 80 mg/mL lysine.
5. Albumin-NaCl (AN) 10Â stock: mix 5 g of BSA (see Note 2),
0.85 g of NaCl and add ddH 2 O to 100 mL. Filter sterilize and
wrap in foil at 4
C.
6. NH 4 Cl 1000Â stock: dissolve 10 g of NH 4 Cl in 100 mL of
ddH 2 O, and filter sterilize.
7. ZnSO 4 10,000Â stock: dissolve 1 g of ZnSO 4 in 100 mL of
ddH 2 O, followed by filter sterilization.
8. 1 M NaNO 2 : dissolve 69 mg of NaNO 2 in 1 mL of ddH 2 O.Prepare a fresh stock of NaNO 2 the day of the experiment.
9. 2% v/v Tyloxapol: dissolve 2 mL of 100% Tyloxapol in 98 mL
of ddH 2 O and filter sterilize. Wrap in foil at 4
C.
10. PBS–Tyloxapol (PBS-Tyl): dissolve 10 mL of 2% v/v Tyloxapol in 1 L of Dulbecco’s PBS without Ca
2+ and Mg
2+ . Filter
sterilize and wrap in foil at 4
C.
11. Modified Sauton’s medium: dissolve 0.5 g of KH 2 PO 4 , 0.5 g
of MgSO 4 , 0.05 g of ferric ammonium citrate in 900 mL of
ddH 2 O. Prepare fresh and use immediately without filter
sterilization.
12. Nonreplicating complete medium (without NaNO 2 ) for
M. tuberculosis wild type: mix 900 mL of modified Sauton’s
medium, 100 mL of AN, 10 mL of 2% Tyloxapol, 100 μL of
10% w/v ZnSO 4 , 1 mL of 10% w/v NH 4 Cl, and 500 μL of
99% w/v butyrate. Adjust pH to 5.0 with 2 N NaOH. Filter
sterilize (see Note 3).
Multi-stress Model for Nonreplicating M. tuberculosis
617