2 Materials
2.1 Hypoxia-Induced
Rich Medium
NRP Assay
1. 20% (w/v) Tween 80: Filter-sterilize with a 0.22-μm sterile
filter or autoclave at 121
C for 15 min.
2. 50% (v/v) Glycerol: Filter-sterilize with a 0.22-μm sterile filter
or autoclave at 121
C for 15 min.
3. ADC enrichment: Combine 5 g bovine serum albumin fraction V, 2 g dextrose and 3 g catalase in 100 mL of dH 2 O. Mix
until all components have dissolved. Filter-sterilize with a 0.22μm sterile filter. Store at 4
C (see Note 1).
4. 7H9-ADC: Combine 4.6 g of Middlebrook 7H9 broth base,
4 mL of 50% (v/v) glycerol, 2.5 mL of 20% (w/v) Tween 80 in
900 mL of dH 2 O. Autoclave at 121
C for 15 min. Cool to
below 50
C in a water bath. Add 100 mL of sterile ADC
enrichment that has been equilibrated to 50
C. Store at 4
C.
5. Cell culture vented flasks (25 cm
2 ).
6. Sterile screw cap glass culture tubes: Capacity 11 mL, length
100 mm, diameter 16 mm.
7. Sterile screw caps: screw caps with inert PTFE lined rubber disk
(see Note 2).
8. 1 mg/mL Methylene blue solution: Filter-sterilize with a 0.22μm sterile filter.
9. Control Compounds: Isoniazid (INH), Rifampicin (RIF) and
Metronidazole (MET). Dissolve in DMSO at concentrations in
Table 1.
10. Test Compounds: Dissolve in DMSO or another suitable solvent and make the stock concentration 100Â that of the highest final concentration, as only 1 μL of drug stock should be
added to a well in a 96-well plate.
11. Optical density meter (CO8000, Biochrom, UK).
12. Paraffin Wax (see Note 3).
13. Oxygen impervious microtiter plate seal (see Note 3).
14. Sterile dH 2 O.
15. Anaerobic Chamber (see Note 4).
Table 1
Stock concentrations and final concentrations of control compounds
Drug
Stock concentration
Final concentration in assay
Isoniazid
1 mg/mL
1 μg/mL
Rifampicin
1 mg/mL
0.5 μg/mL
Metronidazole
10 mg/mL
25 μg/mL
In vitro Drug Screening of Hypoxic NRP Mycobacteria
249
2.1 Hypoxia-Induced
Rich Medium
NRP Assay
1. 20% (w/v) Tween 80: Filter-sterilize with a 0.22-μm sterile
filter or autoclave at 121
C for 15 min.
2. 50% (v/v) Glycerol: Filter-sterilize with a 0.22-μm sterile filter
or autoclave at 121
C for 15 min.
3. ADC enrichment: Combine 5 g bovine serum albumin fraction V, 2 g dextrose and 3 g catalase in 100 mL of dH 2 O. Mix
until all components have dissolved. Filter-sterilize with a 0.22μm sterile filter. Store at 4
C (see Note 1).
4. 7H9-ADC: Combine 4.6 g of Middlebrook 7H9 broth base,
4 mL of 50% (v/v) glycerol, 2.5 mL of 20% (w/v) Tween 80 in
900 mL of dH 2 O. Autoclave at 121
C for 15 min. Cool to
below 50
C in a water bath. Add 100 mL of sterile ADC
enrichment that has been equilibrated to 50
C. Store at 4
C.
5. Cell culture vented flasks (25 cm
2 ).
6. Sterile screw cap glass culture tubes: Capacity 11 mL, length
100 mm, diameter 16 mm.
7. Sterile screw caps: screw caps with inert PTFE lined rubber disk
(see Note 2).
8. 1 mg/mL Methylene blue solution: Filter-sterilize with a 0.22μm sterile filter.
9. Control Compounds: Isoniazid (INH), Rifampicin (RIF) and
Metronidazole (MET). Dissolve in DMSO at concentrations in
Table 1.
10. Test Compounds: Dissolve in DMSO or another suitable solvent and make the stock concentration 100Â that of the highest final concentration, as only 1 μL of drug stock should be
added to a well in a 96-well plate.
11. Optical density meter (CO8000, Biochrom, UK).
12. Paraffin Wax (see Note 3).
13. Oxygen impervious microtiter plate seal (see Note 3).
14. Sterile dH 2 O.
15. Anaerobic Chamber (see Note 4).
Table 1
Stock concentrations and final concentrations of control compounds
Drug
Stock concentration
Final concentration in assay
Isoniazid
1 mg/mL
1 μg/mL
Rifampicin
1 mg/mL
0.5 μg/mL
Metronidazole
10 mg/mL
25 μg/mL
In vitro Drug Screening of Hypoxic NRP Mycobacteria
249