2.2 Hypoxic Minimal
Cholesterol Medium
NRP Assay
1. Minimal Broth Base: Dissolve 4.6 g of Middlebrook 7H9
media in 1 L of dH 2 O. Add a magnetic stirrer bar. Autoclave
at 121
C for 15 min.
2. Cholesterol solution: Make 1 mL of Tyloxapol: ethanol
(50:50, v/v). Mix until homologous and heat to 65
C. Dissolve 100 mg of cholesterol (see Note 5).
3. Cholesterol minimal medium: Heat the broth base and the
dissolved cholesterol solution to 65
C. Place the broth base
on a hot magnetic stirrer. Add the additive to the media whilst
it is hot and stirring. Filter-sterilize media with a 0.22-μm filter
into a clean sterile bottle. Store at 4
C.
4. Cell culture vented flasks (25 cm
2 ).
5. Sterile screw cap glass culture tubes: Capacity 11 mL, length
100 mm, diameter 16 mm.
6. Sterile screw caps: screw caps with inert PTFE lined rubber disk
(see Note 2).
7. 1 mg/mL Methylene blue solution: Filter-sterilize with a 0.22μm sterile filter.
8. Control Compounds: Isoniazid (INH), Rifampicin (RIF) and
Metronidazole (MET). Dissolve in DMSO at concentrations in
Table 1.
9. 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.
10. Optical Density (OD) meter (CO8000, Biochrom, UK).
11. Paraffin Wax (see Note 3).
12. Oxygen impervious microtiter plate seal (see Note 3).
13. Sterile dH 2 O.
14. Anaerobic Chamber (see Note 4).
2.3 High-Throughput
Screening Under NRP
Conditions
1. Sterile, non-treated, flat bottom, clear 96-well microtiter plate.
2. Oxygen impervious plate seal (see Note 3).
3. Spectrophotometric plate reader.
2.4 Resuscitation of
an NRP Culture
1. OADC enrichment: Combine 5 mg oleic acid, 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 0.22-μm sterile filter. Store at 4
C (see
Note 1).
2. 7H11-OADC: Combine 20 g of Middlebrook 7H11 agar base
and 10 mL of 50% (w/v) glycerol in 900 mL. Autoclave at
250
Savannah E. R. Gibson et al.
Cholesterol Medium
NRP Assay
1. Minimal Broth Base: Dissolve 4.6 g of Middlebrook 7H9
media in 1 L of dH 2 O. Add a magnetic stirrer bar. Autoclave
at 121
C for 15 min.
2. Cholesterol solution: Make 1 mL of Tyloxapol: ethanol
(50:50, v/v). Mix until homologous and heat to 65
C. Dissolve 100 mg of cholesterol (see Note 5).
3. Cholesterol minimal medium: Heat the broth base and the
dissolved cholesterol solution to 65
C. Place the broth base
on a hot magnetic stirrer. Add the additive to the media whilst
it is hot and stirring. Filter-sterilize media with a 0.22-μm filter
into a clean sterile bottle. Store at 4
C.
4. Cell culture vented flasks (25 cm
2 ).
5. Sterile screw cap glass culture tubes: Capacity 11 mL, length
100 mm, diameter 16 mm.
6. Sterile screw caps: screw caps with inert PTFE lined rubber disk
(see Note 2).
7. 1 mg/mL Methylene blue solution: Filter-sterilize with a 0.22μm sterile filter.
8. Control Compounds: Isoniazid (INH), Rifampicin (RIF) and
Metronidazole (MET). Dissolve in DMSO at concentrations in
Table 1.
9. 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.
10. Optical Density (OD) meter (CO8000, Biochrom, UK).
11. Paraffin Wax (see Note 3).
12. Oxygen impervious microtiter plate seal (see Note 3).
13. Sterile dH 2 O.
14. Anaerobic Chamber (see Note 4).
2.3 High-Throughput
Screening Under NRP
Conditions
1. Sterile, non-treated, flat bottom, clear 96-well microtiter plate.
2. Oxygen impervious plate seal (see Note 3).
3. Spectrophotometric plate reader.
2.4 Resuscitation of
an NRP Culture
1. OADC enrichment: Combine 5 mg oleic acid, 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 0.22-μm sterile filter. Store at 4
C (see
Note 1).
2. 7H11-OADC: Combine 20 g of Middlebrook 7H11 agar base
and 10 mL of 50% (w/v) glycerol in 900 mL. Autoclave at
250
Savannah E. R. Gibson et al.