4. Middlebrook 7H10 + OADC: To 900 mL of DI water, add
1.5 g disodium phosphate, 1.5 g monopotassium phosphate,
0.5 g L-glutamic acid, 0.5 g ammonium sulfate, 0.4 g sodium
citrate, 40.0 mg ferric ammonium citrate, 25.0 mg magnesium
sulfate, 1.0 mg zinc sulfate, 1.0 mg copper sulfate, 1.0 mg
pyridoxine, 0.5 mg calcium chloride, 0.5 mg biotin, 0.25 mg
malachite green, 5.0 mL glycerol, and 15.0 g agar (see Note 1).
Heat and stir. Autoclave at 121
C for 15 min. Cool agar to
45–50
C before adding aseptically 100 mL of OADC enrichment. Mix and pour into petri dishes. Cool and harden according to instructional guidelines (see Note 4).
2.8 Large-Scale
Growth
of M. tuberculosis
for Research
1. M. tuberculosis, 1 mL frozen stock.
2. 7H11 + OADC large agar plate (15 Â 150 mm): To 900 mL of
DI water, add 5.0 mL glycerol, 1.5 g disodium phosphate,
1.5 g monopotassium phosphate, 1.0 g pancreatic digest of
casein, 0.5 g L-glutamic acid, 0.5 g ammonium sulfate, 0.4 g
sodium citrate, 50.0 mg magnesium sulfate, 40.0 mg ferric
ammonium citrate, 1.0 mg malachite green, 1.0 mg pyridoxine, 0.5 mg biotin, and 15.0 g agar (see Note 1). Heat and stir
to dissolve. Autoclave at 121
C for 15 min. Cool agar to
45–50
C before aseptically adding 100 mL of OADC enrichment (see Subheading 2.7, item 2). Mix and pour into Petri
dishes. For use with large-scale growth for research protocol,
pour into large 15 Â 150 mm plates. Cool and harden according to instructional guidelines.
3. Inoculation loops, 10 μL.
4. Plastic zip freezer storage bags.
5. 2.8 L Fernbach flasks (see Note 5).
6. 1 L sterile glycerol alanine salts (GAS) medium: To 900 mL of
DI water, add 0.3 g Bactocasitone, 0.05 g ferric ammonium
citrate, 4.0 g dibasic anhydrous potassium phosphate, 2.0 g
citric acid, 1.0 g L-alanine, 1.2 g magnesium chloride, 0.6 g
potassium sulfate, 2.0 g ammonium chloride, 1.8 mL sodium
hydroxide 10 M, and 10 mL glycerol (see Note 1). Dissolve
components and bring volume up to 1 L. Adjust pH to 6.6.
Upscale recipe to larger volume as needed and distribute to
flasks once pH adjusted. Autoclave flasks with media on liquid
cycle at 121
C for 45 min.
7. Cell scrapers.
8. Parafilm.
9. Orbital platform shaker with appropriate metal clamps.
10. Serological pipettor with 10 and 50 mL pipettes.
11. 1 L roller bottles with solid plastic caps containing 400 mL
GAS medium (see Note 6).
26
Elizabeth Wallace et al.
1.5 g disodium phosphate, 1.5 g monopotassium phosphate,
0.5 g L-glutamic acid, 0.5 g ammonium sulfate, 0.4 g sodium
citrate, 40.0 mg ferric ammonium citrate, 25.0 mg magnesium
sulfate, 1.0 mg zinc sulfate, 1.0 mg copper sulfate, 1.0 mg
pyridoxine, 0.5 mg calcium chloride, 0.5 mg biotin, 0.25 mg
malachite green, 5.0 mL glycerol, and 15.0 g agar (see Note 1).
Heat and stir. Autoclave at 121
C for 15 min. Cool agar to
45–50
C before adding aseptically 100 mL of OADC enrichment. Mix and pour into petri dishes. Cool and harden according to instructional guidelines (see Note 4).
2.8 Large-Scale
Growth
of M. tuberculosis
for Research
1. M. tuberculosis, 1 mL frozen stock.
2. 7H11 + OADC large agar plate (15 Â 150 mm): To 900 mL of
DI water, add 5.0 mL glycerol, 1.5 g disodium phosphate,
1.5 g monopotassium phosphate, 1.0 g pancreatic digest of
casein, 0.5 g L-glutamic acid, 0.5 g ammonium sulfate, 0.4 g
sodium citrate, 50.0 mg magnesium sulfate, 40.0 mg ferric
ammonium citrate, 1.0 mg malachite green, 1.0 mg pyridoxine, 0.5 mg biotin, and 15.0 g agar (see Note 1). Heat and stir
to dissolve. Autoclave at 121
C for 15 min. Cool agar to
45–50
C before aseptically adding 100 mL of OADC enrichment (see Subheading 2.7, item 2). Mix and pour into Petri
dishes. For use with large-scale growth for research protocol,
pour into large 15 Â 150 mm plates. Cool and harden according to instructional guidelines.
3. Inoculation loops, 10 μL.
4. Plastic zip freezer storage bags.
5. 2.8 L Fernbach flasks (see Note 5).
6. 1 L sterile glycerol alanine salts (GAS) medium: To 900 mL of
DI water, add 0.3 g Bactocasitone, 0.05 g ferric ammonium
citrate, 4.0 g dibasic anhydrous potassium phosphate, 2.0 g
citric acid, 1.0 g L-alanine, 1.2 g magnesium chloride, 0.6 g
potassium sulfate, 2.0 g ammonium chloride, 1.8 mL sodium
hydroxide 10 M, and 10 mL glycerol (see Note 1). Dissolve
components and bring volume up to 1 L. Adjust pH to 6.6.
Upscale recipe to larger volume as needed and distribute to
flasks once pH adjusted. Autoclave flasks with media on liquid
cycle at 121
C for 45 min.
7. Cell scrapers.
8. Parafilm.
9. Orbital platform shaker with appropriate metal clamps.
10. Serological pipettor with 10 and 50 mL pipettes.
11. 1 L roller bottles with solid plastic caps containing 400 mL
GAS medium (see Note 6).
26
Elizabeth Wallace et al.