(0.2 μm filter) (see Note 3). ADC is made by mixing 50 g BSA
fraction V (low heavy metals), 20 g glucose, 8.1 g NaCl in 1 L
water. The ADC is filter-sterilized (0.2 μm filter) and stored at
4
C.
3. 7H9/Glucose/BSA/Tyloxapol broth: Mix 4.7 g Middlebrook
7H9 broth base, 4 g glucose, 5 g BSA fraction V (low heavy
metals), 0.81 g NaCl, and 0.5 mL Tyloxapol (add with a 1-mL
sterile syringe) (see Note 2) in 1000 mL water. The medium is
filter-sterilized (0.2 μm filter) and can be stored at room temperature for 2 weeks.
4. 7H9/dipalmitoyl phosphatidylcholine (DPPC)/cholesterol/
BSA/Tyloxapol broth: Mix 4.7 g Middlebrook 7H9 broth
base, 5 g BSA fraction V (low heavy metals), 0.81 g NaCl in
1000 mL water. Make a 10 mg/mL stock of DPPC in ethanol.
It will dissolve at room temperature upon vortexing. Add
500 μL of this solution to the broth base giving a final DPPC
concentration of 5 μg/mL. To 1 mL ethanol add 500 μL
Tyloxapol, heat at 70
C. Then add 24 mg cholesterol and
heat again until completely dissolved. Add this solution to the
1 L medium described above and filter-sterilize (0.2 μm filter).
This carbon source medium needs to be kept at 37
C and used
within 24 h.
5. 7H9/Glucose/BSA/Tween 80 broth: Mix 4.7 g Middlebrook
7H9 broth base, 4 g glucose, 5 g BSA fraction V (low heavy
metals), 0.81 g NaCl, and 0.5 mL Tween 80 (add with a 1-mL
Table 1
MIC in μM of drugs in series of media
Compounds
Media
7H9/
ADC/
Tw
7H9/
Glucose/
BSA/Tw
7H9/
Glucose/
BSA/Tx
7H9/Glucose/
Casitone/Tx
7H9/
DPPC/
BSA/Tw
7H9/DPPC/
Casitone/Tx
7H11/
OADC
agar
EMB
1
2
2
1
1
1
3.5
SM
0.25
0.5
0.5
0.25
0.5
0.25
0.5
LNZ
1.56
3.1
1.56
1.56
3.1
3.1
1.56
BDQ
1.5
5
1.5
1.5
5
1.5
1.5
MXF
0.1
0.2
0.2
0.2
0.2
0.2
0.2
INH
0.28
0.28
0.28
0.28
0.13
0.13
0.28
PA824
0.1
0.3–0.8
0.3
0.2
0.3
0.2
0.2
RIF
0.005 0.01
0.08
0.08
0.01
0.05
0.16
EMB ethambutol, SM streptomycin, LNZ linezolid, BDQ bedaquiline, MXF moxifloxacin, INH isoniazid, RIF rifampicin, Tw tween 80, Tx tyloxapol
598
Qinglan Wang and Helena I. M. Boshoff
fraction V (low heavy metals), 20 g glucose, 8.1 g NaCl in 1 L
water. The ADC is filter-sterilized (0.2 μm filter) and stored at
4
C.
3. 7H9/Glucose/BSA/Tyloxapol broth: Mix 4.7 g Middlebrook
7H9 broth base, 4 g glucose, 5 g BSA fraction V (low heavy
metals), 0.81 g NaCl, and 0.5 mL Tyloxapol (add with a 1-mL
sterile syringe) (see Note 2) in 1000 mL water. The medium is
filter-sterilized (0.2 μm filter) and can be stored at room temperature for 2 weeks.
4. 7H9/dipalmitoyl phosphatidylcholine (DPPC)/cholesterol/
BSA/Tyloxapol broth: Mix 4.7 g Middlebrook 7H9 broth
base, 5 g BSA fraction V (low heavy metals), 0.81 g NaCl in
1000 mL water. Make a 10 mg/mL stock of DPPC in ethanol.
It will dissolve at room temperature upon vortexing. Add
500 μL of this solution to the broth base giving a final DPPC
concentration of 5 μg/mL. To 1 mL ethanol add 500 μL
Tyloxapol, heat at 70
C. Then add 24 mg cholesterol and
heat again until completely dissolved. Add this solution to the
1 L medium described above and filter-sterilize (0.2 μm filter).
This carbon source medium needs to be kept at 37
C and used
within 24 h.
5. 7H9/Glucose/BSA/Tween 80 broth: Mix 4.7 g Middlebrook
7H9 broth base, 4 g glucose, 5 g BSA fraction V (low heavy
metals), 0.81 g NaCl, and 0.5 mL Tween 80 (add with a 1-mL
Table 1
MIC in μM of drugs in series of media
Compounds
Media
7H9/
ADC/
Tw
7H9/
Glucose/
BSA/Tw
7H9/
Glucose/
BSA/Tx
7H9/Glucose/
Casitone/Tx
7H9/
DPPC/
BSA/Tw
7H9/DPPC/
Casitone/Tx
7H11/
OADC
agar
EMB
1
2
2
1
1
1
3.5
SM
0.25
0.5
0.5
0.25
0.5
0.25
0.5
LNZ
1.56
3.1
1.56
1.56
3.1
3.1
1.56
BDQ
1.5
5
1.5
1.5
5
1.5
1.5
MXF
0.1
0.2
0.2
0.2
0.2
0.2
0.2
INH
0.28
0.28
0.28
0.28
0.13
0.13
0.28
PA824
0.1
0.3–0.8
0.3
0.2
0.3
0.2
0.2
RIF
0.005 0.01
0.08
0.08
0.01
0.05
0.16
EMB ethambutol, SM streptomycin, LNZ linezolid, BDQ bedaquiline, MXF moxifloxacin, INH isoniazid, RIF rifampicin, Tw tween 80, Tx tyloxapol
598
Qinglan Wang and Helena I. M. Boshoff