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.
6. 7H9/cholesterol/BSA/Tyloxapol broth: Mix 4.7 g Middlebrook 7H9 broth base, 5 g BSA fraction V (low heavy metals),
and 0.81 g NaCl in 1000 mL water. Gently warm this medium
to 37
C. To 1 mL ethanol add 500 μL Tyloxapol, heat at
70
C. Then add 96 mg cholesterol and heat again until
completely dissolved. Add this solution to the 1 L prepared
medium and filter-sterilize. This carbon source medium needs
to be kept at 37
C and used within 24 h.
7. 7H9/ DPPC/casitone/Tyloxapol: Mix 4.7 g Middlebrook
7H9 broth base, 0.3 g Bacto casitone, 0.81 g NaCl, and
0.5 mL Tyloxapol (add with a 1-mL sterile syringe) (see Note
2) in 1000 mL water. Make a 14 mg/mL stock of DPPC in
ethanol. It will dissolve at room temperature upon vortexing.
Add 1000 μL of this solution to the broth giving a final DPPC
concentration of 14 μg/mL. Filter-sterilize (0.2 μm filter) and,
although less susceptible to precipitation than cholesterol
media, this medium should be used within a week.
8. 7H9/cholesterol/BSA/Tween 80 broth: Mix 4.7 g Middlebrook 7H9 broth base, 5 g BSA fraction V (low heavy metals),
and 0.81 g NaCl in 1000 mL water. Gently warm this medium
to 37
C. To 1 mL ethanol add 500 μL Tween 80 (see Note 2),
heat at 70
C. Then add 96 mg cholesterol and heat again until
completely dissolved. Add this solution to the 1 L medium and
filter-sterilize. This carbon source medium needs to be kept at
37
C and used within 24 h.
9. 7H9/glucose/ casitone/Tyloxapol medium: Mix 4.7 g Middlebrook 7H9 broth base, 4 g glucose, 0.3 g Bacto casitone,
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 filtersterilized (0.2 μm filter) and can be stored at room temperature
for 2 weeks.
10. 7H9/butyrate/Tyloxapol (pH ¼ 6) medium: Dissolve 4.7 g
Middlebrook 7H9 broth base, 230 μL butyric acid (giving a
final butyrate concentration of 2.5 mM), 5 g BSA fraction V
(low heavy metals), 0.81 g NaCl, and 0.5 mL Tyloxapol in
1000 mL water. The pH of the medium should be 5.95. If the
pH is not around 6, either 6 M HCl or 6 M NaOH is added to
adjust the pH to 6.0. The medium is sterilized by filtration
through a 0.2 μm filter and can be stored at room temperature
for 2 weeks.
11. 7H9/butyrate/nitrite/Tyloxapol (pH ¼ 6) medium: Dissolve
4.7 g Middlebrook 7H9 broth base, 230 μL butyric acid
(giving a final butyrate concentration of 2.5 mM), 5 g BSA
fraction V (low heavy metals), 0.81 g NaCl, and 0.5 mL
MIC Determination for Mycobacterium Tuberculosis
599
filter-sterilized (0.2 μm filter) and can be stored at room temperature for 2 weeks.
6. 7H9/cholesterol/BSA/Tyloxapol broth: Mix 4.7 g Middlebrook 7H9 broth base, 5 g BSA fraction V (low heavy metals),
and 0.81 g NaCl in 1000 mL water. Gently warm this medium
to 37
C. To 1 mL ethanol add 500 μL Tyloxapol, heat at
70
C. Then add 96 mg cholesterol and heat again until
completely dissolved. Add this solution to the 1 L prepared
medium and filter-sterilize. This carbon source medium needs
to be kept at 37
C and used within 24 h.
7. 7H9/ DPPC/casitone/Tyloxapol: Mix 4.7 g Middlebrook
7H9 broth base, 0.3 g Bacto casitone, 0.81 g NaCl, and
0.5 mL Tyloxapol (add with a 1-mL sterile syringe) (see Note
2) in 1000 mL water. Make a 14 mg/mL stock of DPPC in
ethanol. It will dissolve at room temperature upon vortexing.
Add 1000 μL of this solution to the broth giving a final DPPC
concentration of 14 μg/mL. Filter-sterilize (0.2 μm filter) and,
although less susceptible to precipitation than cholesterol
media, this medium should be used within a week.
8. 7H9/cholesterol/BSA/Tween 80 broth: Mix 4.7 g Middlebrook 7H9 broth base, 5 g BSA fraction V (low heavy metals),
and 0.81 g NaCl in 1000 mL water. Gently warm this medium
to 37
C. To 1 mL ethanol add 500 μL Tween 80 (see Note 2),
heat at 70
C. Then add 96 mg cholesterol and heat again until
completely dissolved. Add this solution to the 1 L medium and
filter-sterilize. This carbon source medium needs to be kept at
37
C and used within 24 h.
9. 7H9/glucose/ casitone/Tyloxapol medium: Mix 4.7 g Middlebrook 7H9 broth base, 4 g glucose, 0.3 g Bacto casitone,
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 filtersterilized (0.2 μm filter) and can be stored at room temperature
for 2 weeks.
10. 7H9/butyrate/Tyloxapol (pH ¼ 6) medium: Dissolve 4.7 g
Middlebrook 7H9 broth base, 230 μL butyric acid (giving a
final butyrate concentration of 2.5 mM), 5 g BSA fraction V
(low heavy metals), 0.81 g NaCl, and 0.5 mL Tyloxapol in
1000 mL water. The pH of the medium should be 5.95. If the
pH is not around 6, either 6 M HCl or 6 M NaOH is added to
adjust the pH to 6.0. The medium is sterilized by filtration
through a 0.2 μm filter and can be stored at room temperature
for 2 weeks.
11. 7H9/butyrate/nitrite/Tyloxapol (pH ¼ 6) medium: Dissolve
4.7 g Middlebrook 7H9 broth base, 230 μL butyric acid
(giving a final butyrate concentration of 2.5 mM), 5 g BSA
fraction V (low heavy metals), 0.81 g NaCl, and 0.5 mL
MIC Determination for Mycobacterium Tuberculosis
599