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. On day of MIC set-up,
sodium nitrite is added to a final concentration of 0.1 mM.
Sodium nitrite prepared fresh as a 1 M stock in water, filtersterilized through a 0.2 μm filter, and diluted 1:10,000 in the
7H9/butyrate/Tyloxapol (pH ¼ 6) medium.
12. Sterile 60 mL square polycarbonate bottles.
13. 25 mg/mL kanamycin prepared in water and filter-sterilized.
Store at À20
C as aliquots.
14. Incubator shaker.
2.2 Preparation of
Plates for Liquid MIC
Determination
1. 96-well V-bottom polypropylene plates.
2. DMSO.
3. Drug controls made as DMSO stocks as appropriate (see Note
4) including 10 mM isoniazid stock, 1 mM rifampicin stock,
0.2 M pyrazinamide stock, and/or 10 mM linezolid stock (see
Note 5).
4. 2–20 μL Multichannel Pipette.
2.3 Preparation of
Plates for Solid
Medium MIC
Determination
1. Same materials described in Subheading 2.1.
2. 96-well, U-bottom, microtiter plates with lids.
3. 1000 μL Multichannel Pipette.
4. Sterile basins.
5. 7H11/OADC medium: Add 21 g Middlebrook 7H11 agar
base (Difco), 5 mL glycerol (add with a 6-mL sterile syringe)
with 900 mL water in a clean glass bottle containing a magnetic
stir bar. Autoclave at 15 pounds pressure at 121
C for 15 min.
Cool to 60
C in a warm water bath. Aseptically add 100 mL
oleic acid-dextrose-albumin complex (OADC). OADC consists of 8.1 g NaCl, 50 g BSA fraction V (low heavy metals),
20 g glucose, 30 mL sodium oleate solution and water to a
total volume of 1000 mL. Sodium oleate solution is made by
warming 30 mL water containing 0.19 mL 10 M NaOH to
56
C, adding 0.6 mL oleic acid, and swirling until clear. The
OADC is filter-sterilized (0.2 μm sterile filters) and stored at
4
C.
6. Heating block.
2.4 Measuring
Growth Using an
Inverted Mirror
1. 96-well, U-bottom, sterile microtiter plates with lids.
2. Sterile basins.
3. Enlarging inverted mirror.
4. Sealable plastic bags, e.g., Ziploc bags.
600
Qinglan Wang and Helena I. M. Boshoff
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. On day of MIC set-up,
sodium nitrite is added to a final concentration of 0.1 mM.
Sodium nitrite prepared fresh as a 1 M stock in water, filtersterilized through a 0.2 μm filter, and diluted 1:10,000 in the
7H9/butyrate/Tyloxapol (pH ¼ 6) medium.
12. Sterile 60 mL square polycarbonate bottles.
13. 25 mg/mL kanamycin prepared in water and filter-sterilized.
Store at À20
C as aliquots.
14. Incubator shaker.
2.2 Preparation of
Plates for Liquid MIC
Determination
1. 96-well V-bottom polypropylene plates.
2. DMSO.
3. Drug controls made as DMSO stocks as appropriate (see Note
4) including 10 mM isoniazid stock, 1 mM rifampicin stock,
0.2 M pyrazinamide stock, and/or 10 mM linezolid stock (see
Note 5).
4. 2–20 μL Multichannel Pipette.
2.3 Preparation of
Plates for Solid
Medium MIC
Determination
1. Same materials described in Subheading 2.1.
2. 96-well, U-bottom, microtiter plates with lids.
3. 1000 μL Multichannel Pipette.
4. Sterile basins.
5. 7H11/OADC medium: Add 21 g Middlebrook 7H11 agar
base (Difco), 5 mL glycerol (add with a 6-mL sterile syringe)
with 900 mL water in a clean glass bottle containing a magnetic
stir bar. Autoclave at 15 pounds pressure at 121
C for 15 min.
Cool to 60
C in a warm water bath. Aseptically add 100 mL
oleic acid-dextrose-albumin complex (OADC). OADC consists of 8.1 g NaCl, 50 g BSA fraction V (low heavy metals),
20 g glucose, 30 mL sodium oleate solution and water to a
total volume of 1000 mL. Sodium oleate solution is made by
warming 30 mL water containing 0.19 mL 10 M NaOH to
56
C, adding 0.6 mL oleic acid, and swirling until clear. The
OADC is filter-sterilized (0.2 μm sterile filters) and stored at
4
C.
6. Heating block.
2.4 Measuring
Growth Using an
Inverted Mirror
1. 96-well, U-bottom, sterile microtiter plates with lids.
2. Sterile basins.
3. Enlarging inverted mirror.
4. Sealable plastic bags, e.g., Ziploc bags.
600
Qinglan Wang and Helena I. M. Boshoff