In summary, these methods can be used to evaluate influx and
efflux of fluorescent substrates by mycobacteria, providing valuable
information on cell wall and membrane permeability, efflux inhibitory activity of compounds and, thus, are useful to predict the
contribution of permeability and efflux to the drug resistance
phenotype.
2 Materials
2.1 Determination
of Minimum Inhibitory
Concentration (MIC)
1. Mycobacterial strains. Commonly, laboratory reference strains
such as M. tuberculosis H37Rv can be used for these protocols.
Other strains such as mutants deleted in genes encoding efflux
pumps, clinical isolates or other mycobacterial species can be
used as well (see Note 1).
2. 7H9 medium plus Tween: dissolve 4.7 g Middlebrook 7H9
broth medium in 900 mL water, add Tween 80 to 0.05%
w/v. Autoclave for 15 min at 121
C and add 100 mL of
oleic acid-albumin–dextrose–catalase supplement (OADC)
(Becton Dickinson) (see Note 2).
3. 7H9 medium plus glycerol: dissolve 4.7 g Middlebrook 7H9
broth medium in 900 mL water, add glycerol to 0.5% w/v. Autoclave for 15 min at 121
C and add 100 mL of oleic acidalbumin–dextrose–catalase supplement (OADC) (Becton
Dickinson) (see Note 2).
4. 96-well microtiter plates.
5. EtBr (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide): stock solution of 1 mg/mL in deionized, sterile water.
Store at 4
C and protect from light (see Note 3).
6. Chlorpromazine (CPZ): stock solution of 1 mg/mL in deionized, sterile water. Store at À20
C and protect from light.
7. Thioridazine (TZ): stock solution of 1 mg/mL in deionized,
sterile water. Store at À20
C and protect from light.
8. Verapamil (VP): stock solution of 10 mg/mL in deionized,
sterile water. Store at À20
C and protect from light.
9. Resazurin: stock solution of 0.1 mg/mL in deionized, sterile
water. Store at 4
C and protect from light (see Note 4).
2.2 Preparation
of Cultures
1. 7H9 medium: see Subheading 2.1, items 2 and 3.
2. Phosphate-buffered saline (PBS): dissolve 8 g NaCl, 0.2 g of
KCl, 1.44 g of Na 2 HPO 4 , and 0.24 g of KH 2 PO 4 in 800 mL of
deionized water. Adjust the pH to 7.4 with HCl and the
volume to 1 L with additional deionized water. Sterilize by
autoclaving for 15 min at 121
C (see Note 5).
3. PBS-Tw: PBS (see Subheading 2.2, item 2) plus 0.05% (w/v)
Tween 80.
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Liliana Rodrigues et al.
efflux of fluorescent substrates by mycobacteria, providing valuable
information on cell wall and membrane permeability, efflux inhibitory activity of compounds and, thus, are useful to predict the
contribution of permeability and efflux to the drug resistance
phenotype.
2 Materials
2.1 Determination
of Minimum Inhibitory
Concentration (MIC)
1. Mycobacterial strains. Commonly, laboratory reference strains
such as M. tuberculosis H37Rv can be used for these protocols.
Other strains such as mutants deleted in genes encoding efflux
pumps, clinical isolates or other mycobacterial species can be
used as well (see Note 1).
2. 7H9 medium plus Tween: dissolve 4.7 g Middlebrook 7H9
broth medium in 900 mL water, add Tween 80 to 0.05%
w/v. Autoclave for 15 min at 121
C and add 100 mL of
oleic acid-albumin–dextrose–catalase supplement (OADC)
(Becton Dickinson) (see Note 2).
3. 7H9 medium plus glycerol: dissolve 4.7 g Middlebrook 7H9
broth medium in 900 mL water, add glycerol to 0.5% w/v. Autoclave for 15 min at 121
C and add 100 mL of oleic acidalbumin–dextrose–catalase supplement (OADC) (Becton
Dickinson) (see Note 2).
4. 96-well microtiter plates.
5. EtBr (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide): stock solution of 1 mg/mL in deionized, sterile water.
Store at 4
C and protect from light (see Note 3).
6. Chlorpromazine (CPZ): stock solution of 1 mg/mL in deionized, sterile water. Store at À20
C and protect from light.
7. Thioridazine (TZ): stock solution of 1 mg/mL in deionized,
sterile water. Store at À20
C and protect from light.
8. Verapamil (VP): stock solution of 10 mg/mL in deionized,
sterile water. Store at À20
C and protect from light.
9. Resazurin: stock solution of 0.1 mg/mL in deionized, sterile
water. Store at 4
C and protect from light (see Note 4).
2.2 Preparation
of Cultures
1. 7H9 medium: see Subheading 2.1, items 2 and 3.
2. Phosphate-buffered saline (PBS): dissolve 8 g NaCl, 0.2 g of
KCl, 1.44 g of Na 2 HPO 4 , and 0.24 g of KH 2 PO 4 in 800 mL of
deionized water. Adjust the pH to 7.4 with HCl and the
volume to 1 L with additional deionized water. Sterilize by
autoclaving for 15 min at 121
C (see Note 5).
3. PBS-Tw: PBS (see Subheading 2.2, item 2) plus 0.05% (w/v)
Tween 80.
236
Liliana Rodrigues et al.