2.3 Determination
of the Steady-State
EtBr Concentration
1. Glucose: 20% (w/v) stock solution in deionized, sterile water.
Store in aliquots at 4
C (see Note 6).
2. 0.2-mL microtubes.
3. PBS: see Subheading 2.2, item 2.
4. PBS (see Subheading 2.2, item 2) plus 0.4% (w/v) glucose.
5. EtBr: see Subheading 2.1, item 5.
6. Fluorometer (e.g., Rotor Gene™ 3000/6000) (see Note 7).
2.4 Demonstration
of the Effect of Efflux
Inhibitors and Cell Wall
Permeabilizers on EtBr
Accumulation
1. PBS: see Subheading 2.2, item 2.
2. Glucose: see Subheading 2.3, item 1.
3. PBS (see Subheading 2.2, item 2) plus 0.4% (w/v) glucose.
4. 0.2-mL microtubes.
5. EtBr: see Subheading 2.1, item 5.
6. Fluorometer (e.g., Rotor Gene™ 3000/6000) (see Note 7).
7. CPZ: see Subheading 2.1, item 6.
8. TZ: see Subheading 2.1, item 7.
9. VP: see Subheading 2.1, item 8.
2.5 EtBr Efflux Assay
See Subheading 2.4, items 1–9.
3 Methods
3.1 Determination
of Minimum Inhibitory
Concentration (MIC)
Before starting any EtBr accumulation or efflux assay, the MIC of
EtBr and compounds to be tested must be determined:
1. Grow mycobacterial strains at 37
C in 10 mL of 7H9 medium
plus Tween until an OD 600 of 0.6–0.8.
2. Dilute the mycobacterial cultures in 7H9 medium plus glycerol
in order to obtain a final concentration of 10
5 CFU/mL (see
Note 8).
3. Transfer 100 μL to the wells of a 96-well plate that contain
100 μL of each compound (e.g., EtBr and efflux inhibitors
CPZ, TZ, and VP) at concentrations prepared from two-fold
serial dilutions in 7H9 medium plus glycerol.
4. Incubate the plates at 37
C for the appropriate period of time
(see Note 9).
5. Add 30 μL of resazurin and incubate at 37
C for the appropriate period of time (see Note 10).
6. A change from blue to pink indicates reduction of resazurin
and, therefore, bacterial growth. The MIC is defined as the
lowest concentration of compound that prevents this color
change [34].
Efflux and Permeability
237
of the Steady-State
EtBr Concentration
1. Glucose: 20% (w/v) stock solution in deionized, sterile water.
Store in aliquots at 4
C (see Note 6).
2. 0.2-mL microtubes.
3. PBS: see Subheading 2.2, item 2.
4. PBS (see Subheading 2.2, item 2) plus 0.4% (w/v) glucose.
5. EtBr: see Subheading 2.1, item 5.
6. Fluorometer (e.g., Rotor Gene™ 3000/6000) (see Note 7).
2.4 Demonstration
of the Effect of Efflux
Inhibitors and Cell Wall
Permeabilizers on EtBr
Accumulation
1. PBS: see Subheading 2.2, item 2.
2. Glucose: see Subheading 2.3, item 1.
3. PBS (see Subheading 2.2, item 2) plus 0.4% (w/v) glucose.
4. 0.2-mL microtubes.
5. EtBr: see Subheading 2.1, item 5.
6. Fluorometer (e.g., Rotor Gene™ 3000/6000) (see Note 7).
7. CPZ: see Subheading 2.1, item 6.
8. TZ: see Subheading 2.1, item 7.
9. VP: see Subheading 2.1, item 8.
2.5 EtBr Efflux Assay
See Subheading 2.4, items 1–9.
3 Methods
3.1 Determination
of Minimum Inhibitory
Concentration (MIC)
Before starting any EtBr accumulation or efflux assay, the MIC of
EtBr and compounds to be tested must be determined:
1. Grow mycobacterial strains at 37
C in 10 mL of 7H9 medium
plus Tween until an OD 600 of 0.6–0.8.
2. Dilute the mycobacterial cultures in 7H9 medium plus glycerol
in order to obtain a final concentration of 10
5 CFU/mL (see
Note 8).
3. Transfer 100 μL to the wells of a 96-well plate that contain
100 μL of each compound (e.g., EtBr and efflux inhibitors
CPZ, TZ, and VP) at concentrations prepared from two-fold
serial dilutions in 7H9 medium plus glycerol.
4. Incubate the plates at 37
C for the appropriate period of time
(see Note 9).
5. Add 30 μL of resazurin and incubate at 37
C for the appropriate period of time (see Note 10).
6. A change from blue to pink indicates reduction of resazurin
and, therefore, bacterial growth. The MIC is defined as the
lowest concentration of compound that prevents this color
change [34].
Efflux and Permeability
237