3.2 Preparation
of Cultures
1. Grow mycobacterial strains (see Note 1) at 37
C in 50 mL (see
Note 11) of 7H9 medium plus Tween until they reach OD 600
of 0.6–0.8.
2. Centrifuge cultures at 3000–4000 Â g for 10 min and discard
the supernatant.
3. Wash the pellet in 25 mL of PBS-Tw (see Note 12) and adjust
the OD 600 to 0.8 with PBS (see Note 13).
3.3 Determination
of the Steady-State
EtBr Concentration
1. Add glucose to yield a final concentration of 0.4% (w/v) to the
bacterial suspension prepared in Subheading 3.2 (see Note 6).
2. In 0.2-mL microtubes (see Note 14), prepare serial dilutions of
EtBr from 0.125 to 4 μg/mL (do not exceed ¼ MIC) in PBS
plus 0.4% (w/v) glucose in a volume of 50 μL per tube.
3. Aliquot 50 μL of bacterial suspension into 0.2-mL microtubes
containing the serial dilutions of EtBr (see Note 15). A final
volume of 100 μL should be obtained in each tube.
4. Include the following controls:
Control 1: add 50 μL of PBS to 50 μL of EtBr at the selected
concentrations; no bacterial cells must be added.
Control 2: add 50 μL of PBS and 50 μL of the bacterial
suspension with no EtBr.
5. Place the 0.2-mL microtubes in the Rotor-Gene fluorometer
and program the instrument with the following settings: a
temperature of 37
C; a unit of time for each measurement
(cycle) of 60 s; the number of cycles necessary to obtain the
total period of time (i.e., 60 cycles to obtain a 60 min assay);
and excitation and detection wavelengths of 530 nm and the
590 nm, respectively (see Notes 7 and 16).
6. Select the highest EtBr concentration that allows readable
steady-state equilibrium without compromising bacterial viability, for use in demonstration of the effect of efflux inhibitors
or permeabilizing agents on EtBr accumulation (see Note 7).
3.4 Determination
of the Effect
of Potential Efflux
Inhibitors/
Permeabilizing Agents
on EtBr Accumulation
1. Prepare a dilution of EtBr in PBS plus 0.4% (w/v) glucose to
reach two times the highest EtBr concentration that allows
readable steady-state equilibrium without compromising bacterial viability, selected as described in Subheading 3.3. Transfer
47.5 μL of this EtBr dilution to 0.2-mL microtubes.
2. Add 5 μL of one compound (CPZ, TZ, VP, or any other, see
Note 17) at ¼ MIC.
3. Add 47.5 μL of bacterial suspension prepared as described in
Subheading 3.2.
4. Include the following controls:
238
Liliana Rodrigues et al.
of Cultures
1. Grow mycobacterial strains (see Note 1) at 37
C in 50 mL (see
Note 11) of 7H9 medium plus Tween until they reach OD 600
of 0.6–0.8.
2. Centrifuge cultures at 3000–4000 Â g for 10 min and discard
the supernatant.
3. Wash the pellet in 25 mL of PBS-Tw (see Note 12) and adjust
the OD 600 to 0.8 with PBS (see Note 13).
3.3 Determination
of the Steady-State
EtBr Concentration
1. Add glucose to yield a final concentration of 0.4% (w/v) to the
bacterial suspension prepared in Subheading 3.2 (see Note 6).
2. In 0.2-mL microtubes (see Note 14), prepare serial dilutions of
EtBr from 0.125 to 4 μg/mL (do not exceed ¼ MIC) in PBS
plus 0.4% (w/v) glucose in a volume of 50 μL per tube.
3. Aliquot 50 μL of bacterial suspension into 0.2-mL microtubes
containing the serial dilutions of EtBr (see Note 15). A final
volume of 100 μL should be obtained in each tube.
4. Include the following controls:
Control 1: add 50 μL of PBS to 50 μL of EtBr at the selected
concentrations; no bacterial cells must be added.
Control 2: add 50 μL of PBS and 50 μL of the bacterial
suspension with no EtBr.
5. Place the 0.2-mL microtubes in the Rotor-Gene fluorometer
and program the instrument with the following settings: a
temperature of 37
C; a unit of time for each measurement
(cycle) of 60 s; the number of cycles necessary to obtain the
total period of time (i.e., 60 cycles to obtain a 60 min assay);
and excitation and detection wavelengths of 530 nm and the
590 nm, respectively (see Notes 7 and 16).
6. Select the highest EtBr concentration that allows readable
steady-state equilibrium without compromising bacterial viability, for use in demonstration of the effect of efflux inhibitors
or permeabilizing agents on EtBr accumulation (see Note 7).
3.4 Determination
of the Effect
of Potential Efflux
Inhibitors/
Permeabilizing Agents
on EtBr Accumulation
1. Prepare a dilution of EtBr in PBS plus 0.4% (w/v) glucose to
reach two times the highest EtBr concentration that allows
readable steady-state equilibrium without compromising bacterial viability, selected as described in Subheading 3.3. Transfer
47.5 μL of this EtBr dilution to 0.2-mL microtubes.
2. Add 5 μL of one compound (CPZ, TZ, VP, or any other, see
Note 17) at ¼ MIC.
3. Add 47.5 μL of bacterial suspension prepared as described in
Subheading 3.2.
4. Include the following controls:
238
Liliana Rodrigues et al.