Control 1: add 50 μL of PBS to 50 μL of EtBr at the selected
concentration; no bacterial cells must be added.
Control 2: add 50 μL of PBS and 50 μL of the bacterial
suspension with no EtBr.
Control 3: mix 95 μL of PBS and 5 μL of efflux inhibitor.
Control 4: mix 47.5 μL of PBS, 47.5 μL of EtBr dilution, and
5 μL of efflux inhibitor.
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 compound resulting in the highest accumulation of
EtBr by bacterial cells for being used in EtBr efflux assays.
3.5 EtBr Efflux Assay
1. Prepare 10 mL of bacterial suspension prepared as described in
Subheading 3.2. Add EtBr (at the concentration selected in
Subheading 3.3) and the compound selected in Subheading
3.4, step 6 (see Note 17).
2. Mix and incubate at 25
C for 60 min (see Note 18).
3. Harvest the EtBr-loaded cells at 3000–4000 Â g for 5 min at
4
C and discard the supernatant.
4. Resuspend to an OD 600 of 0.8 in EtBr-free cold PBS (see Note
13).
5. Keep cells on ice until use in order to minimize EtBr efflux;
otherwise, EtBr efflux would promptly start (see Note 19).
Proceed to the next step immediately.
6. Aliquot 95 μL of cells into 0.2-mL microtubes (see Note 20).
7. Add glucose to yield a final concentration of 0.4% and add the
efflux inhibitors at ¼ the MIC in order to obtain the following
conditions: (a) Minimal efflux ¼ bacteria without glucose +
efflux inhibitor; (b) Baseline efflux ¼ bacteria without glucose;
(c) Maximal efflux ¼ bacteria with glucose; and (d) Efflux
inhibitor ¼ bacteria with glucose + efflux inhibitor.
8. 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 Note 21).
Efflux and Permeability
239
concentration; no bacterial cells must be added.
Control 2: add 50 μL of PBS and 50 μL of the bacterial
suspension with no EtBr.
Control 3: mix 95 μL of PBS and 5 μL of efflux inhibitor.
Control 4: mix 47.5 μL of PBS, 47.5 μL of EtBr dilution, and
5 μL of efflux inhibitor.
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 compound resulting in the highest accumulation of
EtBr by bacterial cells for being used in EtBr efflux assays.
3.5 EtBr Efflux Assay
1. Prepare 10 mL of bacterial suspension prepared as described in
Subheading 3.2. Add EtBr (at the concentration selected in
Subheading 3.3) and the compound selected in Subheading
3.4, step 6 (see Note 17).
2. Mix and incubate at 25
C for 60 min (see Note 18).
3. Harvest the EtBr-loaded cells at 3000–4000 Â g for 5 min at
4
C and discard the supernatant.
4. Resuspend to an OD 600 of 0.8 in EtBr-free cold PBS (see Note
13).
5. Keep cells on ice until use in order to minimize EtBr efflux;
otherwise, EtBr efflux would promptly start (see Note 19).
Proceed to the next step immediately.
6. Aliquot 95 μL of cells into 0.2-mL microtubes (see Note 20).
7. Add glucose to yield a final concentration of 0.4% and add the
efflux inhibitors at ¼ the MIC in order to obtain the following
conditions: (a) Minimal efflux ¼ bacteria without glucose +
efflux inhibitor; (b) Baseline efflux ¼ bacteria without glucose;
(c) Maximal efflux ¼ bacteria with glucose; and (d) Efflux
inhibitor ¼ bacteria with glucose + efflux inhibitor.
8. 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 Note 21).
Efflux and Permeability
239