16. When setting up a new protocol in the Rotor-Gene, it may be
necessary to perform the “Gain Optimization” to determine
the best gain setting. This will insure that all data are collected
within the dynamic range of the detector. If the gain is too low,
the signal will be lost in background noise. If it is too high, all
signals will be lost off scale (saturated). In the case of the
Rotor-Gene, the gain range for each channel is À10 to
10, where À10 is the least sensitive and 10 is the most sensitive.
By our experience, a gain of 8 is appropriate for most
experiments.
17. Other compounds such as Phe-Arg-β-naphthylamide (PAβN)
may be used. MICs of the compounds to be tested on each
bacterial strain must be conducted prior to their use, to test for
antibacterial activity. Each inhibitor is used at a final concentration that should not exceed ¼ MIC in order to not compromise the cellular viability.
18. In order to measure EtBr efflux, it is necessary to promote the
maximum accumulation of EtBr. This is accomplished by
exposing the bacteria to EtBr under conditions that limit efflux
to its minimal activity, namely: a temperature of 25
C, absence
of glucose, and the presence of the most effective compound
selected from accumulation experiments. All these conditions
that decrease efflux must be optimized experimentally according to the instrument used to record the fluorescence emitted
by the fluorescent substrate accumulated inside the mycobacterial cells [18, 20, 21].
19. We have found that for most strains, efflux of EtBr takes place
within the first 10–15 min of the assay at 37
C, and this period
of time may be shorter in the case of drug-resistant strains or
strains overexpressing efflux pumps.
20. It is advisable to keep the microtubes or the 96-well plate on
ice until all reagents are added.
21. In the case of strains with overexpression of efflux pumps, it
might be necessary to adjust the measurement time,
performing shorter acquisition cycles (e.g., every 30 s) due to
the increased efflux activity.
22. When assaying different strains and mutants of M. tuberculosis,
the reference condition could be the assay of reference strain
H37Rv, whereas when assaying the effect of a compound in the
accumulation of efflux of EtBr, the reference condition is the
assay in the absence of any such compound. In both cases, high
RFF values indicate that cells accumulate more EtBr under the
tested conditions than under the reference conditions and vice
versa for negative RFF values.
Efflux and Permeability
243
necessary to perform the “Gain Optimization” to determine
the best gain setting. This will insure that all data are collected
within the dynamic range of the detector. If the gain is too low,
the signal will be lost in background noise. If it is too high, all
signals will be lost off scale (saturated). In the case of the
Rotor-Gene, the gain range for each channel is À10 to
10, where À10 is the least sensitive and 10 is the most sensitive.
By our experience, a gain of 8 is appropriate for most
experiments.
17. Other compounds such as Phe-Arg-β-naphthylamide (PAβN)
may be used. MICs of the compounds to be tested on each
bacterial strain must be conducted prior to their use, to test for
antibacterial activity. Each inhibitor is used at a final concentration that should not exceed ¼ MIC in order to not compromise the cellular viability.
18. In order to measure EtBr efflux, it is necessary to promote the
maximum accumulation of EtBr. This is accomplished by
exposing the bacteria to EtBr under conditions that limit efflux
to its minimal activity, namely: a temperature of 25
C, absence
of glucose, and the presence of the most effective compound
selected from accumulation experiments. All these conditions
that decrease efflux must be optimized experimentally according to the instrument used to record the fluorescence emitted
by the fluorescent substrate accumulated inside the mycobacterial cells [18, 20, 21].
19. We have found that for most strains, efflux of EtBr takes place
within the first 10–15 min of the assay at 37
C, and this period
of time may be shorter in the case of drug-resistant strains or
strains overexpressing efflux pumps.
20. It is advisable to keep the microtubes or the 96-well plate on
ice until all reagents are added.
21. In the case of strains with overexpression of efflux pumps, it
might be necessary to adjust the measurement time,
performing shorter acquisition cycles (e.g., every 30 s) due to
the increased efflux activity.
22. When assaying different strains and mutants of M. tuberculosis,
the reference condition could be the assay of reference strain
H37Rv, whereas when assaying the effect of a compound in the
accumulation of efflux of EtBr, the reference condition is the
assay in the absence of any such compound. In both cases, high
RFF values indicate that cells accumulate more EtBr under the
tested conditions than under the reference conditions and vice
versa for negative RFF values.
Efflux and Permeability
243