3.6 Normalization
of Data
and Presentation
of Results
The final results of the accumulation assay (determination of EtBr
steady-state) are calculated as follows:
1. Subtract the EtBr baseline fluorescence obtained in Control
1 (PBS + EtBr, with no bacterial cells) from the one
(s) corresponding to the bacteria + EtBr.
2. Make sure that fluorescence measurements in Control 2 should
be very low, below those in Control 1, with no increases
over time.
3. If the latter is not the case, conditions should be checked.
The results of the accumulation assay in the presence of efflux
inhibitors are calculated as follows:
1. Subtract the EtBr baseline fluorescence obtained in Control
4 (PBS + EtBr + efflux inhibitor, with no bacterial cells) from
the one(s) corresponding to the bacteria + EtBr + efflux
inhibitor.
2. Make sure that fluorescence measurements in Control 3 should
be very low, below those in Control 4, with no increases
over time.
3. If the latter is not the case, conditions should be checked.
In any of these experiments, fluorescence can be expressed in
terms of relative final fluorescence (RFF) at the last time point
(minute 60) of the assay in comparison with reference conditions
by using the formula (RF assay À RF ref )/RF ref , where RF assay is the
relative fluorescence at the last time point of the EtBr accumulation
assay and RF ref is the relative fluorescence at the last time point of
the EtBr accumulation assay under the reference conditions
[23, 35] (see Note 22).
The results of efflux experiments are presented in terms of
relative fluorescence, calculated by comparing the data obtained
for the bacterial population under conditions that allow maximum
efflux (bacteria in the presence of glucose and absence of efflux
inhibitor) against the data obtained from the control that contains
the EtBr-loaded cells under conditions that restrict efflux (presence
of efflux inhibitor and no glucose). The relative fluorescence thus
corresponds to the ratio of fluorescence that remains per unit of
time, relative to the EtBr-loaded cells.
4 Notes
1. Some mycobacterial strains are pathogenic, so cultures and
further manipulations must be done in biosafety level 2 or
3 laboratories according to the requirements for each specific
strain.
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Liliana Rodrigues et al.
of Data
and Presentation
of Results
The final results of the accumulation assay (determination of EtBr
steady-state) are calculated as follows:
1. Subtract the EtBr baseline fluorescence obtained in Control
1 (PBS + EtBr, with no bacterial cells) from the one
(s) corresponding to the bacteria + EtBr.
2. Make sure that fluorescence measurements in Control 2 should
be very low, below those in Control 1, with no increases
over time.
3. If the latter is not the case, conditions should be checked.
The results of the accumulation assay in the presence of efflux
inhibitors are calculated as follows:
1. Subtract the EtBr baseline fluorescence obtained in Control
4 (PBS + EtBr + efflux inhibitor, with no bacterial cells) from
the one(s) corresponding to the bacteria + EtBr + efflux
inhibitor.
2. Make sure that fluorescence measurements in Control 3 should
be very low, below those in Control 4, with no increases
over time.
3. If the latter is not the case, conditions should be checked.
In any of these experiments, fluorescence can be expressed in
terms of relative final fluorescence (RFF) at the last time point
(minute 60) of the assay in comparison with reference conditions
by using the formula (RF assay À RF ref )/RF ref , where RF assay is the
relative fluorescence at the last time point of the EtBr accumulation
assay and RF ref is the relative fluorescence at the last time point of
the EtBr accumulation assay under the reference conditions
[23, 35] (see Note 22).
The results of efflux experiments are presented in terms of
relative fluorescence, calculated by comparing the data obtained
for the bacterial population under conditions that allow maximum
efflux (bacteria in the presence of glucose and absence of efflux
inhibitor) against the data obtained from the control that contains
the EtBr-loaded cells under conditions that restrict efflux (presence
of efflux inhibitor and no glucose). The relative fluorescence thus
corresponds to the ratio of fluorescence that remains per unit of
time, relative to the EtBr-loaded cells.
4 Notes
1. Some mycobacterial strains are pathogenic, so cultures and
further manipulations must be done in biosafety level 2 or
3 laboratories according to the requirements for each specific
strain.
240
Liliana Rodrigues et al.