8. The correlation between CFU/mL and OD 600 measurement
must be determined prior to carrying out the protocol, as
different results may be observed between laboratories depending on the equipment used to measure the OD 600 [36].
9. Incubation time varies according to the mycobacterial species:
6 days in the case of M. tuberculosis and other slow-growing
mycobacteria, 3 days for fast growers like M. smegmatis.
10. Incubation time with resazurin varies according to the mycobacterial species, for example, 2 days in the case of M. tuberculosis and up to 24 h for M. smegmatis [37]. A word of caution
with the readout of the resazurin signal:background, especially
to assess the antimycobacterial activity of compounds that
affect energy/ATP production (e.g., bedaquiline or delamanid), due to the spectral overlap of the oxidized resazurin with
the reduced resorufin product. Direct measurement of intracellular ATP in mycobacterial cells is a confirmatory and more
sensitive and reliable viability readout in these situations [38].
11. A culture of 50 mL will give approximately 35 mL of bacterial
suspension at an OD 600 of 0.8, which is a volume large enough
to test several experimental conditions and to carry out the
control experiments. The volume of the culture can be scaled
up or down depending on the number of experiments to
be done.
12. PBS is supplemented with Tween 80 at a final concentration of
0.05% to get disperse and homogenous suspensions of mycobacterial cells and avoid the formation of bacterial clumps.
13. If using bacterial suspensions adjusted at an OD 600 of 0.8 does
not result in detection of accumulation or efflux, test other
bacterial suspensions adjusted at an OD 600 of 0.4, 0.6, or 1.0.
This optimization may be necessary according to the instrument used to record the fluorescence emitted by the fluorescent substrate accumulated inside the mycobacterial cells.
14. The assay can be done in 0.2-mL microtubes or in 96-well
plates depending on the requirements of the fluorometer to
be used.
15. Other fluorescent compounds such as Hoechst 33342, ethidium monoazide (EMA), rhodamine 123, or orange acridine
can be used instead of EtBr. If so, excitation and emission
wavelengths should be modified, and the levels of relative
fluorescence sensitivity of the assays will also vary according
to the excitability threshold of the compound used for efflux
substrate. Optical density of the bacterial suspension may have
to be adjusted as well.
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Liliana Rodrigues et al.
must be determined prior to carrying out the protocol, as
different results may be observed between laboratories depending on the equipment used to measure the OD 600 [36].
9. Incubation time varies according to the mycobacterial species:
6 days in the case of M. tuberculosis and other slow-growing
mycobacteria, 3 days for fast growers like M. smegmatis.
10. Incubation time with resazurin varies according to the mycobacterial species, for example, 2 days in the case of M. tuberculosis and up to 24 h for M. smegmatis [37]. A word of caution
with the readout of the resazurin signal:background, especially
to assess the antimycobacterial activity of compounds that
affect energy/ATP production (e.g., bedaquiline or delamanid), due to the spectral overlap of the oxidized resazurin with
the reduced resorufin product. Direct measurement of intracellular ATP in mycobacterial cells is a confirmatory and more
sensitive and reliable viability readout in these situations [38].
11. A culture of 50 mL will give approximately 35 mL of bacterial
suspension at an OD 600 of 0.8, which is a volume large enough
to test several experimental conditions and to carry out the
control experiments. The volume of the culture can be scaled
up or down depending on the number of experiments to
be done.
12. PBS is supplemented with Tween 80 at a final concentration of
0.05% to get disperse and homogenous suspensions of mycobacterial cells and avoid the formation of bacterial clumps.
13. If using bacterial suspensions adjusted at an OD 600 of 0.8 does
not result in detection of accumulation or efflux, test other
bacterial suspensions adjusted at an OD 600 of 0.4, 0.6, or 1.0.
This optimization may be necessary according to the instrument used to record the fluorescence emitted by the fluorescent substrate accumulated inside the mycobacterial cells.
14. The assay can be done in 0.2-mL microtubes or in 96-well
plates depending on the requirements of the fluorometer to
be used.
15. Other fluorescent compounds such as Hoechst 33342, ethidium monoazide (EMA), rhodamine 123, or orange acridine
can be used instead of EtBr. If so, excitation and emission
wavelengths should be modified, and the levels of relative
fluorescence sensitivity of the assays will also vary according
to the excitability threshold of the compound used for efflux
substrate. Optical density of the bacterial suspension may have
to be adjusted as well.
242
Liliana Rodrigues et al.