2. According to the manufacturer, OADC must be added to
Middlebrook 7H9 broth medium. However, in many laboratories, ADC (albumin-dextrose-catalase) is used instead of
OADC. The choice between OADC and ADC may depend
on many factors, such as availability, price, and the particular
growth requirements of mycobacterial species or strains to be
assayed.
3. EtBr is a powerful teratogen. Safety precautions must be followed when working with EtBr. Non-absorbent gloves (e.g.,
nitrile) should be worn when handling EtBr and stock solutions should be prepared under a fume hood. Care should be
taken to avoid contaminating the working area. All disposable
materials used for EtBr preparation or manipulation should be
identified as contaminated and disposed according to local laws
and regulations, such as in appropriate containers for
subsequent incineration.
4. Resazurin stock solutions stored at 4
C may be used for no
longer than 2 weeks.
5. Alternatively, some suppliers provide PBS tablets (Sigma
Aldrich). In this case, dissolve one tablet in 200 mL of deionized sterile water, according to the manufacturer’s
instructions.
6. Glucose solutions do not need to be sterilized if used immediately. If they are kept at 4
C, they should be used within 3 days
of preparation. Glucose is added to provide an energy source
for the activity of the efflux systems.
7. This method can be performed using several types of equipment, for example real-time PCR instruments (e.g., RotorGene™ 3000/6000, Corbett Life Science) and 96-well plate
fluorometers (e.g., Safire
2
™, Tecan). Both platforms allow
accurate real-time measurements of accumulation and efflux
of EtBr [18, 20–25]. Other fluorometers may also be used.
In our experience, the Rotor Gene™ 3000/6000 (0.2-mL
microtube system) and the Safire
2
™ (microplate system) are
suitable instruments to be used in these assays. Any equipment
should be set up for excitation at 530 nm and detection of
fluorescence of EtBr at 590 nm. Most fluorometers provide
measurements of fluorescence at every time point during the
course of the experiment, along with a graphical plot of the
increase of fluorescence over time. This allows the real-time
visualization of when the steady-state equilibrium is reached at
a given EtBr concentration (such as that shown in Fig. 1 for a
concentration of 0.125 μg/mL). This should be reached at no
more than 10% of the relative fluorescence that the instrument
can record [21].
Efflux and Permeability
241
Middlebrook 7H9 broth medium. However, in many laboratories, ADC (albumin-dextrose-catalase) is used instead of
OADC. The choice between OADC and ADC may depend
on many factors, such as availability, price, and the particular
growth requirements of mycobacterial species or strains to be
assayed.
3. EtBr is a powerful teratogen. Safety precautions must be followed when working with EtBr. Non-absorbent gloves (e.g.,
nitrile) should be worn when handling EtBr and stock solutions should be prepared under a fume hood. Care should be
taken to avoid contaminating the working area. All disposable
materials used for EtBr preparation or manipulation should be
identified as contaminated and disposed according to local laws
and regulations, such as in appropriate containers for
subsequent incineration.
4. Resazurin stock solutions stored at 4
C may be used for no
longer than 2 weeks.
5. Alternatively, some suppliers provide PBS tablets (Sigma
Aldrich). In this case, dissolve one tablet in 200 mL of deionized sterile water, according to the manufacturer’s
instructions.
6. Glucose solutions do not need to be sterilized if used immediately. If they are kept at 4
C, they should be used within 3 days
of preparation. Glucose is added to provide an energy source
for the activity of the efflux systems.
7. This method can be performed using several types of equipment, for example real-time PCR instruments (e.g., RotorGene™ 3000/6000, Corbett Life Science) and 96-well plate
fluorometers (e.g., Safire
2
™, Tecan). Both platforms allow
accurate real-time measurements of accumulation and efflux
of EtBr [18, 20–25]. Other fluorometers may also be used.
In our experience, the Rotor Gene™ 3000/6000 (0.2-mL
microtube system) and the Safire
2
™ (microplate system) are
suitable instruments to be used in these assays. Any equipment
should be set up for excitation at 530 nm and detection of
fluorescence of EtBr at 590 nm. Most fluorometers provide
measurements of fluorescence at every time point during the
course of the experiment, along with a graphical plot of the
increase of fluorescence over time. This allows the real-time
visualization of when the steady-state equilibrium is reached at
a given EtBr concentration (such as that shown in Fig. 1 for a
concentration of 0.125 μg/mL). This should be reached at no
more than 10% of the relative fluorescence that the instrument
can record [21].
Efflux and Permeability
241