3.3 Measuring
Growth Using
Resazurin
1. Incubate the plates (from Subheading 3.2) at 37
C for 6 days
for M. tuberculosis or M. bovis or 2 days for M. smegmatis.
2. Add 10 μL/well (384-well plate) or 25 μL/well (96-well plate)
of resazurin solution with the Multidrop Combi Reagent Dispenser or with the electronic multichannel pipettes (see Note
13). Mixing is required.
3. Wrap the plates in aluminium foil and place them in a sealed
box to prevent evaporation.
4. Incubate at 37
C for 2 days (M. tuberculosis and M. bovis) or
1 days (M. smegmatis).
5. Measure fluorescence with Excitation 530 nm; Emission
590 nm; (Cut-off 570 nm) (see Notes 14 and 15).
6. Analyze data as described under Subheading 3.7.
3.4 Measuring
Growth by ATP
This assay measures growth by determining the amount of ATP,
which is related to the number of metabolically active bacteria (see
Note 16).
1. Incubate the plates from Subheading 3.2 at 37
C for 7 days
(M. tuberculosis and M. bovis) or for 2 days (M. smegmatis).
2. Remove the plates from the incubator and equilibrate to room
temperature (see Note 17) at least 1 h.
3. Add 25 μL of reconstituted BacTiter-Glo™ reagent (equilibrated to room temperature) to each well for 384-well plates
(100 μL for 96-well plates) (see Notes 18 and 19).
4. Incubate at room temperature: 30 min for M. tuberculosis,
8 min for M. bovis, or 5 min for M. smegmatis (see Note 20).
5. Measure luminescence with integration time 250 ms (endpoint) (see Note 21) in SpectraMax M5.
6. Analyze data as described in Subheading 3.7.
3.5 Measuring
Growth by Optical
Density
1. Incubate the plates from Subheading 3.2 at 37
C for 10 days
(M. tuberculosis or M. bovis) or 2 days (M. smegmatis).
2. Equilibrate the plates to room temperature for 30 min to
eliminate any water condensation on the top of the plates (see
Note 22).
3. Transfer plate to reader and measure OD 600 .
4. Analyze data as described in Subheading 3.7.
3.6 Measuring
Intracellular MICs
1. Culture M. tuberculosis H37Rv:luc in 7H9-ADC-Tyoxapol
until the OD 600 is between 0.5 and 0.8.
2. Divide 160 mL of culture into 4 Â 50 mL conical tubes and
pellet at 4000 Â g for 10 min.
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643
Growth Using
Resazurin
1. Incubate the plates (from Subheading 3.2) at 37
C for 6 days
for M. tuberculosis or M. bovis or 2 days for M. smegmatis.
2. Add 10 μL/well (384-well plate) or 25 μL/well (96-well plate)
of resazurin solution with the Multidrop Combi Reagent Dispenser or with the electronic multichannel pipettes (see Note
13). Mixing is required.
3. Wrap the plates in aluminium foil and place them in a sealed
box to prevent evaporation.
4. Incubate at 37
C for 2 days (M. tuberculosis and M. bovis) or
1 days (M. smegmatis).
5. Measure fluorescence with Excitation 530 nm; Emission
590 nm; (Cut-off 570 nm) (see Notes 14 and 15).
6. Analyze data as described under Subheading 3.7.
3.4 Measuring
Growth by ATP
This assay measures growth by determining the amount of ATP,
which is related to the number of metabolically active bacteria (see
Note 16).
1. Incubate the plates from Subheading 3.2 at 37
C for 7 days
(M. tuberculosis and M. bovis) or for 2 days (M. smegmatis).
2. Remove the plates from the incubator and equilibrate to room
temperature (see Note 17) at least 1 h.
3. Add 25 μL of reconstituted BacTiter-Glo™ reagent (equilibrated to room temperature) to each well for 384-well plates
(100 μL for 96-well plates) (see Notes 18 and 19).
4. Incubate at room temperature: 30 min for M. tuberculosis,
8 min for M. bovis, or 5 min for M. smegmatis (see Note 20).
5. Measure luminescence with integration time 250 ms (endpoint) (see Note 21) in SpectraMax M5.
6. Analyze data as described in Subheading 3.7.
3.5 Measuring
Growth by Optical
Density
1. Incubate the plates from Subheading 3.2 at 37
C for 10 days
(M. tuberculosis or M. bovis) or 2 days (M. smegmatis).
2. Equilibrate the plates to room temperature for 30 min to
eliminate any water condensation on the top of the plates (see
Note 22).
3. Transfer plate to reader and measure OD 600 .
4. Analyze data as described in Subheading 3.7.
3.6 Measuring
Intracellular MICs
1. Culture M. tuberculosis H37Rv:luc in 7H9-ADC-Tyoxapol
until the OD 600 is between 0.5 and 0.8.
2. Divide 160 mL of culture into 4 Â 50 mL conical tubes and
pellet at 4000 Â g for 10 min.
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