add DMSO to columns 6 and 18 at this stage as they are
reserved for controls which are added separately (controls are
added into column 11 and 12 in the 96-well plates).
3. Perform 1:2 or 1:3 serial dilutions across the row from column
1 to 12 (skip over column 6 going directly from column 5 to
column 7). Similarly perform serial dilutions from column
13 to 24, again skipping over column 18, which is reserved
for controls (for 96-well plates only a single set of serial dilutions are performed across the row from column 1 to 10).
4. Add 50 μL of DMSO (control 1) in Column 6 and add 50 μL
of 0.1 μg/mL Rifampicin (control 2) to column 18 in 384-well
plates (for 96-well plates add 50 μL DMSO to column 11 and
50 μL of 0.1 μg/mL Rifampicin to column 12).
5. Include isoniazid starting at 400 μg/mL (the final assay concentration is 4 μg/mL) or another standard antitubercular
drug in one row as a control.
6. Depending on the readout for growth that will be measured
use the appropriate assay plate type (detailed in Subheadings
2.3, 2.4 and 2.5 for the resazurin, ATP and OD readouts,
respectively). Prepare the assay plates by transferring 250 nL
of compound into each well of the 384-well assay plates
(or 2 μL in 96-well plates) (see Note 8).
3.2 Preparation
of Inoculum
1. Inoculate 100 μL of a glycerol stock of M. tuberculosis, M. bovis
or M. smegmatis into 10 mL of 7H9-ADC-Tyloxapol in a
25 cm
2 flask (see Note 9).
2. Incubate standing at 37
C for approximately 15 days for
M. tuberculosis and M. bovis and 3 days for M. smegmatis.
3. Subculture in 10 mL of Middlebrook 7H9-ADC-Tyloxapol to
OD 600 ¼ 0.01 (see Note 10) in 25 cm
2 flask.
4. Incubate at 37
C standing for 4–6 days (M. tuberculosis and
M. bovis) or 16 h (M. smegmatis) (see Note 11).
5. Standardize the inoculum to approximately 1 Â 10
7 CFU/mL
(see Note 12).
6. Dilute 1:200 in 7H9-ADC-Tyloxapol so the final concentration is 5 Â 10
4 CFU/mL.
7. Dispense 25 μL of inoculum into each well across the entire
384-well assay plate (prepared in Subheading 3.1, step 6) with
the Multidrop Combi Reagent Dispenser or with the electronic
multichannel pipettes. For 96-well plates dispense 200 μL of
the inoculum into each well.
8. Wrap the plates in aluminium foil and place them in a sealed
box to prevent evaporation.
642
Esther Pe ´ rez-Herra ´ n and Alfonso Mendoza
reserved for controls which are added separately (controls are
added into column 11 and 12 in the 96-well plates).
3. Perform 1:2 or 1:3 serial dilutions across the row from column
1 to 12 (skip over column 6 going directly from column 5 to
column 7). Similarly perform serial dilutions from column
13 to 24, again skipping over column 18, which is reserved
for controls (for 96-well plates only a single set of serial dilutions are performed across the row from column 1 to 10).
4. Add 50 μL of DMSO (control 1) in Column 6 and add 50 μL
of 0.1 μg/mL Rifampicin (control 2) to column 18 in 384-well
plates (for 96-well plates add 50 μL DMSO to column 11 and
50 μL of 0.1 μg/mL Rifampicin to column 12).
5. Include isoniazid starting at 400 μg/mL (the final assay concentration is 4 μg/mL) or another standard antitubercular
drug in one row as a control.
6. Depending on the readout for growth that will be measured
use the appropriate assay plate type (detailed in Subheadings
2.3, 2.4 and 2.5 for the resazurin, ATP and OD readouts,
respectively). Prepare the assay plates by transferring 250 nL
of compound into each well of the 384-well assay plates
(or 2 μL in 96-well plates) (see Note 8).
3.2 Preparation
of Inoculum
1. Inoculate 100 μL of a glycerol stock of M. tuberculosis, M. bovis
or M. smegmatis into 10 mL of 7H9-ADC-Tyloxapol in a
25 cm
2 flask (see Note 9).
2. Incubate standing at 37
C for approximately 15 days for
M. tuberculosis and M. bovis and 3 days for M. smegmatis.
3. Subculture in 10 mL of Middlebrook 7H9-ADC-Tyloxapol to
OD 600 ¼ 0.01 (see Note 10) in 25 cm
2 flask.
4. Incubate at 37
C standing for 4–6 days (M. tuberculosis and
M. bovis) or 16 h (M. smegmatis) (see Note 11).
5. Standardize the inoculum to approximately 1 Â 10
7 CFU/mL
(see Note 12).
6. Dilute 1:200 in 7H9-ADC-Tyloxapol so the final concentration is 5 Â 10
4 CFU/mL.
7. Dispense 25 μL of inoculum into each well across the entire
384-well assay plate (prepared in Subheading 3.1, step 6) with
the Multidrop Combi Reagent Dispenser or with the electronic
multichannel pipettes. For 96-well plates dispense 200 μL of
the inoculum into each well.
8. Wrap the plates in aluminium foil and place them in a sealed
box to prevent evaporation.
642
Esther Pe ´ rez-Herra ´ n and Alfonso Mendoza