7. Using a multichannel pipettor, gently mix cells by pipetting up
and down, while moving the tip in a circular motion along the
well bottom. Pop any bubbles using a clean tip.
8. For the M. tuberculosis ΔpanCDΔlysA strain, replace lids and
read the OD 580 . For the M. tuberculosis wild-type strain,
replace the lid with an optical quality PCR sticker as described
in Subheading 3.3, step 14 and determine the OD 580 .
3.10 Data Analysis
1. Convert data from OD 580 to % growth inhibition by comparing data points with wells containing positive and negative
controls.
2. Calculate % inhibition as 100 À (100 Â (OD [TEST
AGENT] À OD [CONTROL] )/(OD [DMSO] À OD [CONTROL] ) for
each well, where TEST AGENT ¼ compounds being assayed;
CONTROL ¼ average optical density of wells containing
rifampicin controls; and DMSO ¼ average optical density of
wells containing DMSO controls.
3. The minimal inhibitory concentration (MIC 90 ) is the lowest
concentration of drug that leads to !90% inhibition of growth.
4. Curve fitting, while not necessary, helps streamline highthroughput analysis of data. Curve fitting software that extrapolates MIC 90 values includes Prism 6.0 (www.graphpad.com)
and Collaborative Drug Discovery (www.collaborativedrug.
com).
5. Calculate the ratio of the MIC 90 -replicating and MIC 90 - nonreplicating (replicating/nonreplicating, or, R/NR) (see Note
34).
6. If the nonreplicating MIC 90 was derived from the 384-well
nonreplicating assay format, use the following definitions: a
replicating active has an R/NR of <0.2; a nonreplicating active
has an R/NR >5.0; and a candidate dual active R/NR of
0.2–5.
7. If the nonreplicating MIC 90 was derived from the 96-well
nonreplicating assay format, use the following definitions: a
replicating active has an R/NR of <0.05; a nonreplicating
active has an R/NR >21; and a candidate dual active R/NR
of 0.05–21.
8. Test candidate dual actives by a CFU-based assay (Subheading
3.4) to assess the compound’s ability to kill nonreplicating
M. tuberculosis (see Note 24). Candidate dual active compounds that fail to kill nonreplicating M. tuberculosis in a
CFU-based readout appear active in the nonreplicating assay
as a result of compound carryover in the replicating outgrowth
phase of the assay [31].
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Ben Gold et al.
and down, while moving the tip in a circular motion along the
well bottom. Pop any bubbles using a clean tip.
8. For the M. tuberculosis ΔpanCDΔlysA strain, replace lids and
read the OD 580 . For the M. tuberculosis wild-type strain,
replace the lid with an optical quality PCR sticker as described
in Subheading 3.3, step 14 and determine the OD 580 .
3.10 Data Analysis
1. Convert data from OD 580 to % growth inhibition by comparing data points with wells containing positive and negative
controls.
2. Calculate % inhibition as 100 À (100 Â (OD [TEST
AGENT] À OD [CONTROL] )/(OD [DMSO] À OD [CONTROL] ) for
each well, where TEST AGENT ¼ compounds being assayed;
CONTROL ¼ average optical density of wells containing
rifampicin controls; and DMSO ¼ average optical density of
wells containing DMSO controls.
3. The minimal inhibitory concentration (MIC 90 ) is the lowest
concentration of drug that leads to !90% inhibition of growth.
4. Curve fitting, while not necessary, helps streamline highthroughput analysis of data. Curve fitting software that extrapolates MIC 90 values includes Prism 6.0 (www.graphpad.com)
and Collaborative Drug Discovery (www.collaborativedrug.
com).
5. Calculate the ratio of the MIC 90 -replicating and MIC 90 - nonreplicating (replicating/nonreplicating, or, R/NR) (see Note
34).
6. If the nonreplicating MIC 90 was derived from the 384-well
nonreplicating assay format, use the following definitions: a
replicating active has an R/NR of <0.2; a nonreplicating active
has an R/NR >5.0; and a candidate dual active R/NR of
0.2–5.
7. If the nonreplicating MIC 90 was derived from the 96-well
nonreplicating assay format, use the following definitions: a
replicating active has an R/NR of <0.05; a nonreplicating
active has an R/NR >21; and a candidate dual active R/NR
of 0.05–21.
8. Test candidate dual actives by a CFU-based assay (Subheading
3.4) to assess the compound’s ability to kill nonreplicating
M. tuberculosis (see Note 24). Candidate dual active compounds that fail to kill nonreplicating M. tuberculosis in a
CFU-based readout appear active in the nonreplicating assay
as a result of compound carryover in the replicating outgrowth
phase of the assay [31].
626
Ben Gold et al.