4 Notes
1. M. tuberculosis wild-type (North or ATCC strains) or
M. tuberculosis ΔpanCDΔlysA H37Rv (mc
2 6220) can be used;
the latter can be obtained from Dr. W. Jacobs, Jr. at the Albert
Einstein College of Medicine.
2. Record manufacturer, type of BSA (protease-free or not; heatshock or not), and lot number. The majority of problems we
encountered with cell outgrowth have been traced back to BSA
quality. We have occasionally observed that cells take longer
than 7–10 days to achieve an OD 580 of >0.4 in the outgrowth
phase or completely fail to outgrow. The one-time cells
completely failed to outgrow was traced back to the outgrowth
medium, 7H9-ADN, which had been prepared with a faulty
batch of BSA. We now recommend using Roche BSA Fraction
V heat shock or the Middlebrook OADC supplement. OADC
supplement is more expensive, but the benefits include slightly
faster outgrowth and less variability.
3. Make sure to filter sterilize medium components as directed in
Subheading 2.1. These steps are essential to ensure components do not precipitate during autoclaving.
4. Supplement autoclaved ddH 2 O with “Water bath treatment”
(Andwin, 190009) fungicide at 4 mL/L and use for water pans
for hypoxic chambers and tissue culture incubators.
5. We prefer Greiner black, clear-bottomed tissue culture-treated
plates. These plates encourage uniform M. tuberculosis growth
across well bottoms, which is essential to obtain high-quality
data when using an optical density readout.
6. Prior to use, wash all plastic containers that will hold 96- and
384-well plates in the incubators with 1% Vesphene (Steris)
followed by a second rinse with Spor Klenz (Steris). Allow to
air-dry.
7. Do not keep cultures for longer than a month to avoid
clumping.
8. The exact volume is not essential because cells will ultimately be
further diluted.
9. For CFU-based assays, an OD 580 ¼ 0.1 of a single-cell suspension is the equivalent of ~5 Â 10
7 CFU/mL.
10. Often, this step is unnecessary as the 30-min period overlaps
the time spent dispensing the cells in microplates.
11. We have not yet adapted the nonreplicating assay to 1536- or
3072-well plates but anticipate it may work with significant
optimization to solve liquid evaporation.
Multi-stress Model for Nonreplicating M. tuberculosis
627
1. M. tuberculosis wild-type (North or ATCC strains) or
M. tuberculosis ΔpanCDΔlysA H37Rv (mc
2 6220) can be used;
the latter can be obtained from Dr. W. Jacobs, Jr. at the Albert
Einstein College of Medicine.
2. Record manufacturer, type of BSA (protease-free or not; heatshock or not), and lot number. The majority of problems we
encountered with cell outgrowth have been traced back to BSA
quality. We have occasionally observed that cells take longer
than 7–10 days to achieve an OD 580 of >0.4 in the outgrowth
phase or completely fail to outgrow. The one-time cells
completely failed to outgrow was traced back to the outgrowth
medium, 7H9-ADN, which had been prepared with a faulty
batch of BSA. We now recommend using Roche BSA Fraction
V heat shock or the Middlebrook OADC supplement. OADC
supplement is more expensive, but the benefits include slightly
faster outgrowth and less variability.
3. Make sure to filter sterilize medium components as directed in
Subheading 2.1. These steps are essential to ensure components do not precipitate during autoclaving.
4. Supplement autoclaved ddH 2 O with “Water bath treatment”
(Andwin, 190009) fungicide at 4 mL/L and use for water pans
for hypoxic chambers and tissue culture incubators.
5. We prefer Greiner black, clear-bottomed tissue culture-treated
plates. These plates encourage uniform M. tuberculosis growth
across well bottoms, which is essential to obtain high-quality
data when using an optical density readout.
6. Prior to use, wash all plastic containers that will hold 96- and
384-well plates in the incubators with 1% Vesphene (Steris)
followed by a second rinse with Spor Klenz (Steris). Allow to
air-dry.
7. Do not keep cultures for longer than a month to avoid
clumping.
8. The exact volume is not essential because cells will ultimately be
further diluted.
9. For CFU-based assays, an OD 580 ¼ 0.1 of a single-cell suspension is the equivalent of ~5 Â 10
7 CFU/mL.
10. Often, this step is unnecessary as the 30-min period overlaps
the time spent dispensing the cells in microplates.
11. We have not yet adapted the nonreplicating assay to 1536- or
3072-well plates but anticipate it may work with significant
optimization to solve liquid evaporation.
Multi-stress Model for Nonreplicating M. tuberculosis
627