13. Nonreplicating complete medium (without NaNO 2 ) for
M. tuberculosis ΔpanCDΔlysA: supplement 1 L of nonreplicating medium (see Subheading 2.1, item 12) with 1 mL of
24 mg/mL pantothenate and 3.13 mL of 80 mg/mL lysine.
Adjust pH to 5.0 with 2 N NaOH. Filter sterilize (see Note 3).
14. Biospherix hypoxia chamber with ProOx 110 and ProCO 2
regulators or an O 2 -regulated incubator (Binder GmbH) (see
Note 4).
15. Spectrophotometer.
2.2 Nonreplicating
Protocol for 384-Well
Format
1. See Subheading 2.1, items 1–15.
2. 384-well black, clear flat bottom, tissue culture-treated microplates with lids (see Note 5).
3. Multidrop Combi Reagent Dispenser with small tube cassette.
4. Plastic containers with clip-lids that hold up to twelve 96- or
384-well lidded microplates in two stacks (approximately
10
00
 7
00
 5
00 ; length  width  height) (see Note 6).
5. Cotton swabs (Puritan).
6. Oxyphenbutazone (10 mM) in DMSO.
7. Rifampicin (10 mg/mL) in DMSO.
2.3 Nonreplicating
Protocol for 96-Well
Format
1. See Subheading 2.1, items 1–15.
2. 96-well, sterile tissue culture-treated microplates with lids.
3. Oxyphenbutazone (10 mM) in DMSO.
4. Rifampicin (10 mg/mL) in DMSO.
2.4 Enumeration
of Colony
Forming Units
1. See Subheading 2.1, items 1–15.
2. 96-well, sterile tissue culture-treated microplates.
3. 7H11-OADC agar: dissolve 21 g of 7H11 powder and 5 mL of
100% glycerol in 900 mL of ddH 2 O. Add a large stir bar.
Autoclave, place flask on a magnetic stir plate set at medium
speed, and allow medium to slowly cool to touch. Aseptically
add 100 mL of OADC supplement and gently mix to avoid
introducing bubbles.
4. Tristyle Petri plates containing 7H11-OADC: In a sterile
hood, using a sterile 25 mL pipette, dispense 8 mL of the
7H11-OADC into each section of the triplate and allow to
solidify. Store plates in sterile biosafety hood overnight to dry.
5. Oxyphenbutazone (10 mM) in DMSO.
6. Rifampicin (10 mg/mL) in DMSO.
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Ben Gold et al.
M. tuberculosis ΔpanCDΔlysA: supplement 1 L of nonreplicating medium (see Subheading 2.1, item 12) with 1 mL of
24 mg/mL pantothenate and 3.13 mL of 80 mg/mL lysine.
Adjust pH to 5.0 with 2 N NaOH. Filter sterilize (see Note 3).
14. Biospherix hypoxia chamber with ProOx 110 and ProCO 2
regulators or an O 2 -regulated incubator (Binder GmbH) (see
Note 4).
15. Spectrophotometer.
2.2 Nonreplicating
Protocol for 384-Well
Format
1. See Subheading 2.1, items 1–15.
2. 384-well black, clear flat bottom, tissue culture-treated microplates with lids (see Note 5).
3. Multidrop Combi Reagent Dispenser with small tube cassette.
4. Plastic containers with clip-lids that hold up to twelve 96- or
384-well lidded microplates in two stacks (approximately
10
00
 7
00
 5
00 ; length  width  height) (see Note 6).
5. Cotton swabs (Puritan).
6. Oxyphenbutazone (10 mM) in DMSO.
7. Rifampicin (10 mg/mL) in DMSO.
2.3 Nonreplicating
Protocol for 96-Well
Format
1. See Subheading 2.1, items 1–15.
2. 96-well, sterile tissue culture-treated microplates with lids.
3. Oxyphenbutazone (10 mM) in DMSO.
4. Rifampicin (10 mg/mL) in DMSO.
2.4 Enumeration
of Colony
Forming Units
1. See Subheading 2.1, items 1–15.
2. 96-well, sterile tissue culture-treated microplates.
3. 7H11-OADC agar: dissolve 21 g of 7H11 powder and 5 mL of
100% glycerol in 900 mL of ddH 2 O. Add a large stir bar.
Autoclave, place flask on a magnetic stir plate set at medium
speed, and allow medium to slowly cool to touch. Aseptically
add 100 mL of OADC supplement and gently mix to avoid
introducing bubbles.
4. Tristyle Petri plates containing 7H11-OADC: In a sterile
hood, using a sterile 25 mL pipette, dispense 8 mL of the
7H11-OADC into each section of the triplate and allow to
solidify. Store plates in sterile biosafety hood overnight to dry.
5. Oxyphenbutazone (10 mM) in DMSO.
6. Rifampicin (10 mg/mL) in DMSO.
618
Ben Gold et al.