8. Transfer 10 μL of M. tuberculosis from the assay plate to the
same well location on the outgrowth plate using a 12-channel
multichannel pipette (see Note 24).
9. After each transfer, mix up and down five to six times using the
same pipette tips.
10. Change tips for each row of the 96-well assay plate.
11. Place outgrowth plates into plastic food-storage bag with a
zipper opening that is cracked open at one end. Incubate at
37
C at 20% O 2 and 5% CO 2 for 7–10 days (until the DMSOtreated control wells have grown).
12. At the end of incubation (between 7 and 10 days), remove
plates from the bag in a biosafety hood. Remove lids of the
microplates and allow liquid to equilibrate to room temperature for approximately 20 min.
13. Mix cells by pipetting 150 μL up and down five times as
described in step 7. Pop any large bubbles with sterile tips.
14. Cover plate surface with an optical quality PCR adhesive sticker
(Denville) and seal tightly. Read OD 580 in a
spectrophotometer.
3.4 Enumeration
of Colony
Forming Units
Determination of CFUs is the gold standard to enumerate viable
bacteria. This assay is not subject to carryover of compound from
an NR assay to the R recovery phase and provides a highly accurate
assessment of a compound’s activity on NR M. tuberculosis.
1. Prepare a single-cell suspension of the nonreplicating cells by a
10 min centrifugation at 123 Â g with the brake turned off to
maintain a tight pellet.
2. Without disturbing the pellet, carefully transfer the single-cell
suspension (the supernatant) to a new tube and measure the
OD 580 (see Note 9).
3. Dilute the single-cell suspension of NR M. tuberculosis to an
OD 580 of 0.1 into NR complete medium.
4. To determine the starting inoculum, prepare triplicate tenfold
serial dilutions from the tube containing nonreplicating
M. tuberculosis at an OD 580 of 0.1. Spread 10 μL of 10
À3 ,
10
À4 , and 10
À5 dilutions on 7H11-OADC agar triplates and
enumerate colonies after 2.5–3 weeks of incubation at 37
C at
20% O 2 and 5% CO 2 .
5. Using a 12-channel multichannel pipette, add 200 μL of the
single-cell nonreplicating M. tuberculosis at an OD 580 of 0.1 to
a 96-well clear-bottomed, tissue culture-treated microplate.
6. In triplicate wells, add 2 μL of test agents in DMSO (see Note
13). Mix up/down at least five to ten times while stirring in a
circular motion at the same time to ensure DMSO is adequately
dissolved.
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Ben Gold et al.
same well location on the outgrowth plate using a 12-channel
multichannel pipette (see Note 24).
9. After each transfer, mix up and down five to six times using the
same pipette tips.
10. Change tips for each row of the 96-well assay plate.
11. Place outgrowth plates into plastic food-storage bag with a
zipper opening that is cracked open at one end. Incubate at
37
C at 20% O 2 and 5% CO 2 for 7–10 days (until the DMSOtreated control wells have grown).
12. At the end of incubation (between 7 and 10 days), remove
plates from the bag in a biosafety hood. Remove lids of the
microplates and allow liquid to equilibrate to room temperature for approximately 20 min.
13. Mix cells by pipetting 150 μL up and down five times as
described in step 7. Pop any large bubbles with sterile tips.
14. Cover plate surface with an optical quality PCR adhesive sticker
(Denville) and seal tightly. Read OD 580 in a
spectrophotometer.
3.4 Enumeration
of Colony
Forming Units
Determination of CFUs is the gold standard to enumerate viable
bacteria. This assay is not subject to carryover of compound from
an NR assay to the R recovery phase and provides a highly accurate
assessment of a compound’s activity on NR M. tuberculosis.
1. Prepare a single-cell suspension of the nonreplicating cells by a
10 min centrifugation at 123 Â g with the brake turned off to
maintain a tight pellet.
2. Without disturbing the pellet, carefully transfer the single-cell
suspension (the supernatant) to a new tube and measure the
OD 580 (see Note 9).
3. Dilute the single-cell suspension of NR M. tuberculosis to an
OD 580 of 0.1 into NR complete medium.
4. To determine the starting inoculum, prepare triplicate tenfold
serial dilutions from the tube containing nonreplicating
M. tuberculosis at an OD 580 of 0.1. Spread 10 μL of 10
À3 ,
10
À4 , and 10
À5 dilutions on 7H11-OADC agar triplates and
enumerate colonies after 2.5–3 weeks of incubation at 37
C at
20% O 2 and 5% CO 2 .
5. Using a 12-channel multichannel pipette, add 200 μL of the
single-cell nonreplicating M. tuberculosis at an OD 580 of 0.1 to
a 96-well clear-bottomed, tissue culture-treated microplate.
6. In triplicate wells, add 2 μL of test agents in DMSO (see Note
13). Mix up/down at least five to ten times while stirring in a
circular motion at the same time to ensure DMSO is adequately
dissolved.
622
Ben Gold et al.