121
C for 15 min. Cool to 55
C in a water bath. Add 100 mL
of sterile OADC enrichment that has been equilibrated to
55
C.
3. Sterile phosphate-buffered saline (PBS). Autoclave at 121
C
for 15 min.
2.5 Lysing an NRP
Culture for Further
Analysis
1. 50-mL conical centrifuge tubes.
2. Sterile phosphate-buffered saline (PBS). Autoclave at 121
C
for 15 min.
3. Scalpel.
4. Proteinase inhibitor cocktail tablets.
5. Glass beads (0.1 mm).
6. BeadBeater.
3 Methods
3.1 Hypoxia-Induced
Rich Medium
NRP Assay
1. Dispense 10 mL aliquots of Middlebrook 7H9 media with
ADC supplement (see Subheading 2.1, item 4) into vented
culture flasks.
2. Inoculate with mycobacteria from a frozen glycerol stock kept
at À80
C.
3. Incubate for approximately 2 weeks in at 37
C with 5% CO 2 .
4. Monitor growth until culture reaches a mid-logarithmic phase
of growth (OD 600nm ¼ 0.6).
5. Take mid-log phase culture and dilute to an OD 600nm of 0.3 in
sterilized glass tubes to a total volume of 3 mL using Middlebrook 7H9 media with ADC supplement.
6. Add 4.5 μL methylene blue solution to each culture tube to
reach a final concentration of 1.5 μg/mL (see Note 6).
7. Leaving the lids loose, place into the anaerobic cabinet for
48–72 h to allow a slow degassing of the cultures to take
place (see Note 7). This will be noticeable from the decolorization of the methylene blue. If required, cultures at this point
can be used for high-throughput assay after normalizing optical
density (OD 600nm ¼ 0.3) (see Subheading 3.3).
8. After the culture has entered NRP stage I, dose with chosen
antimicrobials. It is of crucial importance that the culture has
entered NRP stage I as antibiotic susceptibilities vary significantly between aerobic growth and the NRP state (see Note 8).
The frontline drug, INH, and anaerobic antibiotic MET can be
used in this model as negative and positive controls,
respectively.
In vitro Drug Screening of Hypoxic NRP Mycobacteria
251
C for 15 min. Cool to 55
C in a water bath. Add 100 mL
of sterile OADC enrichment that has been equilibrated to
55
C.
3. Sterile phosphate-buffered saline (PBS). Autoclave at 121
C
for 15 min.
2.5 Lysing an NRP
Culture for Further
Analysis
1. 50-mL conical centrifuge tubes.
2. Sterile phosphate-buffered saline (PBS). Autoclave at 121
C
for 15 min.
3. Scalpel.
4. Proteinase inhibitor cocktail tablets.
5. Glass beads (0.1 mm).
6. BeadBeater.
3 Methods
3.1 Hypoxia-Induced
Rich Medium
NRP Assay
1. Dispense 10 mL aliquots of Middlebrook 7H9 media with
ADC supplement (see Subheading 2.1, item 4) into vented
culture flasks.
2. Inoculate with mycobacteria from a frozen glycerol stock kept
at À80
C.
3. Incubate for approximately 2 weeks in at 37
C with 5% CO 2 .
4. Monitor growth until culture reaches a mid-logarithmic phase
of growth (OD 600nm ¼ 0.6).
5. Take mid-log phase culture and dilute to an OD 600nm of 0.3 in
sterilized glass tubes to a total volume of 3 mL using Middlebrook 7H9 media with ADC supplement.
6. Add 4.5 μL methylene blue solution to each culture tube to
reach a final concentration of 1.5 μg/mL (see Note 6).
7. Leaving the lids loose, place into the anaerobic cabinet for
48–72 h to allow a slow degassing of the cultures to take
place (see Note 7). This will be noticeable from the decolorization of the methylene blue. If required, cultures at this point
can be used for high-throughput assay after normalizing optical
density (OD 600nm ¼ 0.3) (see Subheading 3.3).
8. After the culture has entered NRP stage I, dose with chosen
antimicrobials. It is of crucial importance that the culture has
entered NRP stage I as antibiotic susceptibilities vary significantly between aerobic growth and the NRP state (see Note 8).
The frontline drug, INH, and anaerobic antibiotic MET can be
used in this model as negative and positive controls,
respectively.
In vitro Drug Screening of Hypoxic NRP Mycobacteria
251