3.2 Hypoxic Minimal
Cholesterol Medium
NRP Assay
1. Dispense 10 mL aliquots of minimal cholesterol medium (see
Subheading 2.2, item 3) into vented culture flasks.
2. Inoculate with an inoculation loop of 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
sterile 16-mm-diameter glass tubes to a total volume of 3 mL
using minimal cholesterol medium.
6. Add 4.5 μL methylene blue 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 32 h
to allow a slow degassing of the cultures to take place (see Note
10). This will be noticeable from the decolorization of the
methylene blue. If required, cultures at this point can be used
for high-throughput assay (see Subheading 3.3).
8. After the culture has entered NRP I (see Note 8), dose with
chosen antimicrobials. It is of crucial importance that the culture has entered NRP stage I as antibiotic susceptibilities vary
between aerobic growth and the NRP state (see Note 10). In
this assay, neither INH nor MET has antimicrobial activity (see
Note 11).
9. Tighten the screw cap lids and seal around the lid and tube with
parafilm (see Note 9). The cultures can now be removed from
the anaerobic cabinet.
10. Melt paraffin wax by heating to >70
C. Wear thick protective
gloves when handling hot wax.
11. Seal the lids to the tubes with the molten paraffin wax and
insure that there are no bubbles or holes where oxygen may
permeate. Be careful to insure that while you are sealing you do
not introduce oxygen to the hypoxic atmosphere of the culture
tube; if oxygen is introduced, a blue color will start to reappear.
Avoid heat shock by applying molten wax only to the lid and
join to the glass tube, do not cover the entire tube in wax.
12. Incubate in a shaking incubator at 37
C and shaking at
100–120 rpm.
13. Record OD 600nm using an optical density reader every 24 h for
42 days. If the compound is active, there will be a noticeable
decline in OD in a dose-dependent fashion. If the compound is
inactive, there will be no significant difference in OD between a
drug-treated culture and a non-drug-treated control (Fig. 1).
Additional validation can be obtained by conducting CFU/mL
counts (see Subheading 3.4).
In vitro Drug Screening of Hypoxic NRP Mycobacteria
253
Cholesterol Medium
NRP Assay
1. Dispense 10 mL aliquots of minimal cholesterol medium (see
Subheading 2.2, item 3) into vented culture flasks.
2. Inoculate with an inoculation loop of 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
sterile 16-mm-diameter glass tubes to a total volume of 3 mL
using minimal cholesterol medium.
6. Add 4.5 μL methylene blue 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 32 h
to allow a slow degassing of the cultures to take place (see Note
10). This will be noticeable from the decolorization of the
methylene blue. If required, cultures at this point can be used
for high-throughput assay (see Subheading 3.3).
8. After the culture has entered NRP I (see Note 8), dose with
chosen antimicrobials. It is of crucial importance that the culture has entered NRP stage I as antibiotic susceptibilities vary
between aerobic growth and the NRP state (see Note 10). In
this assay, neither INH nor MET has antimicrobial activity (see
Note 11).
9. Tighten the screw cap lids and seal around the lid and tube with
parafilm (see Note 9). The cultures can now be removed from
the anaerobic cabinet.
10. Melt paraffin wax by heating to >70
C. Wear thick protective
gloves when handling hot wax.
11. Seal the lids to the tubes with the molten paraffin wax and
insure that there are no bubbles or holes where oxygen may
permeate. Be careful to insure that while you are sealing you do
not introduce oxygen to the hypoxic atmosphere of the culture
tube; if oxygen is introduced, a blue color will start to reappear.
Avoid heat shock by applying molten wax only to the lid and
join to the glass tube, do not cover the entire tube in wax.
12. Incubate in a shaking incubator at 37
C and shaking at
100–120 rpm.
13. Record OD 600nm using an optical density reader every 24 h for
42 days. If the compound is active, there will be a noticeable
decline in OD in a dose-dependent fashion. If the compound is
inactive, there will be no significant difference in OD between a
drug-treated culture and a non-drug-treated control (Fig. 1).
Additional validation can be obtained by conducting CFU/mL
counts (see Subheading 3.4).
In vitro Drug Screening of Hypoxic NRP Mycobacteria
253