3.3 High-Throughput
Screening Under NRP
Conditions
1. Once methylene blue has decolorized (see Subheading 3.1, step
7 and Subheading 3.2, step 7) after 32–48 h in the anaerobic
cabinet, normalize all cultures to OD 600nm ¼ 0.5, using either
rich or minimal cholesterol medium (see Notes 8, 10, and 12).
After this point, the cultures should be in NRP stage I and
ready to use.
2. Dose each well of a 96-well microtiter plate with 1 μL of either
a single-shot high-throughput screen (Fig. 2a), a dilution series
of a few select compounds (Fig. 2b), or a checkerboard assay
(Fig. 2c).
3. Inside the anaerobic cabinet, inoculate the 96-well plate containing compounds with the NRP mycobacterial culture
(Fig. 2). A ratio of 1 μL:99 μL compound:culture is advised
(see Note 13).
4. Leave for 30 min to insure that each well contains no residual
oxygen. Seal the plate with an oxygen impermeable plate seal
(see Note 3).
5. Incubate the plates in the anaerobic cabinet at 37
C for up to
42 days. Read every 24 h in a spectrophotometric plate reader
for the duration of the assay (see Note 14).
6. Serially dilute the cultures and spread out onto solid media to
conduct an endpoint CFU/mL count (see Subheading 3.4) to
check the nature of activity of the compounds.
7. Generate a growth curve (Fig. 1). 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 nondrug-treated control.
3.4 Resuscitation of
an NRP Culture
1. Remove the culture tube (from Subheading 3.1, step 13 or
Subheading 3.2, step 13) or 96-well microtiter plate (from
Subheading 3.3, step 6) from the anaerobic cabinet and
break open the hypoxic seal under aseptic conditions. To do
this, use a scalpel to score the wax around the screw cap lid and
remove with a sharp twist. Take due care and caution whilst
using a scalpel.
2. To resuscitate in liquid medium, inoculate 2.5 mL of
7H9-ADC with 500 μL of culture in a glass culture tube.
3. Incubate at 37
C in an orbital incubator shaking at 180 rpm.
Read tubes every 24 h in a spectrophotometer at OD 600nm (see
Note 15).
4. To resuscitate on solid medium, plate 50–100 μL onto 7H11OADC. Incubate in a static incubator at 37
C with 5% CO 2
(see Note 15).
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Savannah E. R. Gibson et al.
Screening Under NRP
Conditions
1. Once methylene blue has decolorized (see Subheading 3.1, step
7 and Subheading 3.2, step 7) after 32–48 h in the anaerobic
cabinet, normalize all cultures to OD 600nm ¼ 0.5, using either
rich or minimal cholesterol medium (see Notes 8, 10, and 12).
After this point, the cultures should be in NRP stage I and
ready to use.
2. Dose each well of a 96-well microtiter plate with 1 μL of either
a single-shot high-throughput screen (Fig. 2a), a dilution series
of a few select compounds (Fig. 2b), or a checkerboard assay
(Fig. 2c).
3. Inside the anaerobic cabinet, inoculate the 96-well plate containing compounds with the NRP mycobacterial culture
(Fig. 2). A ratio of 1 μL:99 μL compound:culture is advised
(see Note 13).
4. Leave for 30 min to insure that each well contains no residual
oxygen. Seal the plate with an oxygen impermeable plate seal
(see Note 3).
5. Incubate the plates in the anaerobic cabinet at 37
C for up to
42 days. Read every 24 h in a spectrophotometric plate reader
for the duration of the assay (see Note 14).
6. Serially dilute the cultures and spread out onto solid media to
conduct an endpoint CFU/mL count (see Subheading 3.4) to
check the nature of activity of the compounds.
7. Generate a growth curve (Fig. 1). 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 nondrug-treated control.
3.4 Resuscitation of
an NRP Culture
1. Remove the culture tube (from Subheading 3.1, step 13 or
Subheading 3.2, step 13) or 96-well microtiter plate (from
Subheading 3.3, step 6) from the anaerobic cabinet and
break open the hypoxic seal under aseptic conditions. To do
this, use a scalpel to score the wax around the screw cap lid and
remove with a sharp twist. Take due care and caution whilst
using a scalpel.
2. To resuscitate in liquid medium, inoculate 2.5 mL of
7H9-ADC with 500 μL of culture in a glass culture tube.
3. Incubate at 37
C in an orbital incubator shaking at 180 rpm.
Read tubes every 24 h in a spectrophotometer at OD 600nm (see
Note 15).
4. To resuscitate on solid medium, plate 50–100 μL onto 7H11OADC. Incubate in a static incubator at 37
C with 5% CO 2
(see Note 15).
254
Savannah E. R. Gibson et al.