5. Add 10 μL of Alamar Blue solution to each well.
6. Replace the plates in the sealed bags and incubate for 24 h at
37
C.
7. Record MIC visually. The MIC value is the lowest concentration of compound that inhibits growth as measured by inhibition of blue to pink conversion.
8. Alternatively, the MIC can be determined by measuring the dye
conversion using a plate reader for absorbance at 570 nm and
600 nm or fluorescence using excitation between 530 and
560 nm with emission recorded at 590–595 nm (see Note 6).
3.6 Measuring
Growth Using
Fluorescence of M.
tuberculosis pMSP12::
GFP
The M. tuberculosis pMSP12::GFP strain is used for measuring the
MIC under conditions of highly restricted growth as occurs at low
pH in the presence or absence of nitrite. Pyrazinamide is active
against M. tuberculosis at low pH and is an essential control for MIC
determination in 7H9/butyrate/Tyloxapol (pH ¼ 6) and
7H9/butyrate/nitrite/Tyloxapol (pH ¼ 6) media (see Note 16).
The M. tuberculosis pMSP12::GFP strain can also be used for MIC
determination under any of the above growth conditions in order
to generate growth inhibition graphs for compounds.
1. Prepare the inoculum as described in steps 1–4 of Subheading
3.1.
2. Using a multichannel pipette, add 200 μL of cell suspension to
black microtiter plates containing 2 μL of compound (Subheading 3.2).
3. Mix the cells and compound and transfer 100 μL of the cell/
compound mix to a clean sterile black microtiter plate to generate a technical repeat (see Notes 11 and 15).
4. Replace the lids, place in sealed bags and incubate at 37
C (see
Note 21).
5. At 10 and 20 days, measure fluorescence at an excitation wavelength of 485 nm and emission recorded at 535 nm.
6. Plot growth inhibition curve using the values from the DMSO
control for 0% growth inhibition and 5 μM rifampicin as 100%
growth inhibition. Determine the IC 50 (half maximal inhibitory concentration) and pIC50 (pIC50 ¼ Àlog 10 (IC50) can
be determined. Compound concentrations that result in 90%
and 99% reduction in fluorescence signal can also be
determined.
3.7 Measuring
Growth on Solid
Medium
1. Prepare the inoculum is as described in Subheading 3.1.
2. Using a multichannel pipette, add 2 μL of cell suspension to the
center of the agar in each well to plates prepared as described in
Subheading 3.3.
MIC Determination for Mycobacterium Tuberculosis
603
6. Replace the plates in the sealed bags and incubate for 24 h at
37
C.
7. Record MIC visually. The MIC value is the lowest concentration of compound that inhibits growth as measured by inhibition of blue to pink conversion.
8. Alternatively, the MIC can be determined by measuring the dye
conversion using a plate reader for absorbance at 570 nm and
600 nm or fluorescence using excitation between 530 and
560 nm with emission recorded at 590–595 nm (see Note 6).
3.6 Measuring
Growth Using
Fluorescence of M.
tuberculosis pMSP12::
GFP
The M. tuberculosis pMSP12::GFP strain is used for measuring the
MIC under conditions of highly restricted growth as occurs at low
pH in the presence or absence of nitrite. Pyrazinamide is active
against M. tuberculosis at low pH and is an essential control for MIC
determination in 7H9/butyrate/Tyloxapol (pH ¼ 6) and
7H9/butyrate/nitrite/Tyloxapol (pH ¼ 6) media (see Note 16).
The M. tuberculosis pMSP12::GFP strain can also be used for MIC
determination under any of the above growth conditions in order
to generate growth inhibition graphs for compounds.
1. Prepare the inoculum as described in steps 1–4 of Subheading
3.1.
2. Using a multichannel pipette, add 200 μL of cell suspension to
black microtiter plates containing 2 μL of compound (Subheading 3.2).
3. Mix the cells and compound and transfer 100 μL of the cell/
compound mix to a clean sterile black microtiter plate to generate a technical repeat (see Notes 11 and 15).
4. Replace the lids, place in sealed bags and incubate at 37
C (see
Note 21).
5. At 10 and 20 days, measure fluorescence at an excitation wavelength of 485 nm and emission recorded at 535 nm.
6. Plot growth inhibition curve using the values from the DMSO
control for 0% growth inhibition and 5 μM rifampicin as 100%
growth inhibition. Determine the IC 50 (half maximal inhibitory concentration) and pIC50 (pIC50 ¼ Àlog 10 (IC50) can
be determined. Compound concentrations that result in 90%
and 99% reduction in fluorescence signal can also be
determined.
3.7 Measuring
Growth on Solid
Medium
1. Prepare the inoculum is as described in Subheading 3.1.
2. Using a multichannel pipette, add 2 μL of cell suspension to the
center of the agar in each well to plates prepared as described in
Subheading 3.3.
MIC Determination for Mycobacterium Tuberculosis
603