volume of cells (50 μL) in the respective growth medium
prepared as in Subheading 3.3 is added. The final drug concentration is 50 μM if the drug stock concentration is 10 mM.
16. Pyrazinamide results in 90% growth inhibition at
500–1000 μM at pH ¼ 6.
17. The 7H11/OADC agar medium rapidly solidifies during preparation of the plates. To overcome this, it is advisable to keep
the basin with agar medium on a hot plate and to work rapidly.
In addition, the tips need to be replaced after each agar addition. It is harder to accurately dispense warm agar medium and
as a result, at least four copies are required for each plate.
18. It is recommended to record MIC data by photographing the
plates.
19. Compound precipitation can confound interpretation of
growth. In addition, cholesterol may precipitate out of plates
containing 7H9/cholesterol/BSA/Tyloxapol medium if the
plates cool down at any time point. In these cases, an alternative method of MIC determination using Alamar Blue (Subheading 3.5) or fluorescence of M. tuberculosis pMSP12::GFP
(Subheading 3.6) as a readout is necessary.
20. The standard time period for recording growth inhibition of
compounds to obtain a full MIC value is 1 week. However,
some compounds may act to slow down growth without resulting in complete growth inhibition at any time point. For these
compounds, the level of growth inhibition may be timedependent in which case a 2-week readout is beneficial. Note
that interpretation of growth inhibition of compounds during
extended periods of incubation is affected by compound stability in the medium and to M. tuberculosis metabolism. Compound stability can be determined by measuring compound
concentrations in the wells by, for example, extraction into
acetonitrile followed by liquid chromatography-mass spectrometry analysis.
21. Time points for growth readout under restrictive growth conditions including 7H9/butyrate/Tyloxapol (pH ¼ 6) and
7H9/butyrate/nitrite/Tyloxapol (pH ¼ 6) are longer than
growth conditions that support florid growth. The time points
for these slower growth conditions are typically 10 days as first
time point and 20 days as second time point.
22. Plates are initially incubated upright to ensure that the liquid
from the cell suspension is drawn into the agar. After this,
plates are inverted to prevent condensation from accumulating
in the lids which can fall onto the agar.
MIC Determination for Mycobacterium Tuberculosis
607
prepared as in Subheading 3.3 is added. The final drug concentration is 50 μM if the drug stock concentration is 10 mM.
16. Pyrazinamide results in 90% growth inhibition at
500–1000 μM at pH ¼ 6.
17. The 7H11/OADC agar medium rapidly solidifies during preparation of the plates. To overcome this, it is advisable to keep
the basin with agar medium on a hot plate and to work rapidly.
In addition, the tips need to be replaced after each agar addition. It is harder to accurately dispense warm agar medium and
as a result, at least four copies are required for each plate.
18. It is recommended to record MIC data by photographing the
plates.
19. Compound precipitation can confound interpretation of
growth. In addition, cholesterol may precipitate out of plates
containing 7H9/cholesterol/BSA/Tyloxapol medium if the
plates cool down at any time point. In these cases, an alternative method of MIC determination using Alamar Blue (Subheading 3.5) or fluorescence of M. tuberculosis pMSP12::GFP
(Subheading 3.6) as a readout is necessary.
20. The standard time period for recording growth inhibition of
compounds to obtain a full MIC value is 1 week. However,
some compounds may act to slow down growth without resulting in complete growth inhibition at any time point. For these
compounds, the level of growth inhibition may be timedependent in which case a 2-week readout is beneficial. Note
that interpretation of growth inhibition of compounds during
extended periods of incubation is affected by compound stability in the medium and to M. tuberculosis metabolism. Compound stability can be determined by measuring compound
concentrations in the wells by, for example, extraction into
acetonitrile followed by liquid chromatography-mass spectrometry analysis.
21. Time points for growth readout under restrictive growth conditions including 7H9/butyrate/Tyloxapol (pH ¼ 6) and
7H9/butyrate/nitrite/Tyloxapol (pH ¼ 6) are longer than
growth conditions that support florid growth. The time points
for these slower growth conditions are typically 10 days as first
time point and 20 days as second time point.
22. Plates are initially incubated upright to ensure that the liquid
from the cell suspension is drawn into the agar. After this,
plates are inverted to prevent condensation from accumulating
in the lids which can fall onto the agar.
MIC Determination for Mycobacterium Tuberculosis
607