5. Drug stocks should be stored frozen in aliquots. Although
drugs such as isoniazid, rifampicin, and linezolid can be frozen
and thawed several times without noticeable changes in MIC
values, it is good practice to aliquot drugs as needed. Pyrazinamide is only active against M. tuberculosis grown at pH 6.
6. The Alamar Blue assay is based on reduction of the resazurin
dye to a pink product. Recording of absorbance or fluorescence
using a plate reader enables inhibition curves to be graphed
with determination of pIC50 values. For absorbance data, the
percentage inhibition (PI) can be calculated using the equation
as follows:
PI ¼ 1 À
O2∗A2 À O1∗A1
O2∗P1 À O1∗P2
∗100%
where: O1 ¼ 80,586, molar extinction coefficient (E) of oxidized
resazurin at 570 nm, O2 ¼ 117,216, E of oxidized resazurin at
600 nm, A1 ¼ absorbance of test well (containing drug) at 570 nm,
A2 ¼ absorbance of the same test well at 600 nm, P1 ¼ absorbance
of control well (no drug) at 570 nm, P2 ¼ absorbance of control
well at 600 nm. For fluorescence data, the PI is calculated as
follows:
PI ¼ 1 À
FI 590 of test well
FI 590 of control well
∗100%
where: FI 590 ¼ fluorescent intensity at 590 nm emission (560 nm
excitation). IC 50 is calculated based on the curve generated from
plotting compound concentration vs PI, and pIC50 is calculated
using the equation: pIC50 ¼ À log 10 (IC50).
7. Alamar Blue solution can be made from resazurin powder in
water at a concentration of 0.04%.
8. M. tuberculosis cultures should not be propagated for more
than two to three sub-cultures since in vitro growth conditions
tend to favor the emergence of strains that have lost phthiocerol dimycoserosate (PDIM) [19]. Freezer stocks are made by
freezing sufficient numbers of aliquots of a confirmed PDIMpositive strain [20] to allow reproducible testing over
2–3 years, depending on the estimated throughput and duration of the laboratory or project.
9. For growth in BSA-free conditions and in low pH medium
(7H9/butyrate/Tyloxapol (pH ¼ 6)), it is recommended to
start cultures from freezer stocks in the respective
BSA-containing carbon source medium (pH ¼ 6.8) to an
OD 600 of 0.2–0.4, then dilute cells 1:100 in the respective
BSA-free or 7H9/butyrate/ Tyloxapol (pH ¼ 6) medium (see
step 3 of Subheading 3.1).
MIC Determination for Mycobacterium Tuberculosis
605
drugs such as isoniazid, rifampicin, and linezolid can be frozen
and thawed several times without noticeable changes in MIC
values, it is good practice to aliquot drugs as needed. Pyrazinamide is only active against M. tuberculosis grown at pH 6.
6. The Alamar Blue assay is based on reduction of the resazurin
dye to a pink product. Recording of absorbance or fluorescence
using a plate reader enables inhibition curves to be graphed
with determination of pIC50 values. For absorbance data, the
percentage inhibition (PI) can be calculated using the equation
as follows:
PI ¼ 1 À
O2∗A2 À O1∗A1
O2∗P1 À O1∗P2
∗100%
where: O1 ¼ 80,586, molar extinction coefficient (E) of oxidized
resazurin at 570 nm, O2 ¼ 117,216, E of oxidized resazurin at
600 nm, A1 ¼ absorbance of test well (containing drug) at 570 nm,
A2 ¼ absorbance of the same test well at 600 nm, P1 ¼ absorbance
of control well (no drug) at 570 nm, P2 ¼ absorbance of control
well at 600 nm. For fluorescence data, the PI is calculated as
follows:
PI ¼ 1 À
FI 590 of test well
FI 590 of control well
∗100%
where: FI 590 ¼ fluorescent intensity at 590 nm emission (560 nm
excitation). IC 50 is calculated based on the curve generated from
plotting compound concentration vs PI, and pIC50 is calculated
using the equation: pIC50 ¼ À log 10 (IC50).
7. Alamar Blue solution can be made from resazurin powder in
water at a concentration of 0.04%.
8. M. tuberculosis cultures should not be propagated for more
than two to three sub-cultures since in vitro growth conditions
tend to favor the emergence of strains that have lost phthiocerol dimycoserosate (PDIM) [19]. Freezer stocks are made by
freezing sufficient numbers of aliquots of a confirmed PDIMpositive strain [20] to allow reproducible testing over
2–3 years, depending on the estimated throughput and duration of the laboratory or project.
9. For growth in BSA-free conditions and in low pH medium
(7H9/butyrate/Tyloxapol (pH ¼ 6)), it is recommended to
start cultures from freezer stocks in the respective
BSA-containing carbon source medium (pH ¼ 6.8) to an
OD 600 of 0.2–0.4, then dilute cells 1:100 in the respective
BSA-free or 7H9/butyrate/ Tyloxapol (pH ¼ 6) medium (see
step 3 of Subheading 3.1).
MIC Determination for Mycobacterium Tuberculosis
605