10. For MIC testing in 7H9/butyrate/nitrite/Tyloxapol
(pH ¼ 6) medium, cells are cultured in the absence of nitrite
(in step 3 of Subheading 3.1). When cells are ready, dilute
cells 1:10 in 7H9/butyrate/nitrite/Tyloxapol (pH ¼ 6)
medium for MIC set-up.
11. For 7H9/cholesterol/BSA/Tyloxapol medium, it is important to keep the medium warm during set-up for MIC since
cholesterol rapidly precipitates as the medium cools down.
12. It is important to ensure that pipettes are calibrated. For most
pipettes, there is a 10% pipetting error implying that the error
is larger on smaller volumes. Thus, it is recommended not to
pipette a lower volume than 2 μL using a 10 μL or 20 μL
pipette.
13. Compound stock solutions in DMSO need to be fully dissolved. In some instances, sonication in a water bath or gentle
heating (depending on compound stability to heat) may be
necessary to ensure full dissolution. Compounds that do not
fully dissolve at the initial compound concentration need to
be further diluted with DMSO until fully dissolved. In the
latter case, the highest concentration in the MIC set-up will
be lower. There are certain chemical series that are not stable
or soluble in DMSO. These compounds may be soluble in
solvents such as ethanol or water. In the latter case, stock
solutions need to be filter-sterilized and the serial dilutions
should be performed with respective solvent.
14. Stock concentrations depend on the desired highest concentration in the MIC set-up. As a rule, the DMSO concentration in the MIC set-up should not be higher than 1% after
addition of cells. In the procedure outlined in the chapter, a
stock concentration of 10 mM will give a final concentration
of 100 μM.
15. Dilution of compound in DMSO is important when the
solubility in aqueous medium is unknown. However, for
compounds whose aqueous solubility is not limiting, the
method can be accelerated by performing drug dilutions in
aqueous medium. In this case, 2 μL of drug is added directly
to the first column of the sterile microtiter plate (plate 1)
containing 200 μL of growth medium in column 1 and
50 μL of growth medium in all other wells. Dilutions are
performed by transferring 50 μL from column 1 to column
2 and serially to the next columns ending with column
11 after which the last 50 μL is discarded. A duplicate plate
(plate 2) containing 50 μL of medium in columns 2–12 is
made by transferring 100 μL from column 1 in plate 1 to this
plate and serially diluting as for plate 1. After this, an equal
606
Qinglan Wang and Helena I. M. Boshoff
(pH ¼ 6) medium, cells are cultured in the absence of nitrite
(in step 3 of Subheading 3.1). When cells are ready, dilute
cells 1:10 in 7H9/butyrate/nitrite/Tyloxapol (pH ¼ 6)
medium for MIC set-up.
11. For 7H9/cholesterol/BSA/Tyloxapol medium, it is important to keep the medium warm during set-up for MIC since
cholesterol rapidly precipitates as the medium cools down.
12. It is important to ensure that pipettes are calibrated. For most
pipettes, there is a 10% pipetting error implying that the error
is larger on smaller volumes. Thus, it is recommended not to
pipette a lower volume than 2 μL using a 10 μL or 20 μL
pipette.
13. Compound stock solutions in DMSO need to be fully dissolved. In some instances, sonication in a water bath or gentle
heating (depending on compound stability to heat) may be
necessary to ensure full dissolution. Compounds that do not
fully dissolve at the initial compound concentration need to
be further diluted with DMSO until fully dissolved. In the
latter case, the highest concentration in the MIC set-up will
be lower. There are certain chemical series that are not stable
or soluble in DMSO. These compounds may be soluble in
solvents such as ethanol or water. In the latter case, stock
solutions need to be filter-sterilized and the serial dilutions
should be performed with respective solvent.
14. Stock concentrations depend on the desired highest concentration in the MIC set-up. As a rule, the DMSO concentration in the MIC set-up should not be higher than 1% after
addition of cells. In the procedure outlined in the chapter, a
stock concentration of 10 mM will give a final concentration
of 100 μM.
15. Dilution of compound in DMSO is important when the
solubility in aqueous medium is unknown. However, for
compounds whose aqueous solubility is not limiting, the
method can be accelerated by performing drug dilutions in
aqueous medium. In this case, 2 μL of drug is added directly
to the first column of the sterile microtiter plate (plate 1)
containing 200 μL of growth medium in column 1 and
50 μL of growth medium in all other wells. Dilutions are
performed by transferring 50 μL from column 1 to column
2 and serially to the next columns ending with column
11 after which the last 50 μL is discarded. A duplicate plate
(plate 2) containing 50 μL of medium in columns 2–12 is
made by transferring 100 μL from column 1 in plate 1 to this
plate and serially diluting as for plate 1. After this, an equal
606
Qinglan Wang and Helena I. M. Boshoff