3. Replace the lids, place the plates in sealed bags and incubate for
24 h at 37
C before inverting the plates for further incubation
(see Note 22).
4. Record visual growth after 7 days and 14 days (see Note 18).
5. After 14 days, add 10 μL of Alamar Blue solution to each well.
Once Alamar Blue reagent has been added the plates should
not be inverted.
6. Replace the plates in the sealed bags and incubate for 24 h at
37
C.
7. Record the MIC as the concentration of compound that results
in no visible growth and/or the concentration of compound
that inhibits the reduction of Alamar Blue to pink.
4 Notes
1. Widely used strains of M. tuberculosis including H37Rv (ATCC
27294), Erdman, HN878, and CDC1551 all show reproducible robust growth in the respective media. At times, clinical
strains of M. tuberculosis including drug-resistant isolates may
need to be tested. Clinical isolates tend to have more variable
growth rates and nutrient requirements depending on the
genetic background of the strain and need to be grown accordingly. In addition, many clinical isolates do not grow well in
minimal media preventing their reproducible assessment under
these conditions. The M. tuberculosis pMSP12::GFP is cultured
with 25 μg/mL kanamycin.
2. Glycerol and detergents such as Tween 80 and Tyloxapol are
highly viscous and cannot be dispensed by pipetting unless
diluted solutions are prepared. A syringe with plunger allows
sufficiently accurate dispensing of these for larger volumes of
media. For the preparation of volumes of growth media less
than 1 L, it is preferable to use stocks of 25% glycerol and 25%
Tyloxapol or Tween 80 prepared in water. These stocks should
be filter-sterilized (0.2 μm filter), and a calculated volume is
dispensed accordingly.
3. 7H9 Middlebrook broth base containing glycerol and detergent can be autoclaved prior to addition of ADC.
4. M. tuberculosis including H37Rv (ATCC 27294), Erdman,
HN878, and CDC1551 all have similar MIC values to standard
drugs such as isoniazid and rifampicin. Drug-resistant clinical
isolates have to be tested with the appropriate drug controls
with linezolid (stock concentration of 10 mM) being the most
widely applicable control drug for drug-resistant isolates except
for resistant strains that have been isolated from patients treated with this oxazolidinone.
604
Qinglan Wang and Helena I. M. Boshoff
24 h at 37
C before inverting the plates for further incubation
(see Note 22).
4. Record visual growth after 7 days and 14 days (see Note 18).
5. After 14 days, add 10 μL of Alamar Blue solution to each well.
Once Alamar Blue reagent has been added the plates should
not be inverted.
6. Replace the plates in the sealed bags and incubate for 24 h at
37
C.
7. Record the MIC as the concentration of compound that results
in no visible growth and/or the concentration of compound
that inhibits the reduction of Alamar Blue to pink.
4 Notes
1. Widely used strains of M. tuberculosis including H37Rv (ATCC
27294), Erdman, HN878, and CDC1551 all show reproducible robust growth in the respective media. At times, clinical
strains of M. tuberculosis including drug-resistant isolates may
need to be tested. Clinical isolates tend to have more variable
growth rates and nutrient requirements depending on the
genetic background of the strain and need to be grown accordingly. In addition, many clinical isolates do not grow well in
minimal media preventing their reproducible assessment under
these conditions. The M. tuberculosis pMSP12::GFP is cultured
with 25 μg/mL kanamycin.
2. Glycerol and detergents such as Tween 80 and Tyloxapol are
highly viscous and cannot be dispensed by pipetting unless
diluted solutions are prepared. A syringe with plunger allows
sufficiently accurate dispensing of these for larger volumes of
media. For the preparation of volumes of growth media less
than 1 L, it is preferable to use stocks of 25% glycerol and 25%
Tyloxapol or Tween 80 prepared in water. These stocks should
be filter-sterilized (0.2 μm filter), and a calculated volume is
dispensed accordingly.
3. 7H9 Middlebrook broth base containing glycerol and detergent can be autoclaved prior to addition of ADC.
4. M. tuberculosis including H37Rv (ATCC 27294), Erdman,
HN878, and CDC1551 all have similar MIC values to standard
drugs such as isoniazid and rifampicin. Drug-resistant clinical
isolates have to be tested with the appropriate drug controls
with linezolid (stock concentration of 10 mM) being the most
widely applicable control drug for drug-resistant isolates except
for resistant strains that have been isolated from patients treated with this oxazolidinone.
604
Qinglan Wang and Helena I. M. Boshoff