diphenyltetrazolium bromide (MTT) or related tetrazolium salts),
ATP measurement, and Optical Density (OD). The differences
between the three surrogates of bacterial growth are summarized
in Table 1.
1.1 Methods
for Monitoring Growth
Resazurin is a non-fluorescent blue dye that is reduced to the
fluorescent pink derivative (resorufin) by oxidoreductases within
viable cells [5]. Although inexpensive, resazurin has some inconveniences, such as a low signal-to-noise ratio and frequent problems
associated with the reactivity of the dye and the compounds under
study. Alternative methodologies with higher sensitivity amenable
for use in high-throughput format (384-well) are available. An
alternative method, also dependent on metabolic activity, is measuring ATP levels using the “BacTiter-Glo™ Microbial Cell Viability Assay” [2, 6, 7] (Table 2).
Finally, growth inhibition can also be determined by measuring
the OD of cultures. This is a very simple and cheap method. The
big disadvantages of this method are the low signal-to-background
ratio and the requirement to increase in either the time of incubation or the starting inoculum.
Table 1
Surrogate for growth
Robustness
Interference
False +/Àves
Throughput
Price
Resazurin
√√
√√
√
√√
√√√√
ATP
√√√
√√
√
√√√√
√√
Oxigen
√√√
√√√
√√
√√√
√√
OD
√
√√√√
√√√
√
√√√√
Table 2
MIC 90 of standards under the ATP and resazurin methods
MIC (μg/mL)
INH Rif
Eth Sm Km Mox
Oflo Cipro Line
ATP
M. smegmatis mc
2 155 36
8.77
0.51 0.82 2.12 ND
ND ND
ND
M. bovis BCG
0.23 0.0004 1.23 0.15 2.37 0.036 0.32 ND
0.39
M. tuberculosis H37Rv 0.23 0.004
1.41 0.63 3.25 ND
ND ND
ND
Resazurin M. smegmatis mc
2 155 125 9
0.5
0.9
2.2
ND
ND ND
ND
M. bovis BCG
0.5
0.002
2.7
0.18 1.2
0.14
0.16 0.09 0.4
M. tuberculosis H37Rv 0.25 0.002
2.5
1.25 2.5
0.06
0.6
0.5
0.6
INH isoniazid, Rif rifampicin, Eth ethionamide, Sm streptomycin, Km kanamycin, Mox moxifloxacin, Oflo ofloxacin,
Cipro ciprofloxacin, Line linezolid
Antimicrobial Susceptibility Testing
639
ATP measurement, and Optical Density (OD). The differences
between the three surrogates of bacterial growth are summarized
in Table 1.
1.1 Methods
for Monitoring Growth
Resazurin is a non-fluorescent blue dye that is reduced to the
fluorescent pink derivative (resorufin) by oxidoreductases within
viable cells [5]. Although inexpensive, resazurin has some inconveniences, such as a low signal-to-noise ratio and frequent problems
associated with the reactivity of the dye and the compounds under
study. Alternative methodologies with higher sensitivity amenable
for use in high-throughput format (384-well) are available. An
alternative method, also dependent on metabolic activity, is measuring ATP levels using the “BacTiter-Glo™ Microbial Cell Viability Assay” [2, 6, 7] (Table 2).
Finally, growth inhibition can also be determined by measuring
the OD of cultures. This is a very simple and cheap method. The
big disadvantages of this method are the low signal-to-background
ratio and the requirement to increase in either the time of incubation or the starting inoculum.
Table 1
Surrogate for growth
Robustness
Interference
False +/Àves
Throughput
Price
Resazurin
√√
√√
√
√√
√√√√
ATP
√√√
√√
√
√√√√
√√
Oxigen
√√√
√√√
√√
√√√
√√
OD
√
√√√√
√√√
√
√√√√
Table 2
MIC 90 of standards under the ATP and resazurin methods
MIC (μg/mL)
INH Rif
Eth Sm Km Mox
Oflo Cipro Line
ATP
M. smegmatis mc
2 155 36
8.77
0.51 0.82 2.12 ND
ND ND
ND
M. bovis BCG
0.23 0.0004 1.23 0.15 2.37 0.036 0.32 ND
0.39
M. tuberculosis H37Rv 0.23 0.004
1.41 0.63 3.25 ND
ND ND
ND
Resazurin M. smegmatis mc
2 155 125 9
0.5
0.9
2.2
ND
ND ND
ND
M. bovis BCG
0.5
0.002
2.7
0.18 1.2
0.14
0.16 0.09 0.4
M. tuberculosis H37Rv 0.25 0.002
2.5
1.25 2.5
0.06
0.6
0.5
0.6
INH isoniazid, Rif rifampicin, Eth ethionamide, Sm streptomycin, Km kanamycin, Mox moxifloxacin, Oflo ofloxacin,
Cipro ciprofloxacin, Line linezolid
Antimicrobial Susceptibility Testing
639