in diagnostics and therapeutics. In this chapter, we will describe
how to grow M. tuberculosis under normoxic and hypoxic conditions using a fermentor/bioreactor.
1.17 Quality Control
of Mycobacterial
Cultures
The laboratories that invest some time in practicing quality control
of their cultures ensure that their strains are what they expect them
to be, maximize reproducibility between cultures and experiments,
and minimize issues caused by using organisms that will not provide
the results needed or expected. In collections like ATCC and BEI
Resources, it has been observed that sometimes the material deposited is not what the depositor described. This could be due to
human error, the use of outdated characterization techniques (for
example, next generation sequence allows to differentiate all species, a feat not possible by other means) or simply by lack of basic
microbiological controls during the preservation and maintenance
in the laboratory. The main points to confirm for every culture in
the lab should be:
1.17.1 Purity
It is important to confirm purity by analyzing the presence of a
single organism by standard macroscopic and microscopic observation, as well as molecular assays such as nucleic acid sequencing (for
example, hsp65 gene).
1.17.2 Viability
The ability of the mycobacteria to grow once it has been preserved
is important to assure the strain can be used in the future. The
frequency and process to check viability varies depending on the
way the strain was preserved and stored. For example, cultures
stored in L-J slants might last only a few years, and viability should
be checked more often. A frozen glycerol stock or a lyophilized vial
might not require checking in long periods of time.
1.17.3 Identification
Confirmation of the specimen identity down to the genus and
species level (if known) should be done for each lot. Accurate
identification and characterization of the material is crucial for
compliance with regulations related to restricted agents.
1.17.4 Strain
Characterization
Some strains or isolates have unique characteristics that make them
useful or relevant for future work. These features should be confirmed prior use. Each laboratory might have defined processes to
achieve this. For example, clinical isolates known to have defined
antibiotic resistance patterns might require confirmation of their
susceptibility profiles. Also, these characteristics might change during laboratory passaging. For this reason, it is recommended that a
seed lot is made and preserved minimizing the number of passages.
Future lots will start from these seed lots, and not from cultures
passaged more times. One example of this is the cultures of
M. ulcerans, which are known to lose virulence by passaging
in vitro (usually after 3–4 passages in vitro). In this case, the
Culturing Mycobacteria
23
how to grow M. tuberculosis under normoxic and hypoxic conditions using a fermentor/bioreactor.
1.17 Quality Control
of Mycobacterial
Cultures
The laboratories that invest some time in practicing quality control
of their cultures ensure that their strains are what they expect them
to be, maximize reproducibility between cultures and experiments,
and minimize issues caused by using organisms that will not provide
the results needed or expected. In collections like ATCC and BEI
Resources, it has been observed that sometimes the material deposited is not what the depositor described. This could be due to
human error, the use of outdated characterization techniques (for
example, next generation sequence allows to differentiate all species, a feat not possible by other means) or simply by lack of basic
microbiological controls during the preservation and maintenance
in the laboratory. The main points to confirm for every culture in
the lab should be:
1.17.1 Purity
It is important to confirm purity by analyzing the presence of a
single organism by standard macroscopic and microscopic observation, as well as molecular assays such as nucleic acid sequencing (for
example, hsp65 gene).
1.17.2 Viability
The ability of the mycobacteria to grow once it has been preserved
is important to assure the strain can be used in the future. The
frequency and process to check viability varies depending on the
way the strain was preserved and stored. For example, cultures
stored in L-J slants might last only a few years, and viability should
be checked more often. A frozen glycerol stock or a lyophilized vial
might not require checking in long periods of time.
1.17.3 Identification
Confirmation of the specimen identity down to the genus and
species level (if known) should be done for each lot. Accurate
identification and characterization of the material is crucial for
compliance with regulations related to restricted agents.
1.17.4 Strain
Characterization
Some strains or isolates have unique characteristics that make them
useful or relevant for future work. These features should be confirmed prior use. Each laboratory might have defined processes to
achieve this. For example, clinical isolates known to have defined
antibiotic resistance patterns might require confirmation of their
susceptibility profiles. Also, these characteristics might change during laboratory passaging. For this reason, it is recommended that a
seed lot is made and preserved minimizing the number of passages.
Future lots will start from these seed lots, and not from cultures
passaged more times. One example of this is the cultures of
M. ulcerans, which are known to lose virulence by passaging
in vitro (usually after 3–4 passages in vitro). In this case, the
Culturing Mycobacteria
23