mycobacteria (NTM) from sputum and other clinical specimens]
and research (i.e., NTM from environmental samples) laboratories.
Isolation of mycobacteria from these complex samples must factor
the presence of other microorganisms that may outgrow mycobacteria present in each sample. For that reason, clinical and environmental samples need to be homogenized, fractionated to
discourage growth of non-mycobacterial species, and concentrated.
Selective media are recommended as an initial step for culture
isolation. Once a mycobacterial strain is isolated and determined
to be pure, culturing may be performed in the absence of selective
media. However, in the case of slow-growing strains, it may be
prudent to continue growth on selective media to ensure or maintain purity from contaminating species inadvertently introduced
into the culture. Thus, mycobacteria are typically cultured in selective media. Large-scale growth, particularly of Biosafety level-3
mycobacteria, such as members of the M. tuberculosis complex,
also poses additional biosafety considerations that need to be
taken into account in order to minimize the risk of infection for
laboratory personnel. In this chapter, we gathered a wide variety of
culture procedures that could be used in several settings for different types of applications. Many of the authors of this chapter are
involved in the production of mycobacterial strains and reagents for
distribution to the mycobacteriology research community [1].
Many mycobacterial strains and biological derivatives are available through ATCC (https://www.atcc.org) or BEI Resources
(https://www.beiresources.org). BEI Resources enables access of
strains and reagents to laboratories engaged in mycobacterial and
infectious disease research and is funded by the National Institute
of Allergy and Infectious Diseases (NIAID). These reagents are
authenticated, quality-assured biomaterials characterized by genotypic and phenotypic assays when produced and provide standards
of quality that may not be available from shared laboratory stocks.
Some of the protocols for growth and production of these reagents
are included in this chapter. Mycobacterium leprae is uncultivable on
microbiological culture media and must be grown in animal models. The Health Resources and Services Administration (HRSA)
National Hansen’s Disease Program Laboratory (NHDP) (Baton
Rouge, LA) grows large quantities of M. leprae in nine-banded
armadillos and provides the infected tissues to the Dobos Laboratory at Colorado State University (Fort Collins, CO) for purification of leprosy reagents. The HRSA NHDP also grows M. leprae
and “M. lepromatosis” in athymic nude mouse footpads for provision of live bacilli to researchers [2]. The Dobos Laboratory produces a large number of M. tuberculosis reagents thanks to their
expertise with large-scale growth of different M. tuberculosis strains.
On the clinical side, resources for researchers, including wellcharacterized NTM isolates from individuals with cystic fibrosis
(CF), accompanying whole-genome sequencing information, and
2
Elizabeth Wallace et al.
and research (i.e., NTM from environmental samples) laboratories.
Isolation of mycobacteria from these complex samples must factor
the presence of other microorganisms that may outgrow mycobacteria present in each sample. For that reason, clinical and environmental samples need to be homogenized, fractionated to
discourage growth of non-mycobacterial species, and concentrated.
Selective media are recommended as an initial step for culture
isolation. Once a mycobacterial strain is isolated and determined
to be pure, culturing may be performed in the absence of selective
media. However, in the case of slow-growing strains, it may be
prudent to continue growth on selective media to ensure or maintain purity from contaminating species inadvertently introduced
into the culture. Thus, mycobacteria are typically cultured in selective media. Large-scale growth, particularly of Biosafety level-3
mycobacteria, such as members of the M. tuberculosis complex,
also poses additional biosafety considerations that need to be
taken into account in order to minimize the risk of infection for
laboratory personnel. In this chapter, we gathered a wide variety of
culture procedures that could be used in several settings for different types of applications. Many of the authors of this chapter are
involved in the production of mycobacterial strains and reagents for
distribution to the mycobacteriology research community [1].
Many mycobacterial strains and biological derivatives are available through ATCC (https://www.atcc.org) or BEI Resources
(https://www.beiresources.org). BEI Resources enables access of
strains and reagents to laboratories engaged in mycobacterial and
infectious disease research and is funded by the National Institute
of Allergy and Infectious Diseases (NIAID). These reagents are
authenticated, quality-assured biomaterials characterized by genotypic and phenotypic assays when produced and provide standards
of quality that may not be available from shared laboratory stocks.
Some of the protocols for growth and production of these reagents
are included in this chapter. Mycobacterium leprae is uncultivable on
microbiological culture media and must be grown in animal models. The Health Resources and Services Administration (HRSA)
National Hansen’s Disease Program Laboratory (NHDP) (Baton
Rouge, LA) grows large quantities of M. leprae in nine-banded
armadillos and provides the infected tissues to the Dobos Laboratory at Colorado State University (Fort Collins, CO) for purification of leprosy reagents. The HRSA NHDP also grows M. leprae
and “M. lepromatosis” in athymic nude mouse footpads for provision of live bacilli to researchers [2]. The Dobos Laboratory produces a large number of M. tuberculosis reagents thanks to their
expertise with large-scale growth of different M. tuberculosis strains.
On the clinical side, resources for researchers, including wellcharacterized NTM isolates from individuals with cystic fibrosis
(CF), accompanying whole-genome sequencing information, and
2
Elizabeth Wallace et al.