Chapter 1
Culturing Mycobacteria
Elizabeth Wallace, Debra Hendrickson, Nicholas Tolli, Carolina Mehaffy,
Marı ´a Pen ˜ a, Jerry A. Nick, Phillip Knabenbaur, Jackson Watkins,
Anne Simpson, Anita G. Amin, Delphi Chatterjee, Karen M. Dobos,
Ramanuj Lahiri, Linda Adams, Michael Strong, Max Salfinger,
Rebecca Bradford, Timothy T. Stedman, Marco A. Riojas,
and Manzour Hernando Hazbo ´ n
Abstract
Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists
have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known
mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo
passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for
diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific
purposes. Detailed protocols used for production of M. tuberculosis in large scale (under normoxic and
hypoxic conditions) in bioreactors and for production of obligate intracellular pathogens such as Mycobacterium leprae and “Mycobacterium lepromatosis” using athymic nude mice and armadillos are provided.
Key words Mycobacterium tuberculosis, Mycobacterium leprae, Nontuberculous mycobacteria, Normoxic culture, Hypoxic culture, Large-scale production of mycobacteria
1 Introduction
Culturing mycobacteria is a critical task for both clinical and
research laboratories that either diagnose mycobacterial infections
or study different aspects of mycobacterial physiology. Difficulties
associated with the culture of mycobacteria have historically been
associated with delayed diagnosis of these infections in the clinical
setting and are factors in the slow pace of research on the genus. In
this chapter, we provide protocols for mycobacteria isolation, smallscale growth for diagnostic purposes, and large-scale growth for
research purposes. Isolation of mycobacteria is a requirement for
both clinical [i.e., Mycobacterium tuberculosis and nontuberculous
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
1
Culturing Mycobacteria
Elizabeth Wallace, Debra Hendrickson, Nicholas Tolli, Carolina Mehaffy,
Marı ´a Pen ˜ a, Jerry A. Nick, Phillip Knabenbaur, Jackson Watkins,
Anne Simpson, Anita G. Amin, Delphi Chatterjee, Karen M. Dobos,
Ramanuj Lahiri, Linda Adams, Michael Strong, Max Salfinger,
Rebecca Bradford, Timothy T. Stedman, Marco A. Riojas,
and Manzour Hernando Hazbo ´ n
Abstract
Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists
have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known
mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo
passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for
diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific
purposes. Detailed protocols used for production of M. tuberculosis in large scale (under normoxic and
hypoxic conditions) in bioreactors and for production of obligate intracellular pathogens such as Mycobacterium leprae and “Mycobacterium lepromatosis” using athymic nude mice and armadillos are provided.
Key words Mycobacterium tuberculosis, Mycobacterium leprae, Nontuberculous mycobacteria, Normoxic culture, Hypoxic culture, Large-scale production of mycobacteria
1 Introduction
Culturing mycobacteria is a critical task for both clinical and
research laboratories that either diagnose mycobacterial infections
or study different aspects of mycobacterial physiology. Difficulties
associated with the culture of mycobacteria have historically been
associated with delayed diagnosis of these infections in the clinical
setting and are factors in the slow pace of research on the genus. In
this chapter, we provide protocols for mycobacteria isolation, smallscale growth for diagnostic purposes, and large-scale growth for
research purposes. Isolation of mycobacteria is a requirement for
both clinical [i.e., Mycobacterium tuberculosis and nontuberculous
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
1