Culturing Mycobacteria.

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.

Extraction and Separation of Mycobacterial Proteins.

The extraction and separation of native mycobacterial proteins remain necessary for antigen discovery, elucidation of enzymes to improve rational drug design, identification of physiologic mechanisms, use as reagents for diagnostics, and defining host immune responses. In this chapter, methods for the manipulation of whole mycobacterial cells and culture exudates are described in detail as these methods are the requisite first steps towards native protein isolation. Specifically, several methods for the inactivation of viable Mycobacterium tuberculosis along with qualification assays are provided, as this is key to safe manipulation of cell pastes for downstream processes. Next, the concentration of spent culture filtrate media in order to permit separation of soluble, secreted proteins is described followed by the separation of mycobacteria extracellular vesicles (MEV) from the remaining soluble proteins in spent media. We then describe the generation of whole-cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol-enriched proteins. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided. Finally, methods for separation of hydrophobic and hydrophilic proteins from both whole-cell lysate and spent culture media are included. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins.