403 Ludwig Eichinger and Francisco Rivero (eds.), Dictyostelium discoideum Protocols, Methods in Molecular Biology 983, DOI 10.1007/978-1-62703-302-2_22, © Springer Science+Business Media, LLC 2013 Chapter 22 Setting Up and Monitoring an Infection of Dictyostelium discoideum with Mycobacteria Sonia Arafah*, Sébastien Kicka*, Valentin Trofimov, Monica Hagedorn, Nuria Andreu, Siouxsie Wiles, Brian Robertson, and Thierry Soldati Abstract Mycobacterium marinum is the causative agent of fish and amphibian tuberculosis in the wild. It is a genetically close cousin of Mycobacterium tuberculosis, and thereby the infection process remarkably shares many of the hallmarks of M. tuberculosis infection in human, at both the cellular and organism levels. Therefore, M. marinum is used as a model for the study of mycobacterial infection in various host organ- isms. Recently, the Dictyostelium–M. marinum system has been shown to be a valuable model that reca- pitulates the main features of the intracellular fate of M. marinum, including phagosome maturation arrest, as well as its particular cell-to-cell dissemination mode. We present here a “starter kit” of detailed methods that allows to establish an infection of Dictyostelium with M. marinum and to monitor quantita- tively the intracellular bacterial growth. Key words Phagocytosis, Phagosome maturation, Mycobacteria, Intracellular infection Dictyostelium discoideum is an amoeba model that has been used for decades to investigate development, morphogenesis, social behavior, chemotactic cell motility, and many other aspects of cell biology (1). Its fully sequenced and annotated haploid genome (2), associated with genetics tool, easy manipulation, and real-time live imaging, makes the social amoeba a remarkable experimental model organism. Dictyostelium naturally grazes by extensive phagocytosis on a broad variety of soil bacteria including both Gram-negative and Gram-positive bacteria. The ability of Dictyostelium cells to grow 1 Introduction *Sonia Arafah and Sébastien Kicka contributed equally to the work.