Culturing, Media, and Handling of Legionella
This chapter describes methods for culturing Legionella pneumophila in both complex and defined media. The first protocol describes the use of buffered charcoal yeast extract (BCYE) agar, the solid medium that is most commonly used for culturing L. pneumophila. The next procedure details the cultivation of L. pneumophila in buffered yeast extract (BYE) broth, i.e., the liquid medium version of BCYE agar. We describe how culturing in BYE broth can also be used for investigating proteins that are secreted by the type II secretion system of L. pneumophila. The next part of the chapter explains the cultivation of L. pneumophila in a chemically defined liquid media (CDM). CDM contains a mixture of amino acids, metals, α-ketoglutarate, and pyruvate. Because of its defined nature, CDM provides a simple means for controlling the concentration of nutrients and thereby allows for investigations of physiology and metabolism. To illustrate this point, the use of deferrated CDM for the purpose of assessing Legionella siderophore production is outlined. Finally, the chapter ends with a brief discussion of the storage and shipping of L. pneumophila.
Key wordsLegionella pneumophila Complex media Defined media BCYE BYE CDM Type II protein secretion Protease Iron Siderophore
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