Analysis of Lipid Biosynthesis and Location

A procedure for metabolic labeling of all cellular lipids starting with a culture of mycobacteria is described in this chapter using either a pulse-chase or a simple labeling experimental design. Three fractions are produced for subsequent lipid analysis: (1) the culture filtrate; (2) a readily released surface lipid fraction; and (3) the killed, labeled bacteria. A standardized, TLC-based method for general lipid analysis that can be used to quantify the labeling of all the mycobacterial lipids is given as well as a protocol for analyzing the fatty acyl moieties of the lipids.