How is the bacterial chromosome organized within the bacterial cell? Over the last 60 years, a variety of approaches have been used to investigate this question. More recently, the parallel development of epifluorescence microscopy and genetic tools has enabled the direct visualization of the intracellular positioning of DNA sequences in live cells and has consequently revolutionized our view of the architecture of the nucleoid in vivo. In this chapter I present a comprehensive methodology designed to characterize the architecture of the nucleoid DNA and the positioning of specific DNA sequences in live Escherichia coli cells. DNA localization systems, preparation of stable agarose-mounted microscopy slides, and basic image analysis tools are mentioned.
Keywords: Bacterial chromosome; DNA architecture and dynamics; Epifluorescence microscopy; Live cell imaging.