Fluorescence and Brightfield Cytology of Live M. tuberculosis Cells

Although light microscopy fell out of favor as a research tool in prokaryotic biology in the 1980s, advances in the reagents available for cell labeling (staining) and in the user-friendliness of microscopes were underpinning a revolution in eukaryotic cell biology. The development of epifluorescence hardware, particularly confocal microscopy and low-light imaging systems, and computational deblurring and video enhancement methodologies, substantially extended the range of potential applications. These developments now enable us to detect weaker signals at higher levels of resolution than was previously possible. Finally the personal computer and related software developments have brought image analysis within affordable range for many laboratories and facilitate quantitation of cellular properties on an objective basis. We have sought to apply these advances across a range of prokaryotic applications and here we describe the methods we have applied to live Mycobacterium tuberculosis cells. Although we have principally been concerned with two applications, the determination of viability at the cellular level (see Note 1) and the nature and distribution of lipid domains, more general aspects of light microscopic cytological analyses are discussed below.