Arne Rietsch writes: > >I'd like to use FACS to analyze the DNA content of bacterial cells in a >population (the question is if the bacterium we're studying segregates >daughter cells carrying only one of the chromosomes under starvation >conditions. These cells should be detectable as a population with >dramatically reduced DNA content.) >I was wondering if there are protocolls out there for staining bacteria >for FACS analysis. Preferably a stain that does not overlap with the >emisssion spectrum of GFP. Also the FACS that is available to me does not >have a UV laser, i.e. DAPI will not work. Unfortunately, if you want to measure DNA accurately in bacteria, you will almost certainly have to find a machine with either UV or blue-violet (436 to 457 nm) excitation, because only DAPI and the Hoechst dyes (UV-excited) and the mithramycin-ethidium combination (blue-violet excited) appear to provide adequate DNA specificity. Other dyes (and ethidium when used alone) stain double-stranded RNA as well as DNA; while RNAse treatment removes the interference from RNA in mammalian cells, it has, as far as I know, not been possible to permeabilize bacterial cells sufficiently to allow RNAse digestion of the RNA while preserving the DNA. While 7-aminoactinomycin D is excitable at 488 nm and DNA-specific, it does not give good stoichiometric staining and has a low quantum efficiency, eliminating it from consideration thus far for quantitative work on bacteria. There are several machines in the Harvard medical area which could provide UV or blue-violet excitation; staining protocols appear in an article by Steen et al in Volume 42 of Methods in Cell Biology. -Howard
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