In our experience, it is possible to stain and quantitate bacterial DNA content using propidium iodide after ethanol fixation and RNAase treatment. Durodie et al. (pp 95-110, Flow cytometry in Microbiology, David Lloyd ed., Springer-Verlag, 1993) have published a protocol for measuring ploidy of E.coli cells. We have optimized a protocol for DNA staining of Gram positive streptococcal species (J. Micro. Meth. 34(3):223-233, 1999). The critical steps appear to be lysozyme and mutanolysin treatment after ethanol fixation, and using DNAase-free RNAase A for removing ds RNA prior to PI staining (we use the protocol from Sambrook et al., Volume III, 2nd. edition, 1989, for making our RNAaseA DNAase-free). Using our protocol, we were able to discern peaks in DNA distributions of streptococci growing in chains that corresponded to integral multiples of chromosome equivalents. As always, controls (such as antibiotic-treated cells containing an integral number of chromosomes) are essential for correct interpretation of data. In an aside, we have noticed that ethanol fixation significantly reduces green fluorescence intensity from Gfp in S.cerevisiae cells (J. Biotechnol. 62(1):29-45, 1998), although we have not done a similar study in bacterial cells. Arvind Natarajan University of Minnesota _______________________________________________________________________________ Howard Shapiro writes: 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-aminoactinomycinD 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. _______________________________________________________________________________ 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.
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