Sue DeMaggio asks: >Has anyone used Hoechst to stain bacteria? or have a procedure for it? I >have a client asking. Live or fixed? Hoechst will get into some live bacteria; so will DAPI; both will stain a broader range of bacteria after ethanol fixation. Many years ago, the Livermore group used staining with Hoechst 33258 and chromomycin A3 to discriminate bacterial species on the basis of the A-T/G-C ratio in DNA (Van Dilla MA, Langlois RG, Pinkel D et al: Bacterial characterization by flow cytometry. Science 220:620, 1983). However, a recent paper (Walberg M, Gaustad P, Steen HB: Uptake kinetics of nucleic acid targeting dyes in S. aureus, E. faecalis, and B. Cereus: a flow cytometric study. J Microbiol Methods 1999; 35:167-176) indicates that staining of bacteria by Hoechst dyes and most other nucleic acid stains can vary considerably from species to species, and may be affected by the presence or absence of efflux pumps (in live cells), as well as by the fixative or permeabilizing agent used. With a consistent preparative procedure, one ought to be able to compare DNA contents of different cells from a single species; comparing species could be trickier. It is becoming increasingly evident that, in terms of their behavior when exposed to the fluorescent dyes we know and love, bacteria aren't just little eukaryotes. -Howard
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