Sue, we have similar experiences with fluorescence variability among bacterial species as described by Howard, also, with repect to efflux pumps. In addition, a remark on the UV- laser output: In the past we used a FACS II (YES, some of them are still alive) with a 200 mW water-cooled laser for Hoechst 33342 fluorescence in live bacteria; we got clear fluorescence separation of unstained / stained populations. In contrast, the 20 mW 323nm air-cooled laser of our Elite now gives big overlaps of pos./negatives. We came to the point that we somewhat question the use of Hoechst 33342 as live stain for bacteria, especially with low-output UV-lasers or in combination with (0.1 µm-filtered) minimal media; the number of events accounting for background fluorescence is always hard to assess. Joerg Ueckert Unilever Research, NL -----Original Message----- From: Howard Shapiro [SMTP:hms@shapirolab.com] Sent: Friday, February 18, 2000 1:23 AM To: Cytometry Mailing List Subject: Re: Hoechst and microbiology 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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