Re: bacteria and flow
Howard Shapiro (hms@shapirolab.com)
Sat, 12 Oct 1996 21:22:00 -0400 (EDT)
>
>Staining live, unfixed bacteria with Hoechst 33342 (or another DNA stain
with similar spectral characteristics). I tried loading bacteria with H
33342 but they did not take up the dye. Do I need to use EDTA or some other
agent to get the H 33342 into the bacteria?
>
DAPI generally gets in more readily than Hoechst 33342. You might need EDTA
for Gram-negative bacteria. It is now established that some bacteria have
glycoprotein efflux pumps which will pump Hoechst 33342 and many other dyes
out, although you can usually stain cells which have efflux pumps if you use
a high enough concentration of dye. Steen et al (Methods in Cell Biology
Vol. 41) use a mixture of EDTA and azide to get ethidium into unfixed cells,
but that might not be compatible with viable sorting.
-Howard
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