>
> I am looking for advise on a stain/dye that will penetrate cells
> (specifically bacteria) or bind with the surface membrane and not
> have to be washed off before acquisition. The stain/dye must excite
> with a 488nm laser and need either no fixation or a fixative that is
> mixed with the dye and does not need washing before acquisition.
FITC may do the trick - we have used this with unfixed Micrococcus
luteus using a stock of 1mg/ml FITC in acetone and adding it to the
bacterial suspension. A final concentration of 5ug/ml is an
approximate ball park figure - you may have to adjust this according
to your samples. I can't remember whether the staining took longer
with unfixed cells but we certainly got a fluorescence histogram not
dissimilar to that for fixed bacteria. It was mentioned briefly in
"Davey, H. M., Davey C. L. and Kell D.B. (1993). On the determination
of the size of microbial cells using flow cytometry. Flow cytometry
in microbiology. D. Lloyd. London, Springer-Verlag: 49-65." (On a
different thread David Lloyd's book is an excellent book for anyone
working on flow cytometry of microbes.....)
Haze.
----------------------------------------------------------
| Hazel Marie Davey hlr@aber.ac.uk |
|Sefydliad y Gwyddorau Biolegol*Inst. Biological Sciences|
|Prifysgol Cymru * University of Wales|
| ABERYSTWYTH, Ceredigion, CYMRU / WALES SY23 3DA |
| http://pcfcij.dbs.aber.ac.uk/index.htm |