Re: bacteria and flow

Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
14 Oct 1996 10:00:52 +0100

Supravital DNA stains are in most cases actively pumped out
of the bacteria. You can inhibit the pumps using 0.2% azide
or use high dye concentrations. Incubation at 40oC also
helps influx of the dyes they get pumped out within minutes
if you lower the dye concentration outside. The buffer
composition can also inhibit the pumps i.e. deenergize the
cells if they are washed often enough.

Both, azide and EDTA are compatible with viable sorting for
short incubation times (about 2 hours) as long as your cells
are not injured.

Sorting literature on bugs is so far pretty rare, but you
might want to get in touch with Rob Campbell at BD (1993,
Proc. Natl.Acad.Sci Vol 90 p10444-10448), Roger W.Pickup at
the Institute of Freshwater Ecology at Lake Windermere UK
(1993, Appl. Environmental Microbiology Vol 59,No 10
p3327-3333) or myself for further information. You can find
some nice images about direct sorting onto plates and some
information on viable staining on the next purdue CD or on
ftp://ftp.cyto.purdue.edu/cdupload/gerhard as long as it is
still there.

Regards

Gerhard Nebe-v.Caron
Unilever Research, Colworth,
Sharnbrook, Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
gerhard.nebe-von-caron@unilever.com

______________________________ Reply Separator _________________________________
Subject: bacteria and flow
Author: knappt@aurorabio.com at INTERNET
Date: 12/10/96 03:56

Hello all,

I am looking for references and/or advice on the following:

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?

Does anyone have any tips or good references in general on sorting bacteria
using a flow cytometer.

Thanks in advance,

Tom Knapp
Aurora Biosciences Corporation
11149 N. Torrey Pines Road
La Jolla, California 92037 USA

email "knappt@aurorabio.com"
Web Site "http://www.aurorabio.com"
(619) 452-5000 ex 125

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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone: (765)-494-0757; FAX(765) 494-0517; Web http://www.cyto.purdue.edu , EMAIL cdrom3@flowcyt.cyto.purdue.edu