[Cytometry] stain specific for bacteria vs marine populations

John Casey john.casey at bios.edu
Mon Feb 1 19:56:29 EST 2010

Hi Rochelle,
    Since you're at Caltech I'm just going to wing-it and say that you're looking at E coli from sewage outflows in coastal water?  
    We often analyze phytoplankton and bacterioplankton from natural seawater and there are a dizzying number of approaches (see some of my favorites below:)- your choice will largely depend on your instrument and the complexity of the natural assemblage where your client sampled. I find that SYTO 13 (Invitrogen S7575) works best with 'blue water' bacteria (mostly alphaproteobacteria like SAR11 etc) and gives better discrimination and CV's than does SYBR G1, however you must take care to filter the stain through a 0.2um or better as there is a problem with free stain binding to detritus and pieces of cells which can overlap the so-called 'low nucleic acid bacteria' population. If you absolutely need to sort both phytoplankton and bacteria at the same time, you may want to go with a fluor that will not bleed into the autofluorescence bands of chlorophyll, PE, APC, etc such as Hoechst 33342 or DAPI, though you will want to do some calibrations between these and a cyanine based dye because there is a considerable difference in the binding properties. As far as discriminating E. coli from other bacteria, you may want to go the route of the CARD-FISH but remember this will mean you can't do FCM counts (need to resuspend the cells off a filter and/or pelletize). Good luck and feel free to give a shout!

T. GUINDULAIN, J. COMAS, AND J. VIVES-REGO (1997) Use of Nucleic Acid Dyes SYTO-13, TOTO-1, and YOYO-1 in the Study of Escherichia coli and Marine Prokaryotic Populations by Flow Cytometry. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 63(11);4608-4611

DOMINIQUE MARIE, FREDERIC PARTENSKY, STEPHAN JACQUET, AND DANIEL VAULOT (1997) Enumeration and Cell Cycle Analysis of Natural Populations of Marine Picoplankton by Flow Cytometry Using the Nucleic Acid Stain SYBR Green I. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 63(1);186-193

Raju Sekar, Bernhard M. Fuchs, Rudolf Amann, and Jakob Pernthaler (2004) Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 70(1);6210–6219


John Casey
Marine Particle Imaging Lab
Bermuda Institute of Ocean Sciences
17 Biological Lane
Ferry Reach, St. George's Parish
Bermuda, GE-01
(441) 297-1880 ext. 731
john.casey at bios.edu 

----- Original Message -----
From: "Rochelle A. Diamond" <diamond at its.caltech.edu>
To: cytometry at flowcyt.cyto.purdue.edu
Sent: Monday, February 1, 2010 4:56:07 PM
Subject: [Cytometry] stain specific for bacteria vs marine populations

HI Everyone,
I have a client that sorts marine phytoplankton specimens. We are 
looking for a stain or dye that we could use to label E.coli cells 
that would make them distinct from her natural seawater cells, so 
that I could sort them out concurrently with the phytoplankton 
populations that she is interested in.  I was wondering if you have 
any suggestions of a stain or dye that we could use that would have 
distinct fluorescent characteristics from the populations I have been 
sorting for thus far.

Ideas anyone?

Thanks for any suggestions,
Rochelle (Shelley) Diamond
Caltech Flow Cytometry Cell Sorting Facility
diamond at its.caltech.edu

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