[Cytometry] Log phase e.coli uptake of PI?

Reed, Douglas S dsreed at pitt.edu
Tue Feb 24 09:21:39 EST 2009


Have you tried determining how your flow cytometry counts compare to what can be quantitated by culturing on solid agar or counting under a microscope? I'm curious as to whether these cells are indeed viable/culturable (keeping in mind that viable does not necessarily equal culturable). I know you say that you expect only a few % to be dead but that might not be the case; they could indeed be dead.

I'd be curious how the staining looks if you tried earlier or later points in the growth curve.

Sincerely,
Doug


Douglas S. Reed, Ph.D.
Aerobiology Manager
Regional Biocontainment Laboratory
Assistant Professor
Department of Immunology
University of Pittsburgh
8037 BST3, 3501 Fifth Avenue
Pittsburgh, PA 15261
(412) 648-9290
(412) 648-0050 lab
(412) 648-8917 fax
dsreed at cvr.pitt.edu




-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Brit Johansen
Sent: Monday, February 23, 2009 9:57 AM
To: cytometry at lists.purdue.edu
Subject: [Cytometry] Log phase e.coli uptake of PI?


Hi all

One question for you: Is there a chance that healthy e.coli cells in
exponential phase might incorporate PI?

I'm using Live/Dead BacLight kit.

In my experiment I stain a growing population of e.coli cells with syto9 and
PI. Looking at the results it appears that the PI histogram population is
split in two, and that one peak has a higher PI fluorescence intensity than
the other. I would expect only a few % dead cells, but if I should judge by
the peak with the highest fluorescence intensity I have over 50 %...

In the literature this is found for g+ and g- cells ( but not coli)(sorry I
got the quotes from someone else and I don't have the references):

"PI uptake depended on the physiological state of the bacterial cells.
Unexpectedly, up to 40% of both strains were stained by PI during
exponential growth on glucose when compared to 2-5% of cells in the early
stationary phase of growth". 

" exponentially-growth-phase E. coli cells stained with a combination of
SYBR green and PI displaied higher green fluorescent intensity levels than
did stationary phase bacteria. This result might be due to cell envelope
alterations.(...)Additionally, exponential cells are believed to have higher
contents of RNA due to increased metabolic activity, which can also lead to
enhanced green fluorescence intensity".

Any suggestions?

Thanks

Brit R. Johansen
Hedmark University College
Norway

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