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

Nebe-Von-Caron, G g.nebe-von-caron at spdspark.com
Tue Feb 24 06:49:40 EST 2009


Just to clarify that propidium iodide measures membrane integrity not
viability which is a much more complex subject. 

The reference that you stated 
(97.	Shi, L., Günther, S., Hübschmann, T., Wick, L.Y., Harms,H. and
Müller ,S. (2007) Limits of propidium iodide as a cell viability
indicator for environmental bacteria. Cytometry, online publication
April 2007 
http://www3.interscience.wiley.com/journal/114208367/abstract
Shows the same problem of PI uptake in exponential growth. 

Ever since I explain membrane integrity stains I add the caveat that
there are conditions of temporary permeabilisation even to the level of
PI uptake. Osmotic shock is a classical example as is electroporation. I
always state that one has to remove the cells from the stress before
testing for membrane integrity. Now it only seems natural that
exponential growth can be also be stress (and which parent would
disagree here?) and the cells might even show active uptake of the dyes
together with some substrate, but the key point is not to stress them
any further. My macroscopic analogy to the vulnerability of fast
replicating bacteria is that humans are also most vulnerable when caught
with their trousers down. 

In the recent issue in cytometry
http://www3.interscience.wiley.com/cgi-bin/fulltext/121577773/PDFSTART I
show the example of sonication with PI added before and after the
sonication just to raise awareness of the impact of the staining
protocol. Ideally you dilute the cells (if you have to) in the stuff
they just have been in (only spun clean) to have least perturbance or
otherwise in a nice buffer but avoid stressful environments such as
distilled water. If anything make them feel happy, dilute them with
Champaign so they do not feel the pain. Sorry could not resist this old
joke


All the best from

Gerhard
   
Perhaps I did take one pill to many this morning


Gerhard Nebe-von-Caron 
Research Scientist and Biomedical Engineer 
SPDspark, www.swissprecisiondiagnostics.com
SPD Development Company Limited
Priory Business Park
Stannard Way
Bedford    MK44 3UP
 
Company Registration No.  6032177
Mob+44(0)7792-116609
Tel+44(0)1234-835474 
Fax+44(0)1234-835006
mailto:g.nebe-von-caron at spdspark.com 
 

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Brit Johansen
Sent: 23 February 2009 14:57
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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