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

Brit Johansen brit.johansen at hihm.no
Mon Feb 23 09:56:33 EST 2009


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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