[Cytometry] Log phase e.coli uptake of PI?
hms at shapirolab.com
Mon Feb 23 23:39:33 EST 2009
Brit Johansen wrote:
> One question for you: Is there a chance that healthy e.coli cells in
> exponential phase might incorporate PI?
Yes. See below.
> 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?
During the past few years a number of labs have reported uptake of PI by
apparently viable cells. The difference between propidium (PI) and
ethidium (EB) is that in ethidium, the side chain on the
phenanthridinium ring nitrogen is an uncharged ethyl group, whereas in
propidium, the side chain is a propyl group with a quaternary ammonium
substituent on the end opposite the ring. Although it is often
erroneously substituted for propidium as a putative indicator of
nonviability due to membrane damage, ethidium, with its single
delocalized positive charge, readily crosses the membrane and enters
many eukaryotic and prokaryotic cell types but is normally pumped out.
Propidium, with an extra, localized positive charge in addition to the
charge on the ring, is largely excluded by cells with intact cytoplasmic
membranes; it will get through damaged membranes, producing red staining
of double-stranded nucleic acids within the cell.
A high percentage of cells in biofilms have been reported to take up
propidium; although some of them may be dead, the overall growth rate is
too high for the population to be maintained only by the fraction of the
population that does not take up propidium.
In experiments with several types of bacteria, we have established,
using a flow cytometer with 488 and 633 nm excitation beams, that the
red-excited dye TO-PRO-3, which is analogous to propidium by virtue of
having a ring with a delocalized positive charge and a side chain with a
quaternary ammonium group, behaves as does propidium; cells either take
up both dyes or neither. That allowed us to do experiments in which we
simultaneously measured membrane potential, using an accurate and
precise ratiometric method with DiOC2(3) (available as a kit from
Molecular Probes), and permeability, using TO-PRO-3. We had not and have
not found an effective substitute for DiOC2(3) and related
oxacarbocyanines in the ratiometric potential measurement; since one of
the wavelengths at which we measure DiOC2(3) in that method is the peak
emission wavelength for PI, we could not use PI directly for the
We used the proton ionophore carbonyl cyanide chlorophenylhydrazone
(CCCP), which carries protons across the membrane but does not form
pores, to depolarize bacteria; this agent reduced membrane potential to
zero but did not make cells permeable to TO-PRO-3. Gramicidin, which
forms pores about 5 Angstroms in diameter, also depolarized cells
without inducing permeability to TO-PRO-3. Nisin, which forms pores
about 8 Angstroms in diameter, both depolarized cells and induced
permeability to TO-PRO-3. The uptake of TO-PRO-3 we observed in bacteria
with nonzero membrane potentials could not be explained by membrane
damage, because Gramicidin-induced pores, which compromise the membrane
sufficiently to reduce membrane potential to zero, do not permit the dye
to enter. TO-PRO-3 uptake can also not be explained by the hypothesis
that the dye does readily enter cells but is pumped out, as is the case
with ethidium; according to that hypothesis, one would expect cells
treated with CCCP, which interferes with energy metabolism but does not
produce breaches in the membrane, to retain the dye due to interference
with the efflux pump, and this is not the case. Although some events in
which DiOC2(3) fluorescence indicates the presence of a membrane
potential and TO-PRO-3 fluorescence indicates uptake are explainable as
aggregates of live cells with intact membranes and dead cells with
damaged membranes, the overall growth rate of the culture cannot be
accounted for by reproduction of only those cells not exhibiting
TO-PRO-3 uptake. I should point out that we are not growing cells in the
presence of TO-PRO-3, and that TO-PRO-3 is normally nontoxic to cells
because it does not get in; however, when the dye *is* taken up by
cells, it is toxic, as would be expected of a nucleic acid-binding
compound. The conclusion drawn from our experiments is that under
certain conditions, bacteria produce a transporter, the biological
substrate of which is presumably of some nutritive value, that will
carry TO-PRO-3 or PI into the organisms across intact cytoplasmic
membranes; thus, in bacteria, uptake of TO-PRO-3, PI, and, presumably,
other dyes with similar charge characteristics cannot always be taken as
an indicator of "nonviability."
These findings also suggested a previously unexplored path toward
development of antimicrobial agents; the relevant U. S. Patent is No.
The original references on ratiometric membrane potential and
permeability measurement are:
Novo D, Perlmutter NG, Hunt RH, Shapiro HM: Accurate flow cytometric
membrane potential measurement in bacteria using diethyloxacarbocyanine
and a ratiometric technique. Cytometry. 1999: 35:55-63.
Novo D, Perlmutter NG, Hunt RH, Shapiro HM: Multiparameter flow
cytometric analysis of antibiotic effects on membrane potential,
membrane permeability, and bacterial counts of Staphylococcus aureus and
Micrococcus luteus. Antimicrob Agents Chemother. 2000; 44:827-834
These are available online; a newer article on methodology is not, but I
will be happy to send anyone interested a .pdf file on request.
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