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

Howard Shapiro 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 
permeability measurement.

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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