Dead Cell Marker on 633 nm

Douglas B. Kell dbk at aber.ac.uk
Mon Nov 11 15:49:14 EST 1996


>           Despite my favour of membrane integrity measurements to
>           the detection of bacterial death, membrane
>           permeabilisation can be transient, so its measurement is
>           only relevant under defined conditions. Electroporation or
>           chemoporation are prime examples and solvents like DMSO
>           are quite helpfull to get molecules across intact
>           membranes as well.
> 
>           EB is a supravital stain that indeed undergoes active
>           transport that can be inhibited by starvation or for
>           example azide. When we sort single EB+ bacteria directly
>           onto agar plates (using our inferior Coulter Elite with
>           Autoclone) they do indeed divide.
> 
>           Bacteria that take up BIS-Oxonol (a dead cell marker
>           supposingly superior to propidium iodide) can also grow
>           out as we could also show by direct sorting of such
>           populations onto agar plates. Simultanious staining with
>           both dyes indeed shows that there is a (resuscitable)
>           population of BOX+,PI- cells, a population that also
>           occurs naturally in the early stages of sporulation.
>

Gerhard - Fascinating, and as I suspected, you have done the 
pertinent expts. You must tell us more, esp. if it's published or is
about to be. I thought you'd probably assist the discussion; this
was my reply to Tom Frey (but not the group) yesterday:

From:                 Self <Single-user mode>
To: cyto-inbox
Subject:          Re: Re[4]: Dead Cell Marker on 633 nm
Copies to:        dbk
Send reply to:    dbk at aber.ac.uk
Date sent:        Sun, 10 Nov 1996 19:23:29 +0000

> Yes, I read your "gold standard" question as related to concerns
> about the quality of the TO-PRO-3 that you were using, not the true
> biology.  I included the apoptosis remark because I was peripherally
> aware (you can't keep up on everything) that bug workers found these
> intermediate states also, and were begining to debate the
> relationship to apoptosis mechanisms.  

That's certainly the case, though (apart from some rather special
cases of suicide genes) there's little if any evidence for the
necrosis/apoptosis distinction commonly described in higher cells.

>I've run out of remarks that
> are knowledgable enough for public posting, but maybe some of the
> other bacteria researchers will take up your gold standard
> challenge.
> 

Actually we are just about to send off an invited minireview of this
general problem; even the insiders are faced with a ghastly and
confusing literature, and the outsiders have no chance of seeing what
the real issues are by reading the sort of literature mainly
available. The conclusion is that no optical probe makes general sense
for discriminating live, dead and in-between; only in specified
strains and condiitons can one sometimes get decent correlations with
division. Maybe Gerhard Nebe von Caron will take up the cudgels for
the flowcyt group!

Best wishes,
Douglas.
---
 
>           If I guess it correctly TO-PRO is thiazolorange-protein
>           conjugate, where the protein could also give rise to a
>           binding reaction, in particular with bacteria. 

I don't think so, but surely the information is available 
somewhere. PO-PRO is  
 4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-1-(3'-
trimethylammonium propyl)-pyridinium diiodide which I must have got 
off MP's data sheet. Either way we should be able to find out from 
Mol Probes. Proteases would plausibly turn TOPRO --> TO and surely 
that would not be good!?


>Thus to
>           probe for the intracellular binding it would either need
>           high resolution microscopy or perhaps some clever
>           quenching experiment. In the case of TO-PRO3 dissociation
>           Martin might give us perhaps some guidance in what
>           spectral shift to expect if the protein would start to
>           'dissociate'.
> 
>           With respect to resuscitation or the population effect on
>           apoptosis we should perhaps be able to get a very
>           interresting thread to start. Perhaps there is some
>           relevance in the bacterial cell death compared to the
>           behaviour of mitochondria in the apoptotic pathway.
> 
>           Gerhard Nebe-v.Caron
>           Unilever Research, Colworth,
>           Sharnbrook, Bedfordshire
>           GB - MK44 1LQ
>           Tel:    +44(0)1234-222066
>           FAX:    +44(0)1234-222344
>           gerhard.nebe-von-caron at unilever.com
> 
> 
> ______________________________ Reply Separator
> _________________________________ Subject: Re: Re[2]: Dead Cell
> Marker on 633 nm Author:  dbk at aber.ac.uk at INTERNET Date:   
> 11/11/96 02:21
> 
> 
> Tom Frey writes:
> 
> > In response to professor Kell's post, I advance the following
> > personal opinions.
> >
> 
> [munch]
> 
> > > Out of interest, what is here the  'gold standard'? We in Aber
> > > are beginning to have suspicions that supposedly pure(ish) 
> > > TO-PRO[3], or likely a metabolite / degradation product thereof,
> > > penetrates (and ergo stains) ""live"" [i.e. not all that
> > > knackered] cells (bugs). What say you, and netters to whom this
> > > is copied?
> >
> > I mention in passing in Cytometry 21:265 that TO-PRO-3 enters
> > apoptotic thymocytes more rapidly than the normal cells in the
> > same sample.  PI and EB are well know to behave the same.  While
> > "apoptotic" may or may not mean something to bugs, I would
> > hesitate to suggest problems with TO-PRO-3 unless I knew that PI
> > and EB do not show the same effect.  You may, of course, know
> > this.
> 
> This is the basic problem. Of course in the case of apoptosis in
> eukaryotes there is presumably a state of commitment fairly early on
> by when the apoptotic 'programme' is set and canot be reversed, all
> cells reaching that point going on to self-immolate (or die by
> necrosis if they meet something even nastier, presumably). At any
> rate it is easy soon after to classify them as dead, or not alive
> (in a 2-valued logic system).
> 
> But in bugs in particular there are 'in-between' states in which
> vital stains do not necessarily discriminate populations which
> conveniently classify themselves by OUR gold standard method (which
> is ability to divide), and cells which fall in the nominally 'dead'
> populations may in fact be dormant and can be resuscitated. See a
> series of papers by Arseny Kaprelyants and me, e.g.
> 
> Kell, D.B., Ryder, H.M., Kaprelyants, A.S. & Westerhoff, H.V. (1991)
> Quantifying heterogeneity: flow cytometry of bacterial cultures.
> Antonie van Leeuwenhoek 60, 145- 158.
> 
> Kaprelyants, A.S. & Kell, D.B. (1992) Rapid assessment of bacterial
> viability and vitality using rhodamine 123 and flow cytometry. J.
> Appl. Bacteriol., 72, 410-422.
> 
> Kaprelyants, A.S. & Kell, D.B. (1993) Dormancy in stationary-phase
> cultures of Micrococcus luteus: flow cytometric analysis of
> starvation and resuscitation. Appl. Env. Microbiol. 59, 3187-3196.
> 
> Kaprelyants, A.S., Mukamolova, G.V. & Kell, D.B. (1994) Estimation
> of dormant Micrococcus luteus cells by penicillin lysis and by
> resuscitation in cell-free spent culture medium at high dilution.
> FEMS Microbiol. Lett., 115, 347-352.
> 
> Votyakova, T.V., Kaprelyants, A.S. & Kell, D.B. (1994) Influence of
> viable cells on the resuscitation of dormant cells in Micrococcus
> luteus cultures held in extended stationary phase. The population
> effect. Appl. Env. Microbiol. 60, 3284-3291.
> 
> See also various papers by Mike Barer (Newcastle) using image
> cytometry.
> 
> >
> > This does suggest my opinion of a gold standard.  TO-PRO-3 is only
> > available from Molecular Probes, and should be compared to similar
> > molecules like ethidium and propidium which have long histories
> > and multiple sources.
> 
> So I perhaps should have clarified here; the gold standard would for
> a bug person normally be division, colony formation and the like,
> and not some other (and perhaps equally questionable) optical probe.
> EB for instance is actively pumped out of various Gram-negative
> cells (Jernaes, M. W. & Steen, H. B. (1994). Staining of Escherichia
> coli for flow cytometry: influx and efflux of ethidium bromide.
> Cytometry 17, 302-309).
> 
> Best wishes,
> Douglas.
> (Prof) Douglas B. Kell
> Edward Llwyd Building,
> Institute of Biological Sciences
> University of Wales,
> Aberystwyth SY23 3DA, UK
> Tel +44 1970 622334
> Fax +44 1970 622354
> dbk at aber.ac.uk
> http://gepasi.dbs.aber.ac.uk/home.htm
(Prof) Douglas B. Kell
Edward Llwyd Building, 
Institute of Biological Sciences
University of Wales,
Aberystwyth SY23 3DA, UK
Tel +44 1970 622334
Fax +44 1970 622354
dbk at aber.ac.uk
http://gepasi.dbs.aber.ac.uk/home.htm



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