Dead Cell Marker on 633 nm

Gerhard Nebe-von-Caron Gerhard.Nebe-von-Caron at unilever.com
Mon Nov 11 08:33:17 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.

          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. 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



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