I don't know what the structures of the "ChemChrome dyes" are but suspect that
one at least is a misnamed carboxyfluorescein diacetate succinimidyl ester
("CFSE"). That is because of the following abstract:
FEMS Microbiol Lett 1999 Sep 15;178(2):219-26
Effectiveness of CSE to counterstain particles and dead
bacterial cells with permeabilised membranes:
application to viability assessment in waters.
Catala P, Parthuisot N, Bernard L, Baudart J, Lemarchand
K, Lebaron P.
Observatoire Oceanologique, Centre National de la
Recherche Scientifique UMR7621, Institut National des Sciences de l'Univers
et Universite Pierre et Marie Curie, Banyuls-sur-Mer,
France.
The CSE dye (Chemunex, Maisons-Alfort, France) was
combined with an activity marker to improve bacterial activity assessment
in natural waters. Its effectiveness to counterstain dead
cells with permeabilised membranes was investigated on live and dead
cells of a variety of strains from collections or
isolated from the natural environment. Cells were killed by heat treatment. For
all
strains tested, the fluorescent dye showed an intense
staining of killed cells having permeabilised membranes while no
significant signal was detected when applied to live
cells. Furthermore, the CSE dye had no toxicity on viable cells. Then, CSE
was combined with the ChemChrome V6 dye (Chemunex) to
assess the activity of bacterial cells in different waters. Both
fluorescences were analysed simultaneously by solid-phase
cytometry. The active cell counts were sometimes lower when both
dyes were combined suggesting that CSE was able to
counterstain cells having a residual esterase activity and compromised
membranes. These cells were subtracted from the active
cell counts determined with ChemChrome V6. In most samples, active
cell counts were congruent with those determined by the
direct viable count method.
I bet that "CSE" = CFSE
The following abstract indicates that both ChrmChrome dyes are fluorescein
derivatives
J Appl Microbiol 2000 Aug;89(2):370-80
Evaluation of ChemChrome V6 for bacterial viability
assessment in waters.
Parthuisot N, Catala P, Lemarchand K, Baudart J, Lebaron
P.
Observatoire Oceanologique, Centre National de la
Recherche Scientifique, Institut National des Sciences de l'Univers et
Universite Pierre et Marie Curie, Banyuls-sur-Mer,
France.
The efficiency of ChemChrome B (CB) and ChemChrome V6
(CV6) dyes to stain viable bacterial cells in water was compared.
Both dyes are fluorogenic esters converted to free
fluorescein by esterase activity. The dyes were applied to a wide variety of
bacterial species, including those poorly stained by CB,
and to natural waters. Some species tested gave unacceptable low
fluorescence intensities by being inefficiently or
non-labelled with the CB. In contrast, CV6-stained bacteria were easily detected
by both flow cytometry and solid-phase cytometry. As a
consequence, higher viable cell counts were found with CV6 compared
with CB in natural waters. Viable counts determined by
CV6 staining were always higher than cfu counts. In contrast, respiring
cell counts (CTC) were always lower than CV6 counts and,
in the case of tap and mineral waters, they were lower than cfu
counts.
although the "conversion to fluorescein" is probably not correct because they
are propably substituted fluoresceins.
There are few options for esterase substrates for bacteria but probably the best
will be our CellTracker Green CMFDA (because it is very membrane permeant and
stays in cells following fixation)
http://www.probes.com/servlets/product?region=USA&item=2925
with this reference particularly pertinent
J Microbiol Methods 2000 May;40(3):265-74
Fluorescent labelling of intracellular bacteria in living
host cells.
Boleti H, Ojcius DM, Dautry-Varsat A.
Unite de Biologie des Interactions Cellulaire, Institut
Pasteur, URA CNRS 1960, 25 rue du Dr Roux, 75724, Paris, France.
hboleti@mail.pasteur.gr
The fluorescent reagent, CellTracker, labels
metabolically-active cells and was used here to label Chlamydia in vivo during
their
exponential phase of growth in infected cells. HeLa cells
infected with C. psittaci were labelled with the CellTracker reagents
between 15 and 48 h post-infection. The fluorescent label
accumulated in the host-cell membrane compartment (inclusion) within
which Chlamydia reside and replicate, and was also
incorporated by the bacteria. Labelling with the CellTracker affected neither
the growth nor the differentiation of the chlamydiae, and
labelled chlamydiae isolated from infected cells were infectious. Our
results demonstrate that the CellTracker could become a
valuable tool for in vivo labelling of obligate intracellular parasites for
which no genetic tools exist.
However, esterase substrate (like CFSE and CMFDA) are used much less in bacteria
than in eukaryotic cells.
I would have expected CFSE to have worked too, however. If it became fluorescent
without cells it was deteriorating in your buffer. If you had carrier proteins
in your buffer that would also be a problem, as would pH and possibly thiols.
This is a very reliable reagent for eukarotic cells but has rarely been used in
bacteria, except in the following:
Appl Environ Microbiol 2000 Oct;66(10):4486-96
Development of a vital fluorescent staining method for
monitoring bacterial transport in subsurface
environments.
Fuller ME, Streger SH, Rothmel RK, Mailloux BJ, Hall JA,
Onstott TC, Fredrickson JK, Balkwill DL, DeFlaun MF.
Envirogen, Inc., Princeton Research Center,
Lawrenceville, New Jersey 08648, USA. fuller@envirogen.com
Previous bacterial transport studies have utilized
fluorophores which have been shown to adversely affect the physiology of
stained cells. This research was undertaken to identify
alternative fluorescent stains that do not adversely affect the transport or
viability of bacteria. Initial work was performed with a
groundwater isolate, Comamonas sp. strain DA001. Potential compounds
were first screened to determine staining efficiencies
and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate,
succinimidyl ester (CFDA/SE) efficiently stained DA001
without causing undesirable effects on cell adhesion or viability.
Members of many other gram-negative and gram-positive
bacterial genera were also effectively stained with CFDA/SE. More
than 95% of CFDA/SE-stained Comamonas sp. strain DA001
cells incubated in artificial groundwater (under no-growth
conditions) remained fluorescent for at least 28 days as
determined by epifluorescent microscopy and flow cytometry. No
differences in the survival and culturability of
CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment
microcosms were detected. The bright, yellow-green cells
were readily distinguished from autofluorescing sediment particles by
epifluorescence microscopy. A high throughput method
using microplate spectrofluorometry was developed, which had a
detection limit of mid-10(5) CFDA-stained cells/ml; the
detection limit for flow cytometry was on the order of 1,000 cells/ml. The
results of laboratory-scale bacterial transport
experiments performed with intact sediment cores and nondividing DA001 cells
revealed good agreement between the aqueous cell
concentrations determined by the microplate assay and those determined
by other enumeration methods. This research indicates
that CFDA/SE is very efficient for labeling cells for bacterial transport
experiments and that it may be useful for other microbial
ecology research as well.
Hoefel Daniel wrote:
> Dear List,
>
> I recently tried using 5-(and 6-) carboxyfluoresceine diacetate,
> succinimidyl ester (CFDA/SE) for flow cytometric detection of esterase
> active bacteria in water samples (ie. as an indication of bacterial
> viability). Unfortunately, for various reasons, the assay was unsuccessful.
>
> I am currently searching for another method for detecting esterase active
> bacteria. I have read many papers that have successfully used
> carboxyfluoresceine diacetate (CFDA), although I am a bit reluctant to try
> this as it is a similar compound to CFDA/SE which I have already had trouble
> with.
>
> I am also thinking of trying ChemChrome B and/or ChemChrome V6, although I
> have been told by the manufacturer (Chemunex) that they wont sell these
> reagents unless they are to be used on their own instrumentation.
>
> Therefore, I would appreciate hearing from anyone who has successfully used
> any of the reagents mentioned above. I would also like any
> comments/suggestions for other compounds that people may have used for the
> detection of esterase active bacteria.
>
> Regards,
>
> Daniel Hoefel
>
> E-mail: daniel.hoefel@sawater.sa.gov.au
>
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