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Microbes and Infection, 2, 2000, 1523−1535

© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved



Use of fluorescent probes to assess

physiological functions of bacteria
at single-cell level
Fabien Joux, Philippe Lebaron*
Observatoire océanologique, université Pierre et Marie Curie, UMR-7621 CNRS, Institut national des sciences de l’Univers,
BP 44, F-66651 Banyuls-sur-Mer cedex, France

ABSTRACT – A wide diversity of fluorescent probes is currently available to assess the

physiological state of microorganisms. The recent development of techniques such as solid-phase
cytometry, the increasing sensitivity of fluorescence tools and multiparametric approaches
combining taxonomic and physiological probes have improved the effectiveness of direct methods in
environmental and industrial microbiology. © 2000 Éditions scientifiques et médicales Elsevier
bacteria / fluorescent probes / physiology / cytometry

1. Introduction fluorescent dye technology offering probes for a variety of

cellular functions. The VNC state was initially suggested
One of the most basic questions that microbiologists by Roszak et al. [4] for readily culturable bacteria which
address is whether a bacterial cell is alive or dead. The under particular circumstances become non-culturable
answer to this question is far from simple and the question but retain ‘viability’. Nevertheless, the viability of VNC
remains unanswered after 20 years of intense research and cells was never defined by culture but only by the detec-
permanent controversy [1]. Life is generally characterised tion of some forms of activity in cells, which are not
by: (i) the presence of structure; (ii) changeable genetic directly related to the capacity of these cells to reproduce.
information; (iii) metabolism or functional activity, and (iv) For these reasons, the term ‘active but non-culturable’
the ability to reproduce and grow [2]. However, living (ANC) might be more precise than viable but non-
bacteria are generally only characterised by their ability to culturable [1].
reproduce, probably because the capacity of a cell to Fluorescence-based methods have remained very use-
multiply as determined by culture has long been the single ful for a wide diversity of applications ranging from indus-
method available to microbiologists for bacterial viability trial to environmental microbiology. These tools are used
assessment. Therefore, culturability and viability are often for viability/activity assessment in food, pharmaceutical
considered synonymous terms. and cosmetic industries, and in the natural environment,
During the last two decades, an increasing need for including fresh and marine waters. The increased use of
alternative techniques has been stimulated by: (i) the need fluorescent probes is also due to improvements in the
for real-time monitoring methods for viability assessment quantitative and qualitative sensitivity of instruments. The
[3]; (ii) the existence of injured and ‘viable but non- aim of this review is to describe the different strategies
culturable’ (VNC) cells sometimes encountered when cells used for the detection of cell fluorescence, the different
are stressed (e.g. heat, oxidative, osmotic stress, or starva- cellular target sites and fluorescent probes which are
tion) [1, 4], and (iii) the discovery of an important diversity actually used in activity assays, and the main applications
of species for which suitable in vitro culture conditions of these probes.
have not yet been defined and thus, which cannot be
recognised as ‘alive or dead’ by the culturable/non-
culturable dichotomy [5]. The development of these alter-
native methods was also concomitant with advances in
2. Analytical instruments
Instrumentation strategy is of great importance, since
* Correspondence and reprints. all instruments have different ranges of application,
E-mail address: (Philippe Lebaron). depending on their quantitative and qualitative sensitivi-

Microbes and Infection 1523

2000, 1523-1535
Review Joux and Lebaron

Table I. Characteristics of the different fluorescent dyes exposed in the text.

Characteristic Absorption (nm) Emission (nm) Molecular weight
Dehydrogenase activity (hydrolysis product)
CTC (CTC formazan, CTF) 450 580–660 332
Esterase activity (hydrolysis product)
FDA (fluorescein) 473 514 416
CFDA (carboxyfluoroscein) 492 517 460
CFDA-AM (carboxyfluoroscein) 492 517 532
BCECF-AM (BCECF) 482 520 615
Calcein-AM (calcein) 494 517 995
ChemChrome (*) 488 520 *
Membrane potential
Rh123 507 529 381
DiOC6(3) 484 501 573
DiBAC4(3) 493 516 517
Oxonol VI 599 634 316
Probe efflux
Ethidium bromide 518 605 394
Membrane integrity
SYTO-9 (membrane permeant stain) * (blue) * (green) *
SYTO-13 (membrane permeant stain) 488 509 ∼ 400
Propidium iodide 535 617 668
Sytox Green 502 523 600
PO-PRO-3 539 567 605
CSE * (blue) * (orange) *
* Unspecified by the manufacturer. Source of information: Chemunex (Ivry-sur-Seine, Paris, France), Molecular Probes (Eugene, Oreg., USA), Polysciences
Europe (Germany).

ties. The quantitative limits, accuracy, speed of analysis, [20]. FCM allows the rapid and simultaneous measure-
type of samples which can be analysed and light sources ment of various cell parameters with a precision of a few
of these instruments are important selection criteria. All per cent. Analyses are commonly performed at a flow rate
fluorochromes are characterised by their absorption and between 10 and 100 µL per min and count rates ranging
emission spectra (table I). The characteristics of selected from 100 to 1 000 cells/s. Most flow cytometers are
dyes must be compatible with those of the instrumentation equipped to sort cells with specific characteristics for
used for fluorescence detection. further analyses. However, FCM is not adapted to the
Epifluorescence microscopy (EFM) of fluorochrome- detection of rare events since it is difficult to detect less
stained cells has become the standard method by which than 100 bacteria per mL, which is still 10–100-fold better
bacteria are enumerated. Most microscopes are equipped than current microscopic procedures. For instance, at a
with a mercury or a mercury–xenon lamp. The xenon concentration of 100 bacteria per mL and at a mean flow
lamp provides a continuous spectrum, whereas the mer- rate of 50 µL/min, 20 min are needed to detect 100 events.
cury lamp emits spectral lines (365, 405, 435, 546 and Furthermore, when targeted cells represent less than one
578 nm) with a weak background continuum at other cell in one thousand or one cell in one million of non-
wavelengths. Xenon lamps should be used in preference targeted cells, flow cytometric analysis may range from
to mercury lamps only for dyes whose absorption spectra difficult to impossible [7].
does not match any of the strong mercury lines. The main Laser scanning cytometer (LSC) offers the advantage of
limitation in the use of microscopy is that it is time con- rapid detection of bacteria on filters. LSC is a microscope-
suming and subjective [6]. Moreover, the detection limit of based cytofluorometer which possesses the attributes of
microscopy is low and it becomes very tedious to detect both flow and image cytometry [8]. A 10-µm laser spot
less than 103 targeted cells per cm2 of filter – this limit allows the measurement of emitted fluorescence in addi-
corresponding to the detection of one cell per microscopic tion to forward scattered light every 5 or 10 µm over
field. Thus, bacteria often have to be much more concen- sequential fields. The rapid scanning speed (0.3 m/s) and
trated for microscopic examination than for plate counting the low-level resolution enable faster processing of the
after filtration. This can be quite problematic for samples data. However, the field-by-field scanning is an intrinsic
containing non-cellular particles and when targeted cells limitation, and the 10-µm step does not always allow
are diluted within non-targeted cells. Therefore, micro- resolution of individual cells. This is probably why this
scopic examination does not facilitate the simple detec- instrument has received little application in bacteriology.
tion of rare events. Recently, a solid-phase cytometer (SPC) equivalent to a
Flow cytometry (FCM) has became more popular and rapid laser scanning device (ChemScan; Chemunex, Ivry
offers the ability to perform analysis on individual cells at sur Seine, France) has been developed [9, 10]. SPC differs
numbers which begin to be more representative of nature from LSC in that the former is not a microscope-based
1524 Microbes and Infection
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Fluorescent physiological probes for bacteria Review

instrument. Cells are concentrated on a membrane filter as filterability of the products to be analysed will also influ-
for microscopy, and sample illumination and data collec- ence the choice of the instrument. The lack of consistency
tion are achieved without the use of an objective. This sometimes reported between microscopic and flow cytom-
allows high scanning speeds (1 m/s) and scanning of the etry counts of total bacteria in microcosm studies and
whole membrane (25-mm diameter) in one scan pattern mainly the higher variations of FCM counts ([13], unpub-
with a fully overlapping laser beam. The scanning is lished data from our group) may be due to day-to-day
performed in under 3 min and any event detected in the fluctuations in flow cytometry operations, which require
scanning procedure can be positioned for observation in strong quality controls. When controls are made, EFM,
the visual field of a conventional epifluorescence micro- FCM and SPC provide accurate counts of bacterial cells
scope in which the stage is driven by the instrument’s ([9] Lebaron, unpublished data).
computer [11]. This procedure is of great interest since it
allows the possibility of an immediate visual control and
confirmation of the result. The system is also able to
differentiate between labelled microorganisms and autof-
luorescent particles present in the sample based on the 3. Definition of terms
optical and electronic characteristics of the generated
signals. SPC is increasingly used in routine water analyses
for the detection of rare events (Escherichia coli, Defining cell death and cell viability is philosophically
Cryptosporidium spp., Giardia spp.), in some industrial and experimentally difficult. A viable cell should be con-
applications for real-time control of pharmaceutical-grade sidered a cell capable of fission to produce similarly viable
water and in different histological and cytological controls progeny under realisable culture conditions [1]. More
[11]. This technique allows the detection of a single cell on generally, the term ‘viable’ should be restricted to circum-
a 25-mm diameter membrane after filtration. stances where evidence that a cell is able to divide is a
Confocal scanning laser microscopy (CSLM) is a com- priori or a posteriori provided. From an operational point
puterised microscope that couples a laser light source to a of view, viability is demonstrated by culture, and cell
light microscope. This technique allows for the generation proliferation can be detected at both macroscopic (mac-
of three-dimensional digital images of microorganisms. rocolonies or turbidity) or microscopic (microcolonies)
This technique combined with the use of fluorescent scale. The main advantage of direct methods based on
probes has proven useful for the analyses of the three- fluorescent probes is the lack of incubation. Thus, there is
dimensional distribution of total and active cells in solid clear evidence that methods based on the use of physi-
structures (e.g., biofilms, aggregates, endosymbionts, ological probes cannot directly address cell division but
rhizophere and soil) [12]. However, CSLM is neither only the presence of some active functions or the integrity
adapted to routine applications for cell counting nor to the of cell structures. Therefore, cells in which any kind of
detection of rare events. metabolic activity can be detected should be called active
The argon ion laser is the most widely used light source cells. When metabolic activity is not detectable but integ-
for FCM, LSC, SPC and CSLM. Argon ion lasers provide rity of essential envelope structures is preserved, cells are
emission lines at several wavelengths ranging from 351.1 called inactive cells. These cells may be active but not
to 514.5 nm: the most widely used is the single line at enough to be detected as active cells or may be dying cells
488 nm. In the case of instruments equipped with a single with intact membranes. Lastly, cells with damaged mem-
laser source, the excitation wavelength is fixed and the branes are considered dead cells, whereas those with
strategy for staining is limited to the range of probes and intact membranes are considered intact cells. Dead cells
stains excitable at this wavelength. The use of probes with can be detected until cell lysis and are called ‘ghost’ cells
contrasting wavelengths is usually required for multipa- when cellular envelopes are maintained, whereas the
rameter measurement (e.g., combination of a nucleic acid nucleoid is lost [14]. Most of these cellular categories were
dye for the quantification of total bacteria with physiologi- already described by Neve-von Caron et al. [15].
cal and taxonomic probes) (see section 6). In this instance, The question of whether the active but non-culturable
contrasting wavelength means a combination of excita- (ANC) cells are able or not to return to a growing state
tion and emission wavelengths, which allows discrimina- when their environment becomes favourable is at the
tion of each probe in the presence of the others. Double centre of the current debate on the ANC state (for a
staining procedures with a single laser excitation source complete review, see [1]). The demonstration of the revers-
(often 488 nm) are limited since both dyes may have a ibility of this non-growing state is based on resuscitation
common excitation wavelength and different emission studies but most of these are open to criticism. In most
wavelengths with a minimal overlap. studies, it is generally uncertain whether the ANC cells are
The choice of an instrument is primarily driven by its capable of resuscitation or whether the increase in cell
technical characteristics but also by its cost. EFM is now numbers reported in a large number of publications on this
considered traditional equipment for fluorescence appli- issue is merely the result of growth of a few viable cells [1].
cations. FCM is more frequently used not only for research This is due to the existence of an important heterogeneity
but also in many industrial applications. CSLM is expen- of physiological states within a population and to our
sive and not adapted to routine bacterial analyses. Other inability to provide the proof that a cell in a given cellular
instruments are still under validation for different applica- state as determined by any given method is able to grow
tions. The quantitative sensitivity of the instrument and the and to divide.

Microbes and Infection 1525

2000, 1523-1535
Review Joux and Lebaron

Figure 1. Different cellular target sites for physiological and taxonomic fluorescent dyes.

4. Physiological target sites Rhodamine 123 (Rh123) is a lipophilic, cationic dye

commonly used to detect MP [16]. However, staining with
This section presents the different probes used to assess Rh123 often requires a pretreatment step of the cells,
different physiological functions and cellular structures. generally performed by adding EDTA, to permeabilise the
The interpretation of these dyes in terms of cellular activity outer membrane of Gram-negative bacteria [17]. When
or viability is discussed in more details in sections 5 and 6. antibiotic or disinfectant treatments are studied, the per-
Figure 1 summarises the different physiological target sites meabilisation step can introduce some bias by enhancing
of these probes. the toxic effects of these compounds (i.e. they penetrate
more easily inside the cells). Furthermore, the pretreat-
4.1. Membrane potential ment conditions may vary depending on the environment
The electrochemical potential occurring through the (e.g., salinity) and between species. This is why MP dyes
plasma membrane of metabolising bacteria is generated have received little applications at the community level.
by respiration or by ATP hydrolysis. It results from the An additional problem is that Rh123 staining requires
selective permeability of biological membranes to a vari- several cell-washing steps, which are time consuming and
ety of cations and anions leading to a difference of electric may result in cell losses. The cationic carbocyanine dyes
potential across the membrane. Inside, the cell is nega- (e.g., 3,3'-dihexyloxacarbocyanine; DiOC6(3)) have also
tively charged compared with outside the cell, and mem- been used to estimate MP of bacteria. Advantages of these
brane potential (MP) plays a central role in different cell- dyes are the absence of permeabilization and washing
life processes (ATP synthesis, active transport, mobility, steps. However, nonspecific binding of carbocyanine dyes
regulation of intracellular pH, etc.). Voltage-sensitive dyes to hydrophobic regions of the cell and quenching of the
have been developed to measure MP in bacteria. Depend- fluorescence of intracellular dye have been reported [17].
ing on the charge of the dye, they are accumulated in Thus, careful calibration of the staining procedure is
polarised (cationic dyes) or depolarised (anionic dyes) required to avoid false positive signals.
cells. Reliability of staining is confirmed by observing if MP can also be determined by the anionic lipophilic
dye uptake is sensitive to uncouplers (e.g., carbonyl cya- oxonols. Accumulation inside bacterial cells is favoured
nide m-chlorophenyl hydrazone; CCCP) or ionophores by a reduction in the magnitude of the MP, allowing dye
(e.g., nigericin, valinomycin). In appropriate conditions, molecules to concentrate within the cell, and bind to
the amount of dye taken up can be directly related to the lipid-rich components. Bis-(1,3-dibutylbarbituric acid) tri-
level of energy metabolism in the cell. methine oxonol [DiBAC4(3)] (BOX) has been reported to
1526 Microbes and Infection
2000, 1523-1535
Fluorescent physiological probes for bacteria Review

be useful to detect depolarised cells of numerous Gram- only a single strain did not reduce CTC over a large
positive and Gram-negative bacterial species. In some number of marine bacterial strains examined (n = 27). A
protocols, oxonol dyes combine a pretreatment with EDTA similar trend was reported from the correlation found
or EGTA 1–5 mM to increase membrane permeability [15, between total and CTC counts in deep sediment samples,
18]. Without permeabilisation, uptake of oxonols would which suggests that CTC also stains anaerobic bacteria
be more related to membrane integrity rather than mem- [34]. In contrast, the lack of universality of CTC was
brane depolarisation [15]. Staining only dead cells with pointed out by Yamaguchi and Nasu [35] for environmen-
oxonols may be difficult when the treatments studied lead tal applications. Toxic effects of CTC on bacterial metabo-
to the disruption of the cells (e.g., surfactants, antibiotics). lism have been reported [36], and this toxicity may explain
In these cases, it is important to determine total cell counts why CTC counts are sometimes lower than active counts
and then to deduce the remaining fraction of living cells. determined by other metabolic assays (e.g., microautora-
For instance, Comas and Vives-Rego [19] suggested the diographic counts, or esterase activity) [36, 37].
simultaneous detection of depolarised and total cell popu-
lation by combining BOX with the nucleic acid dye SYTO- 4.2.2. Esterase activity
17. Detection of esterase activity is measured using lipo-
Applications of MP dyes to natural samples are scarce philic, uncharged and non-fluorescent fluorogenic sub-
and inconclusive [20], probably because of both the effects strates. Once within active cells, the substrate is cleaved
of the permeabilisation step, which vary depending on by non-specific esterases releasing a polar fluorescent
species and the interaction between charged dyes and product (fluorescein or fluoroscein derivatives) retained
chemical compounds present in the sample. inside cells having an intact membrane. Contrary to
eukaryotic cells, Gram-negative bacteria are generally
4.2. Enzyme activity impermeable to the lipophilic fluorogenic probes, and a
4.2.1. Dehydrogenase activity permeabilisation step of the outer membrane is required.
Cell-specific assays to detect the respiratory activity of Esterases are present in all living organisms, and these
bacteria have been developed based on the use of different enzymes can be used to provide information on the meta-
tetrazolium salts. Tetrazolium dyes are reduced from a bolic state of bacterial cells. Although enzyme synthesis
colourless complex to a brightly coloured, intracellular, requires energy, the enzyme–substrate reaction does not,
formazan precipitate by components of the electron trans- and this assay alone should be considered energy inde-
port system and/or a variety of dehydrogenase enzymes pendent. However, dead or dying cells with damaged
present in active bacterial cells. Since electron transport is membranes rapidly leak the dye, even if they retain some
directly related to cellular energy metabolism in respiring residual esterase activity. Consequently, fluorogenic sub-
cells, the ability of cells to reduce tetrazolium compounds strates for esterases often serve as activity and cell integrity
can be considered an indicator of bacterial activity. A probes that measure both enzymatic activity, which is
variant approach is the use of the redox dye 5-cyano-2,3- required to activate their fluorescence, and cell-membrane
ditolyl tetrazolium chloride (CTC). CTC is reduced by integrity, which is required for intracellular retention of
bacteria to a water-insoluble, red fluorescent formazan their fluorescent products.
product. It allows the quantification of the metabolic Fluorescein diacetate (FDA) is known to give weak
activity of bacteria under both aerobic and different anaero- fluorescence signals, since fluorescein is poorly retained
bic conditions [21, 22]. However, different factors may inside the cells [17]. In contrast, hydrophobic FDA deriva-
affect the CTC reduction during incubation [23–25]. tives are cleaved into hydrophilic products that are retained
Although CTC counts are commonly determined by epif- more efficiently inside cells with an intact membrane.
luorescence microscopy, flow cytometry can be used when Among these, acetoxymethyl ester (calcein-AM) was
possible, to overcome the rapid fading of the fluorescence shown ineffective to label different species, with the excep-
signal [26], yielding equal or higher counts [25, 27]. In tion of Staphylococcus aureus [38, 39]. Comparison made
contrast to other probes, samples can be fixed after stain- by Jepras et al. [40] of different fluorogenic esters shows
ing and stored at –20 °C until analysis. that carboxyfluorescein diacetate (CFDA) is superior to
The addition of nutrients during incubation with CTC is FDA (fluorophore retention problems) and carboxyfluo-
sometimes used to improve the fluorescent signal and/or rescein diacetate acetoxy methyl ester (CFDA-AM) (solu-
the number of respiring cells [28]. In this case, controls are bility problems). The ability of a range of fluorogenic esters
needed to ensure that growth does not occur during incu- to stain several bacterial species was investigated by Dia-
bation or that growth can be controlled by means of per and Edwards [38]. They found that ChemChrome B
antibiotics (e.g., cephalexin) without interfering with meta- (Chemunex) (a commercial preparation of unknown for-
bolic activity [18, 29]. Total cell counts can be determined mulation) is superior to FDA, CFDA, and 2',7'-bis-
by counterstaining the cells with a fluorescent nucleic (2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxy
acid stain (commonly DAPI). methyl ester (BCECF-AM), as it stains the widest diversity
CTC is commonly used in microbial ecology, for both of Gram-negative and Gram-positive species.
aquatic and terrestrial systems. Applications include drink- The main limitations encountered with the fluorogenic
ing water [30], biofilms [31], lake and seawater [32], esterases are related to a poor dye uptake, an active dye
sediments [33, 34], and subsurface soils [33]. However, extrusion (see section 4.3) [41, 42], and problems of low
the universality of CTC to detect respiring cells in natural labelling efficiency for some bacterial species in pure
samples remains controversial. Sherr et al. [32] found that cultures such as Pseudomonas spp. [41, 43]. Most of these

Microbes and Infection 1527

2000, 1523-1535
Review Joux and Lebaron

limitations were recently overcome by the ChemChrome intact and dead cells. Loss of membrane integrity as mea-
V6 staining kit (Chemunex) which combines a counter- sured by uptake of membrane-impermeant dyes is gener-
stain to reduce non-specific fluorescence and a specific ally considered irreversible. However restoration of the
buffer to reduce dye extrusion [44]. membrane integrity has already been reported by Votya-
Porter et al. [20] found that BCECF-AM and Calcein-AM kova et al. [47] (revealed by the use of the membrane-
were inappropriate dyes to enumerate viable bacteria in impermeant fluorescent DNA stain PO-PRO-3) during
freshwater environments. Inversely, these authors observed resuscitation of starved Micrococcus luteus.
a clear signal of fluorescence for active cells stained with A wide diversity of impermeant nucleic acid stains can
CFDA and ChemChrome B, and the latter was found more be used; Molecular Probes (Eugene, Oreg., USA) offers a
efficient with river water samples. Universality and high large variety of dyes with molecular weights ranging from
quality of staining was reported for CFDA with both pol- 402–1 355, among which propidium iodide (PI) is the
luted and non-polluted river waters [35]. Reynolds and most commonly used. In order to simultaneously detect
Fricker [43] used ChemChrome B to determine the frac- dead and intact cells, Molecular Probes has developed the
tion of living bacteria in a large number of potable waters Live/Dead BacLight kit containing two nucleic acid stains
and they found that this fluorogenic ester was always (SYTO-9 and PI) which differ in their spectral characteris-
superior to plate counts, and often significantly higher tics and their ability to penetrate intact bacterial mem-
than CTC counts. The application of ChemChrome V6 to branes. SYTO-9 penetrates inside cells with both intact or
different water samples was recently reported by Catala et damaged membranes, staining the cells green, whereas PI
al. [44]. only penetrates cells with damaged membranes, staining
the cells red. When the dyes are used in combination, cells
4.3. Pump activity with intact membrane show a green fluorescence while
Several observations have confirmed that different dyes cells with damaged membranes show a red fluorescence
can be loaded into bacteria and are subsequently actively (SYTO-9 emission contributes to the excitation of PI by
removed by energised cells. This is the case for Rh123 energy transfer). According to the manufacturer, SYTO-9
[45], BCECF (from BCECF-AM) [41], carboxyfluoroscein should penetrate intact membranes of a large number of
(from CFDA) [42], and ethidium bromide [15]. Active Gram-negative and Gram-positive bacteria. However,
extrusion of these dyes can lead to biased results. To limit Langsrud and Sundheim [48] found that 30% of Pseudomo-
pump interference, Nebe-von Caron et al. [15] suggested nas aeruginosa strains tested (n = 18) did not accumulate
the addition of sodium azide in staining solutions. SYTO-9. To overcome this problem, other membrane-
When active dye extrusion is not inhibited, probe efflux permeable stains have been tested in combination with PI
can be used as an additional measure of cell activity [15, (e.g., SYTO-13 [19]).
42]. Actively pumping cells are determined using suitable The Live/Dead BacLight staining kit was recently used
growth conditions and are sometimes detected using by different authors to estimate the number of live cells in
ethidium bromide at the single-cell level [15]. However, marine planktonic environments using epifluorescence
pump activity assays have only been applied to a few microscopy [49, 50]. The highly variable percentage of
species in culture and are not universal enough to be viable cells reported from these studies may result from
applied to environmental samples. cells exhibiting a continuum of in-between colours. In
order to obtain a better discrimination between green and
4.4. Membrane integrity red fluorescent cells, Gasol et al. [50] recommended the
The loss of membrane integrity represents significant filter should be rinsed with isopropanol after staining.
damage for cells due to multiple functions linked to the Recently, Boulos et al. [30] reported the application of the
plasma membrane (permeability barrier, transport, respi- Live/Dead BacLight kit to drinking water and similar trends
ratory activity, etc.). The maintenance of membrane integ- were found between Live/Dead BacLight and CTC in the
rity is commonly measured in eukaryotic cells as an indi- absence of stress. In contrast, stress results in a more rapid
cator of cell damage or cell death [46]. Assessment of decrease of viable counts as determined by CTC com-
membrane integrity of bacteria is complicated due to their pared to Live/Dead BacLight kit. Defives et al. [51] used
complex membrane structure. The envelope of Gram- the Live/Dead BacLight kit to determine intact and dead
negative bacteria consists of three interacting layers: the cell concentrations in natural mineral water during storage
outer membrane, the rigid petidoglycan layer and the and mechanical bottling. Results suggest that the Live/
inner membrane (plasma membrane). For Gram-positive Dead BacLight kit is a reliable stain for both autochtho-
cells the outer membrane is absent. Additionally, some nous and allochthonous bacterial strains introduced by
Gram-negative and Gram-positive bacteria have a highly the bottling system.
hydrated polysaccharide layer outside the cell, called the Recently, Molecular Probes has introduced a new
capsule. Membrane integrity analysis is based on the impermeant nucleic acid dye (Sytox Green). According to
capacity of the cells to exclude fluorescent dye com- the manufacturer, Sytox Green can be used to detect both
pounds, which when used at low concentrations do not Gram-negative and Gram-positive bacteria with damaged
normally cross intact membranes. Most of the membrane membranes. However, Lebaron et al. [52] reported limita-
integrity assays use nucleic acid stains, due to the high tions in the use of Sytox Green alone for death assessment
concentrations of nucleic acids within the cells and the in the case of treatment leading to DNA degradation (e.g.,
large fluorescence enhancement exhibited by nucleic acid long-term starvation). In this case, cells with damaged
stains upon binding, leading to a clear separation between membranes and damaged DNA display a low fluores-
1528 Microbes and Infection
2000, 1523-1535
Fluorescent physiological probes for bacteria Review

cence signal due to the degradation of target sites and may discriminating live and dead cells and more recently, to
be considered cells having intact membrane and intact investigate if at least one assay could be used to detect and
DNA since these cells are, as living cells, not stained with to enumerate growing cells responsible for the production
the dye. This limitation may exist for all membrane- of bacterial biomass. Both types of studies have given rise
impermeant DNA stains. to important semantic confusion around the definition of
activity and viability as already discussed in section 3.
4.5. Nucleic acids
The detection of damaged DNA, such as breaks in the 5.1. Heterogeneity at the population level
DNA strands, is often used to characterise apoptosis in Probes targeting different cellular functions and the
eukaryotic cells [46]. Application of this method to bacte- technique of flow cytometry have been used to analyse the
ria may be biased by the maintenance of intact DNA for physiological heterogeneity of bacterial cells within tar-
extended periods after cell death has occurred [28, 53]. geted populations in different stress conditions. Many of
For instance, changes in the topology of the DNA mol- these dyes share common excitation/emission properties,
ecule such as those occurring in starved cells may be prohibiting simultaneous labelling of samples and there-
misinterpreted as DNA damage [28, 53]. Changes in the fore the collection of multiple parameters on a given cell.
fluorescence of DAPI-stained bacteria have been used to However, the speed of flow cytometry allows the staining
estimate the efficiency of drinking water disinfection by of identical sub-samples by several dyes and then the
sodium hypochlorite [54]. However, death was not a comparison of results. Figure 2 illustrates the succession of
determining factor in the loss of DAPI fluorescence for all cell changes that occurred at different rates in a Salmo-
disinfection treatments, because bacteria killed at high nella typhimurium population during starvation in artifi-
doses of monochloramine were still fluorescent after DAPI cial seawater. The proportion of culturable cells decreased
staining [54]. Recently, evidence has been presented that rapidly (state 1) followed by a decrease in the proportion
bacterial abundance in seawater enumerated by conven- of respiring cells without or with nutrient addition (states 2
tional DAPI staining and EFM include cells that do not and 3, respectively). After the loss of these functions, cell
contain a genome (‘ghost’ cells) due to unspecific binding integrity is maintained for a few days (state 4) and, after the
of DAPI [14]. Using a destaining procedure with isopro- loss of membrane integrity, cells with intact DNA (state 5)
panol to eliminate non-specific binding of DAPI, Zweifel are rapidly subjected to an apparent DNA degradation
and Hasgtröm [14] found that a large fraction (68–92%) of (state 6) following lysis or formation of non-nucleoid-
total counts can be scored as ghost cells. However, doubts containing bacteria (state 7). According to the definition of
persist about the significance of ghost cells [55]. physiological states given in section 3, cells in states 2 and
The cellular rRNA content of bacterial cells can be 3 may be considered ANC, those in state 4 intact cells, in
quantified by fluorescence in situ rRNA hybridization states 5 and 6 dead cells and in state 7 ghost cells.
(FISH) of oligonucleotides carrying a fluorochrome. These results and those from many other studies suggest
Because rRNA content in many bacterial species varies that there is a strong heterogeneity of physiological states
depending on their growth rate and decrease rapidly in within bacterial populations subjected to environmental
inactive cells, FISH has been proposed to estimate the stress, e.g., [28, 29, 47, 53, 60, 61]. Because each cellular
physiological state of cells [56]. Moreover, in the case of function is lost at a different rate, the activity of non-
complex communities, this assay could be developed to culturable cells is represented by a decreasing number of
detect activity of specific bacteria using appropriate oligo- active physiological functions during the time of stress and
nucleotide probes [12]. However, in dynamic environ- the fraction of active cells generally decreases up to the
ments and when cells are submitted to stress treatment loss of membrane integrity. Today, the significance of each
(e.g., cold stress, acetic acid, or ethanol), the rRNA content cellular state in terms of viability remains unclear, mainly
is a poor indicator of activity due to the high stability of because of our inability to analyse the recovery of specific
rRNA [57]. The recent development of FISH techniques cells under favourable growth conditions. In a recent study
using mRNA or pre-rRNAs (precursors in rRNA synthesis) combining flow cytometry and cell sorting, it was shown
as target molecules and those which determine the expres- that S. typhimurium cells with depolarised membranes
sion of specific functional genes may provide more reli- could be recovered after cell sorting [15]. It suggests that
able methods to assess the activity of individual cells depolarisation is a sensitive measure of cell damage but a
within complex bacterial communities [58, 59]. poor indicator of cell death. The loss of membrane integ-
rity as determined by the penetration of impermeant
nucleic acid dyes is more often used as an indicator of
5. Heterogeneity of physiological states death, but inversely, the integrity of the membrane cannot
at population and community levels be considered alone as an indicator of viability but is
commonly used as an indicator of cell integrity [62, 63].
Most investigations at the population level were per- Interpretation of results may also vary depending on the
formed in microcosm, to further understand the cellular conditions affecting the physiological state of the cells. For
and molecular responses of targeted species to different instance, cell death occurs rapidly after exposure to UV or
stress conditions. An important part of these studies aimed gamma radiation due to DNA damage, but the radiation
to identify a definite live versus dead state. Studies at the does not affect dehydrogenase activity, membrane poten-
community level were performed to characterise the physi- tial, membrane integrity and β-D-galactosidase activity
ological structure of natural communities with the aim of [64]. Therefore, this implies a delay following death before

Microbes and Infection 1529

2000, 1523-1535
Review Joux and Lebaron

gens and should be determined for each species and for a

wide variety of stress conditions. If ANC forms are present
and are able to persist in the natural environment, then it
will be important to further evaluate the public health
significance of this state.
5.2. Heterogeneity at the community level
In natural waters, culture methods give microorganism
counts that represent less than 1% of the total bacterial
cells and overlook an unknown but probably large propor-
tion of living cells (that do not grow on the conventional
substrates). On the other hand, total counts determined by
nucleic acid dyes (such as DAPI or acridine orange) include
Figure 2. Evolution of the different cell states in a S. typhimurium
dead or inactive cells. The detection and enumeration of
population during starvation survival in artificial seawater. Each
active cells (living bacteria with any detectable metabolic
cell state (or lysed/non-nucleoid-containing cells) is expressed as a
activity), growing cells (bacteria which participate in the
percentage of the original total cell count. The methods used are
production of biomass) and dead cells (representing
developed in the text. Results published in Joux et al. [28].
organic particles) in natural bacterial assemblages would
State 1: culturable cells as determined on nutrient agar
be of great help and most useful in aquatic microbial
(bioMérieux, France) plates.
ecology. It would help to better understand the mecha-
State 2: non-culturable cells showing real respiration by the CTC
nisms behind the regulation of bacterial activity. Different
(Polysciences Europe, Germany) method.
probes and methods have been used to estimate the pro-
State 3: non-culturable cells showing no real respiration but
portion of both active and dead cells and only a few recent
potential respiration as determined by the CTC method after
investigations have tried to detect and to enumerate grow-
nutrient (1/10 R2A broth) supply.
ing cells. However, consensus on what each of the probes
State 4: non-culturable cells showing no respiration but mem-
tested in these studies measures has not yet been reached.
brane integrity maintenance as determined by the Live/Dead
BacLight kit (Molecular Probes). The detection of active cells within complex commu-
State 5: non-culturable cells showing loss of membrane integrity nities requires the use of universal probes which target the
with no DNA change as determined by Hoechst 33342 (Sigma) widest diversity of species. Therefore, only a few probes
staining. (formazan salt reduction, esterase activity, and rRNA
State 6: non-culturable cells showing cellular integrity with probes) have been applied to natural communities to
DNA change determined by Hoechst staining. describe the physiological heterogeneity of bacterial cells.
State 7: lysed or non-nucleoid-containing cells. Despite the different drawbacks of CTC reported in section
4.2.1, studies suggest that CTC provides an ecologically
meaningful measure of bacterial activity [26, 67]. Sherr et
enzymes are degraded and membranes are damaged. In al. [32] suggested that CTC-positive cells represent those
this case, there is no protocol available to rapidly assess bacteria characterised by a high level of metabolic activ-
cell viability and the use of culture should be also consid- ity, and that cells which show no apparent reduction of
ered with caution, since DNA repair mechanisms may CTC have either low or no metabolic activity. This assump-
restore viability [65]. Some recent and promising investi- tion is far from clear, and the interpretation of CTC counts
gations have been made to improve direct methods by remains controversial. The recent and increasing use of
combining traditional probes targeting enzyme activities CTC counts in microbial ecology and at the same time the
or membrane integrity with a probe targeting DNA dam- well-known limitations of the method illustrate the impor-
age such as fragmentation [66]. tant need, for microbial ecologists, to identify which cells
The heterogeneity of cellular physiological states are responsible for bacterial community activity. As an
reported in most microcosm-stressed populations is always alternative to CTC, growing cells can also be determined
observed by using a large inoculum of cells (> 105 cells per by flow cytometry as the fraction of cells with a high DNA
mL) because of the low quantitative sensitivity of both flow content [68]. The fraction of cells with a high DNA content
cytometry and microscopy. As a consequence, high cell is easily discriminated by flow cytometric analysis of SYBR
densities may introduce a bias in the temporal evolution of Green-stained cells, and in a recent study, Gasol et al. [50]
the physiological states of individual cells due to the lysis indicated that high-DNA bacteria are the dynamic mem-
of some cells and the use of released organic compounds bers of the bacterial assemblage. General acceptance of
by the most metabolically active cells. Consequently, some this approach to determine the dynamic members of natu-
physiological states may exist at smaller time scales than ral communities will require further investigation to under-
those found in most laboratory studies. The recent devel- stand how it works and what its results mean.
opment of more sensitive instruments such as solid-phase Studies which perform a comparison of methods are
cytometry may help to provide a more accurate descrip- scarce and more intercalibration between methods is cur-
tion of the temporal variations in physiological states, and rently needed. Karner and Fuhrman [37] have applied
therefore to measure the persistence time of active but different methods to assess bacterial activity in seven
non-culturable cells within stressed populations. This infor- different marine water samples and they found an average
mation remains of great importance in the case of patho- proportion of 56% of cells containing detectable rRNA,
1530 Microbes and Infection
2000, 1523-1535
Fluorescent physiological probes for bacteria Review

49% of cells able to take up added radiolabelled com- (BOX) or cell polarisation (Rh123) [18]. The CV6/CSE
pounds, 29% of nucleoid-containing cells and only 0.7% labelling kit from Chemunex offers the possibility of simul-
of respiring cells. Parthuisot et al. [69] found that active taneously detecting cells with an esterase activity (CV6)
bacteria determined by esterase activity measurements in and membrane integrity (CSE). Application of this staining
different water samples are higher than those determined procedure to S. typhimurium cells starved in artificial sea-
by CTC and CFU and that CTC counts are sometimes water allowed a more accurate assessment of active cell
lower than CFU. From these results and other studies [55, counts by counterstaining cells with permeabilised mem-
70], there is clear evidence that there is an important branes [44]. A nice illustration of multiple-staining assays
heterogeneity of physiological states within natural com- was provided by Nebe-von Caron et al. [15] using a
munities. However, each method yields different estimates combination of three fluorescent dyes to measure mem-
of the proportion of bacterial cells that are active. The brane potential (BOX), pump activity (ethidium bromide)
physiological and ecological significance of each cell and membrane integrity (PI).
state remains unclear and the answer, if it exists, to this Most of these studies remained methodological studies
question will require further investigations to relate a given and suffer from a lack of validation. Therefore, the signifi-
cellular physiological state to additional information such cance of these methods and their possible use for viability
as its growth potential, enzyme activity or taxonomic assessment is unknown. ChemChrome V6 from Chemu-
identity. nex is one of the most commonly used methods which
combines a fluorogenic ester for esterase activity assess-
ment with a counterstain which penetrates inside cells
6. Combination of fluorescent probes with permeabilised membranes [44, 69]. This method is
widespread in different industrial applications (pharma-
The combination of fluorescent probes in single assays ceutical, food and cosmetic industries) and when coupled
provides confident tools and some of these are actually with flow cytometry provide real-time estimation of active
under validation for industrial applications such as water cell counts. Although active counts are sometimes signifi-
quality assessment in the pharmaceutical industry [43]. cantly higher than viable counts determined by CFU, they
However, although physiological probes are very useful are considered very useful to detect production problems
for different industrial and environmental applications, and perhaps yield a more accurate estimation of viable
they cannot address the detection of specific active cells counts than do culture methods.
such as pathogenic microorganisms. Most of these appli-
cations are based on the combination of physiological and 6.2. Combination of physiological
and taxonomic fluorescent probes
taxonomic probes and require instruments which can
detect rare events. In the case of complex communities, physiological
fluorescent probes provide useful information on the per-
6.1. Combination of different centage of active, inactive or dead cells of the overall
physiological fluorescent probes community. However, detection of the activity of specific
A more accurate evaluation of cell activity should be bacteria is of primary importance in different microbio-
obtained by combining physiological probes targeting logical areas. For instance, it could be used to detect active
different cellular functions. Assays in which both pathogens in food and waters, or to detect active specific
membrane-based and metabolism-based probes are used populations implicated in different transformations (biore-
simultaneously provide information on whether the dye actors, environment). Fluorescent antibodies and fluores-
assays accurately reflect cell activity. Probes should be cent oligonucleotide rRNA are taxonomic probes that can
selected with excitation and emission wavelengths which be used at the single-cell level to detect bacteria of interest
allow discrimination of each probe in the presence of the including pathogens. One problem encountered with
other(s). Therefore, dyes with a narrow emission spectrum hybridisation of fluorescent oligonucleotide rRNA con-
may be better suited to multicolour applications. The cerns the permeabilisation of cells and amplification of
selection will also be dependent on the instrumentation fluorescence signals. The combination of taxonomic and
used for fluorescence analysis (see section 2). One should physiological fluorescent probes is a key challenge for
also keep in mind that the selection of fluorophores in microbiologists since there is an increasing need to pro-
multiple-staining procedures may take into account vide numbers of specific cells, depending on their physi-
molecular interactions which can result in quenching, a ological state (at least viable or active state).
decrease in the fluorescence signal. This decrease is due to Whiteley et al. [72] described the combination of fluo-
changes in the excited states of the fluorophores, resulting rescent in situ hybridisation (rRNA probes) with the cyto-
in energy dissipation by non-radiative transitions. These chrome oxidase assay. Similar combinations with a tetra-
physico-chemical changes may be caused by the interac- zolium salt necessitate a fastidious sequential procedure,
tion between fluorophores. inconceivable in routine, in which tetrazolium reduction
In multiple-staining assays, PI (dead cells, red fluores- is evaluated prior to probe hybridisation [72]. Different
cence) is often combined with CFDA (cells with esterase authors have demonstrated the feasibility of combining
activity, green fluorescence) [35, 42], Rh123 (polarised immunostaining with CTC [73], Rh123 [74] and Chem-
cells, green fluorescence) [18, 71] or BOX (depolarised Chrome staining [75]. The fluorochrome conjugated to the
cells, green fluorescence) [18, 71]. The CTC dye can also monoclonal antibody must be carefully chosen in order to
be combined with the detection of cell depolarisation avoid fluorescence overlap with the physiological dye.

Microbes and Infection 1531

2000, 1523-1535
Review Joux and Lebaron

Recently, Pyle et al. [76] combined an immunomagnetic vidual bacterial cell. However, despite the continuous
separation (IMS) with the analysis of respiratory activity development of new fluorescent dyes, no universal stain
(CTC staining) to assess the number of active E. coli or staining procedure that is suitable for all applications
O157:H7 present in meat and water. In this procedure, has been found, and will probably never be found. The
E. coli O157:H7 cells are isolated from the sample by efficiency of dyes and protocols needs to be evaluated for
using a magnetic particle concentrator after addition of each application and studies at the population level remain
immunomagnetic beads coated with anti-O157 antibody. essential to understand how different species react in a
Then, cells are incubated with CTC (3 h), stained with given assay and how the response varies depending on the
FITC-labelled antibody (20 min) and filtered for micro- stress conditions applied to these species.
scopic enumeration or solid-phase laser cytometry. This The development of multiparameter analysis which
procedure results in high rates of recovery but cell injury is combines at least two physiological probes is very prom-
observed [76]. However, the combination of taxonomic ising but requires further investigations and validation
probes with the CTC dye has little interest since CTC work to ensure that these methods are reproducible and
counts are well known to underestimate the number of robust enough for routine applications. Validation should
viable targeted cells (see sections 4.2.1 and 5.2). Further- be made in regard to specific applications since no method,
more, most of these assays were performed by using an as is the case for culturability, can be robust enough to be
artificial mixture of strains or artificial contamination. universal and suitable for all applications. For instance,
Such a combination should be treated cautiously for envi- activity assessment after UV or gamma radiation requires
ronmental monitoring, due to additional manipulative methods which are not based on membrane integrity or
steps that could reduce cell recovery. enzyme activities. Inversely, methods based on a combi-
One additional limitation to the actual use of these nation of probes which target membrane permeability and
methods is that in most cases, cells of interest are highly enzyme activity can be very useful and are already rou-
diluted within autochthonous populations, and field appli- tinely used to quantify active cells in water, food products,
cations have long been limited because instruments such drugs and cosmetics, to control the efficiency of freeze-
as microscopy and flow cytometry do not allow the detec- drying procedures in bacterial collections and probably
tion of rare events [77]. Detection of bacterial cells at a other applications. Differences between active and cultur-
frequency of 10–5 to 10–7 is a challenge in various fields of able cell counts do not constitute a limitation to the use of
microbiology (industry, medicine, environment, etc.). The physiological probes if active cell counts are obtained
recent development of solid-phase cytometry has covered rapidly and allow the detection of any interpretable
this gap and this technology offers promising perspectives. changes in terms of quality control. In these cases, limits
The ChemScan technology is now increasingly used in the that are based on the standard cultivation techniques,
industry for the rapid and accurate detection of E. coli, when they exist, should be reconsidered in favour of
Cryptosporidium spp. and Giardia spp. in waters with a alternative methods.
high sensitivity of detection as well as for the quantifica- The traditional debate and conflict around viability and
tion of active bacteria in process waters (pharmaceutical activity as assessed by physiological probes is somehow
industries). For instance, it is possible to detect a single cell paradoxical. On the one hand, traditional culture methods
of E. coli or any other species for which specific antibodies are based on the dividing capacity of a cell which requires
are available in 1 L of tap water and in the presence of a few hours and sometimes a few days to be detected and
106–108 non-targeted cells (Lebaron, unpublished data). on the other hand, physiological probes which have been
This detection limit is similar to that of culture techniques developed for real-time monitoring of cell viability do not
which combine a filtration step. Furthermore, SPC offers allow the control of cell division. Therefore, viability
the advantage that it does not require the use of selective assessment cannot be achieved directly by physiological
growth conditions and thus, allows the detection of injured probes. When assays based on the use of physiological
cells which are viable cells not detected on selective probe(s) are available, they should be evaluated and com-
media. However, although the instrument can detect three pared to standard methods. However, these probes have
fluorescences, applications are still limited because it is never been applied to the detection of rare events such as
equipped with a single source of excitation. Therefore, pathogens because the instrumentation used for fluores-
today the use of at least two probes for physiological cence analysis was until recently inappropriate for the
assessment and an additional probe (taxonomic) for the detection of rare events. This last point as well as the price
detection of specific cells remains difficult if not impos- of these instruments probably explains why alternative
sible. Many applications will require the use of a second methods have received little field application and valida-
source of light. tion and why it is difficult if not impossible today to
provide an accurate evaluation of the potential use of
these probes as alternative methods to the standard culti-
7. Conclusions and future prospects vation techniques.
As stated above, the adoption of these new tools will
There is clear evidence that the application of fluores- also require new standards in bacterial quality assessment
cent dyes will provide microbiologists with a set of valu- since, in most applications, counts are generally higher
able tools to complement existing molecular and conven- than CFU counts which were used to define the actual
tional methods by providing information concerning standards. The development of new fluorescent dyes more
specific physiological activities at the level of the indi- closely related to cellular energy metabolism and assays
1532 Microbes and Infection
2000, 1523-1535
Fluorescent physiological probes for bacteria Review

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