2000 Use of Fluorescent Probes to Assess Physiological Functions of Bacteria at Single-cell Level

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Review 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 SAS bacteria / fluorescent probes / physiology / cytometry 1. Introduction One of the most basic questions that microbiologists address is whether a bacterial cell is alive or dead. The answer to this question is far from simple and the question remains unanswered after 20 years of intense research and permanent controversy [1]. Life is generally characterised by: (i) the presence of structure; (ii) changeable genetic information; (iii) metabolism or functional activity, and (iv) the ability to reproduce and grow [2]. However, living bacteria are generally only characterised by their ability to reproduce, probably because the capacity of a cell to multiply as determined by culture has long been the single method available to microbiologists for bacterial viability assessment. Therefore, culturability and viability are often considered synonymous terms. During the last two decades, an increasing need for alternative techniques has been stimulated by: (i) the need for real-time monitoring methods for viability assessment [3]; (ii) the existence of injured and ‘viable but non- culturable’ (VNC) cells sometimes encountered when cells are stressed (e.g. heat, oxidative, osmotic stress, or starva- tion) [1, 4], and (iii) the discovery of an important diversity of species for which suitable in vitro culture conditions 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 fluorescent dye technology offering probes for a variety of cellular functions. The VNC state was initially suggested by Roszak et al. [4] for readily culturable bacteria which under particular circumstances become non-culturable but retain ‘viability’. Nevertheless, the viability of VNC cells was never defined by culture but only by the detec- tion of some forms of activity in cells, which are not directly related to the capacity of these cells to reproduce. For these reasons, the term ‘active but non-culturable’ (ANC) might be more precise than viable but non- culturable [1]. Fluorescence-based methods have remained very use- ful for a wide diversity of applications ranging from indus- trial to environmental microbiology. These tools are used for viability/activity assessment in food, pharmaceutical and cosmetic industries, and in the natural environment, including fresh and marine waters. The increased use of fluorescent probes is also due to improvements in the quantitative and qualitative sensitivity of instruments. The aim of this review is to describe the different strategies used for the detection of cell fluorescence, the different cellular target sites and fluorescent probes which are actually used in activity assays, and the main applications of these probes. 2. Analytical instruments Instrumentation strategy is of great importance, since all instruments have different ranges of application, depending on their quantitative and qualitative sensitivi- * Correspondence and reprints. E-mail address: [email protected] (Philippe Lebaron). Microbes and Infection, 2, 2000, 1523-1535 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457900013071/REV Microbes and Infection 2000, 1523-1535 1523

Transcript of 2000 Use of Fluorescent Probes to Assess Physiological Functions of Bacteria at Single-cell Level

Page 1: 2000 Use of Fluorescent Probes to Assess Physiological Functions of Bacteria at Single-cell Level

Review

Use of fluorescent probes to assessphysiological functions of bacteria

at single-cell levelFabien 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 thephysiological state of microorganisms. The recent development of techniques such as solid-phasecytometry, the increasing sensitivity of fluorescence tools and multiparametric approachescombining taxonomic and physiological probes have improved the effectiveness of direct methods inenvironmental and industrial microbiology. © 2000 Éditions scientifiques et médicales ElsevierSAS

bacteria / fluorescent probes / physiology / cytometry

1. IntroductionOne of the most basic questions that microbiologists

address is whether a bacterial cell is alive or dead. Theanswer to this question is far from simple and the questionremains unanswered after 20 years of intense research andpermanent controversy [1]. Life is generally characterisedby: (i) the presence of structure; (ii) changeable geneticinformation; (iii) metabolism or functional activity, and (iv)the ability to reproduce and grow [2]. However, livingbacteria are generally only characterised by their ability toreproduce, probably because the capacity of a cell tomultiply as determined by culture has long been the singlemethod available to microbiologists for bacterial viabilityassessment. Therefore, culturability and viability are oftenconsidered synonymous terms.

During the last two decades, an increasing need foralternative techniques has been stimulated by: (i) the needfor real-time monitoring methods for viability assessment[3]; (ii) the existence of injured and ‘viable but non-culturable’ (VNC) cells sometimes encountered when cellsare stressed (e.g. heat, oxidative, osmotic stress, or starva-tion) [1, 4], and (iii) the discovery of an important diversityof species for which suitable in vitro culture conditionshave not yet been defined and thus, which cannot berecognised as ‘alive or dead’ by the culturable/non-culturable dichotomy [5]. The development of these alter-native methods was also concomitant with advances in

fluorescent dye technology offering probes for a variety ofcellular functions. The VNC state was initially suggestedby Roszak et al. [4] for readily culturable bacteria whichunder particular circumstances become non-culturablebut retain ‘viability’. Nevertheless, the viability of VNCcells was never defined by culture but only by the detec-tion of some forms of activity in cells, which are notdirectly related to the capacity of these cells to reproduce.For these reasons, the term ‘active but non-culturable’(ANC) might be more precise than viable but non-culturable [1].

Fluorescence-based methods have remained very use-ful for a wide diversity of applications ranging from indus-trial to environmental microbiology. These tools are usedfor viability/activity assessment in food, pharmaceuticaland cosmetic industries, and in the natural environment,including fresh and marine waters. The increased use offluorescent probes is also due to improvements in thequantitative and qualitative sensitivity of instruments. Theaim of this review is to describe the different strategiesused for the detection of cell fluorescence, the differentcellular target sites and fluorescent probes which areactually used in activity assays, and the main applicationsof these probes.

2. Analytical instruments

Instrumentation strategy is of great importance, sinceall instruments have different ranges of application,depending on their quantitative and qualitative sensitivi-

* Correspondence and reprints.E-mail address: [email protected] (Philippe Lebaron).

Microbes and Infection, 2, 2000, 1523−1535© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

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ties. The quantitative limits, accuracy, speed of analysis,type of samples which can be analysed and light sourcesof these instruments are important selection criteria. Allfluorochromes are characterised by their absorption andemission spectra (table I). The characteristics of selecteddyes must be compatible with those of the instrumentationused for fluorescence detection.

Epifluorescence microscopy (EFM) of fluorochrome-stained cells has become the standard method by whichbacteria are enumerated. Most microscopes are equippedwith a mercury or a mercury–xenon lamp. The xenonlamp provides a continuous spectrum, whereas the mer-cury lamp emits spectral lines (365, 405, 435, 546 and578 nm) with a weak background continuum at otherwavelengths. Xenon lamps should be used in preferenceto mercury lamps only for dyes whose absorption spectradoes not match any of the strong mercury lines. The mainlimitation in the use of microscopy is that it is time con-suming and subjective [6]. Moreover, the detection limit ofmicroscopy is low and it becomes very tedious to detectless than 103 targeted cells per cm2 of filter – this limitcorresponding to the detection of one cell per microscopicfield. Thus, bacteria often have to be much more concen-trated for microscopic examination than for plate countingafter filtration. This can be quite problematic for samplescontaining non-cellular particles and when targeted cellsare diluted within non-targeted cells. Therefore, micro-scopic examination does not facilitate the simple detec-tion of rare events.

Flow cytometry (FCM) has became more popular andoffers the ability to perform analysis on individual cells atnumbers which begin to be more representative of nature

[20]. FCM allows the rapid and simultaneous measure-ment of various cell parameters with a precision of a fewper cent. Analyses are commonly performed at a flow ratebetween 10 and 100 µL per min and count rates rangingfrom 100 to 1 000 cells/s. Most flow cytometers areequipped to sort cells with specific characteristics forfurther analyses. However, FCM is not adapted to thedetection of rare events since it is difficult to detect lessthan 100 bacteria per mL, which is still 10–100-fold betterthan current microscopic procedures. For instance, at aconcentration of 100 bacteria per mL and at a mean flowrate of 50 µL/min, 20 min are needed to detect 100 events.Furthermore, when targeted cells represent less than onecell in one thousand or one cell in one million of non-targeted cells, flow cytometric analysis may range fromdifficult to impossible [7].

Laser scanning cytometer (LSC) offers the advantage ofrapid detection of bacteria on filters. LSC is a microscope-based cytofluorometer which possesses the attributes ofboth flow and image cytometry [8]. A 10-µm laser spotallows the measurement of emitted fluorescence in addi-tion to forward scattered light every 5 or 10 µm oversequential fields. The rapid scanning speed (0.3 m/s) andthe low-level resolution enable faster processing of thedata. However, the field-by-field scanning is an intrinsiclimitation, and the 10-µm step does not always allowresolution of individual cells. This is probably why thisinstrument has received little application in bacteriology.

Recently, a solid-phase cytometer (SPC) equivalent to arapid laser scanning device (ChemScan; Chemunex, Ivrysur Seine, France) has been developed [9, 10]. SPC differsfrom LSC in that the former is not a microscope-based

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 332Esterase activity (hydrolysis product)FDA (fluorescein) 473 514 416CFDA (carboxyfluoroscein) 492 517 460CFDA-AM (carboxyfluoroscein) 492 517 532BCECF-AM (BCECF) 482 520 615Calcein-AM (calcein) 494 517 995ChemChrome (*) 488 520 *

Membrane potentialRh123 507 529 381DiOC6(3) 484 501 573DiBAC4(3) 493 516 517Oxonol VI 599 634 316

Probe effluxEthidium bromide 518 605 394

Membrane integritySYTO-9 (membrane permeant stain) * (blue) * (green) *SYTO-13 (membrane permeant stain) 488 509 ∼ 400Propidium iodide 535 617 668Sytox Green 502 523 600PO-PRO-3 539 567 605CSE * (blue) * (orange) *

* Unspecified by the manufacturer. Source of information: Chemunex (Ivry-sur-Seine, Paris, France), Molecular Probes (Eugene, Oreg., USA), PolysciencesEurope (Germany).

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instrument. Cells are concentrated on a membrane filter asfor microscopy, and sample illumination and data collec-tion are achieved without the use of an objective. Thisallows high scanning speeds (1 m/s) and scanning of thewhole membrane (25-mm diameter) in one scan patternwith a fully overlapping laser beam. The scanning isperformed in under 3 min and any event detected in thescanning procedure can be positioned for observation inthe visual field of a conventional epifluorescence micro-scope in which the stage is driven by the instrument’scomputer [11]. This procedure is of great interest since itallows the possibility of an immediate visual control andconfirmation of the result. The system is also able todifferentiate between labelled microorganisms and autof-luorescent particles present in the sample based on theoptical and electronic characteristics of the generatedsignals. SPC is increasingly used in routine water analysesfor the detection of rare events (Escherichia coli,Cryptosporidium spp., Giardia spp.), in some industrialapplications for real-time control of pharmaceutical-gradewater and in different histological and cytological controls[11]. This technique allows the detection of a single cell ona 25-mm diameter membrane after filtration.

Confocal scanning laser microscopy (CSLM) is a com-puterised microscope that couples a laser light source to alight microscope. This technique allows for the generationof three-dimensional digital images of microorganisms.This technique combined with the use of fluorescentprobes has proven useful for the analyses of the three-dimensional distribution of total and active cells in solidstructures (e.g., biofilms, aggregates, endosymbionts,rhizophere and soil) [12]. However, CSLM is neitheradapted to routine applications for cell counting nor to thedetection of rare events.

The argon ion laser is the most widely used light sourcefor FCM, LSC, SPC and CSLM. Argon ion lasers provideemission lines at several wavelengths ranging from 351.1to 514.5 nm: the most widely used is the single line at488 nm. In the case of instruments equipped with a singlelaser source, the excitation wavelength is fixed and thestrategy for staining is limited to the range of probes andstains excitable at this wavelength. The use of probes withcontrasting wavelengths is usually required for multipa-rameter measurement (e.g., combination of a nucleic aciddye for the quantification of total bacteria with physiologi-cal and taxonomic probes) (see section 6). In this instance,contrasting wavelength means a combination of excita-tion and emission wavelengths, which allows discrimina-tion of each probe in the presence of the others. Doublestaining procedures with a single laser excitation source(often 488 nm) are limited since both dyes may have acommon excitation wavelength and different emissionwavelengths with a minimal overlap.

The choice of an instrument is primarily driven by itstechnical characteristics but also by its cost. EFM is nowconsidered traditional equipment for fluorescence appli-cations. FCM is more frequently used not only for researchbut also in many industrial applications. CSLM is expen-sive and not adapted to routine bacterial analyses. Otherinstruments are still under validation for different applica-tions. The quantitative sensitivity of the instrument and the

filterability of the products to be analysed will also influ-ence the choice of the instrument. The lack of consistencysometimes reported between microscopic and flow cytom-etry counts of total bacteria in microcosm studies andmainly the higher variations of FCM counts ([13], unpub-lished data from our group) may be due to day-to-dayfluctuations in flow cytometry operations, which requirestrong quality controls. When controls are made, EFM,FCM and SPC provide accurate counts of bacterial cells([9] Lebaron, unpublished data).

3. Definition of terms

Defining cell death and cell viability is philosophicallyand experimentally difficult. A viable cell should be con-sidered a cell capable of fission to produce similarly viableprogeny under realisable culture conditions [1]. Moregenerally, the term ‘viable’ should be restricted to circum-stances where evidence that a cell is able to divide is apriori or a posteriori provided. From an operational pointof view, viability is demonstrated by culture, and cellproliferation can be detected at both macroscopic (mac-rocolonies or turbidity) or microscopic (microcolonies)scale. The main advantage of direct methods based onfluorescent probes is the lack of incubation. Thus, there isclear evidence that methods based on the use of physi-ological probes cannot directly address cell division butonly the presence of some active functions or the integrityof cell structures. Therefore, cells in which any kind ofmetabolic activity can be detected should be called activecells. When metabolic activity is not detectable but integ-rity of essential envelope structures is preserved, cells arecalled inactive cells. These cells may be active but notenough to be detected as active cells or may be dying cellswith intact membranes. Lastly, cells with damaged mem-branes are considered dead cells, whereas those withintact membranes are considered intact cells. Dead cellscan be detected until cell lysis and are called ‘ghost’ cellswhen cellular envelopes are maintained, whereas thenucleoid is lost [14]. Most of these cellular categories werealready described by Neve-von Caron et al. [15].

The question of whether the active but non-culturable(ANC) cells are able or not to return to a growing statewhen their environment becomes favourable is at thecentre of the current debate on the ANC state (for acomplete review, see [1]). The demonstration of the revers-ibility of this non-growing state is based on resuscitationstudies but most of these are open to criticism. In moststudies, it is generally uncertain whether the ANC cells arecapable of resuscitation or whether the increase in cellnumbers reported in a large number of publications on thisissue is merely the result of growth of a few viable cells [1].This is due to the existence of an important heterogeneityof physiological states within a population and to ourinability to provide the proof that a cell in a given cellularstate as determined by any given method is able to growand to divide.

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4. Physiological target sites

This section presents the different probes used to assessdifferent physiological functions and cellular structures.The interpretation of these dyes in terms of cellular activityor viability is discussed in more details in sections 5 and 6.Figure 1 summarises the different physiological target sitesof these probes.

4.1. Membrane potential

The electrochemical potential occurring through theplasma membrane of metabolising bacteria is generatedby respiration or by ATP hydrolysis. It results from theselective permeability of biological membranes to a vari-ety of cations and anions leading to a difference of electricpotential across the membrane. Inside, the cell is nega-tively charged compared with outside the cell, and mem-brane potential (MP) plays a central role in different cell-life processes (ATP synthesis, active transport, mobility,regulation of intracellular pH, etc.). Voltage-sensitive dyeshave been developed to measure MP in bacteria. Depend-ing on the charge of the dye, they are accumulated inpolarised (cationic dyes) or depolarised (anionic dyes)cells. Reliability of staining is confirmed by observing ifdye uptake is sensitive to uncouplers (e.g., carbonyl cya-nide m-chlorophenyl hydrazone; CCCP) or ionophores(e.g., nigericin, valinomycin). In appropriate conditions,the amount of dye taken up can be directly related to thelevel of energy metabolism in the cell.

Rhodamine 123 (Rh123) is a lipophilic, cationic dyecommonly used to detect MP [16]. However, staining withRh123 often requires a pretreatment step of the cells,generally performed by adding EDTA, to permeabilise theouter membrane of Gram-negative bacteria [17]. Whenantibiotic or disinfectant treatments are studied, the per-meabilisation step can introduce some bias by enhancingthe toxic effects of these compounds (i.e. they penetratemore easily inside the cells). Furthermore, the pretreat-ment conditions may vary depending on the environment(e.g., salinity) and between species. This is why MP dyeshave received little applications at the community level.An additional problem is that Rh123 staining requiresseveral cell-washing steps, which are time consuming andmay result in cell losses. The cationic carbocyanine dyes(e.g., 3,3'-dihexyloxacarbocyanine; DiOC6(3)) have alsobeen used to estimate MP of bacteria. Advantages of thesedyes are the absence of permeabilization and washingsteps. However, nonspecific binding of carbocyanine dyesto hydrophobic regions of the cell and quenching of thefluorescence of intracellular dye have been reported [17].Thus, careful calibration of the staining procedure isrequired to avoid false positive signals.

MP can also be determined by the anionic lipophilicoxonols. Accumulation inside bacterial cells is favouredby a reduction in the magnitude of the MP, allowing dyemolecules to concentrate within the cell, and bind tolipid-rich components. Bis-(1,3-dibutylbarbituric acid) tri-methine oxonol [DiBAC4(3)] (BOX) has been reported to

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

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be useful to detect depolarised cells of numerous Gram-positive and Gram-negative bacterial species. In someprotocols, oxonol dyes combine a pretreatment with EDTAor EGTA 1–5 mM to increase membrane permeability [15,18]. Without permeabilisation, uptake of oxonols wouldbe more related to membrane integrity rather than mem-brane depolarisation [15]. Staining only dead cells withoxonols may be difficult when the treatments studied leadto the disruption of the cells (e.g., surfactants, antibiotics).In these cases, it is important to determine total cell countsand then to deduce the remaining fraction of living cells.For instance, Comas and Vives-Rego [19] suggested thesimultaneous detection of depolarised and total cell popu-lation by combining BOX with the nucleic acid dye SYTO-17.

Applications of MP dyes to natural samples are scarceand inconclusive [20], probably because of both the effectsof the permeabilisation step, which vary depending onspecies and the interaction between charged dyes andchemical compounds present in the sample.

4.2. Enzyme activity

4.2.1. Dehydrogenase activityCell-specific assays to detect the respiratory activity of

bacteria have been developed based on the use of differenttetrazolium salts. Tetrazolium dyes are reduced from acolourless complex to a brightly coloured, intracellular,formazan precipitate by components of the electron trans-port system and/or a variety of dehydrogenase enzymespresent in active bacterial cells. Since electron transport isdirectly related to cellular energy metabolism in respiringcells, the ability of cells to reduce tetrazolium compoundscan be considered an indicator of bacterial activity. Avariant approach is the use of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). CTC is reduced bybacteria to a water-insoluble, red fluorescent formazanproduct. It allows the quantification of the metabolicactivity of bacteria under both aerobic and different anaero-bic conditions [21, 22]. However, different factors mayaffect the CTC reduction during incubation [23–25].Although CTC counts are commonly determined by epif-luorescence microscopy, flow cytometry can be used whenpossible, to overcome the rapid fading of the fluorescencesignal [26], yielding equal or higher counts [25, 27]. Incontrast to other probes, samples can be fixed after stain-ing and stored at –20 °C until analysis.

The addition of nutrients during incubation with CTC issometimes used to improve the fluorescent signal and/orthe number of respiring cells [28]. In this case, controls areneeded to ensure that growth does not occur during incu-bation or that growth can be controlled by means ofantibiotics (e.g., cephalexin) without interfering with meta-bolic activity [18, 29]. Total cell counts can be determinedby counterstaining the cells with a fluorescent nucleicacid stain (commonly DAPI).

CTC is commonly used in microbial ecology, for bothaquatic and terrestrial systems. Applications include drink-ing water [30], biofilms [31], lake and seawater [32],sediments [33, 34], and subsurface soils [33]. However,the universality of CTC to detect respiring cells in naturalsamples remains controversial. Sherr et al. [32] found that

only a single strain did not reduce CTC over a largenumber of marine bacterial strains examined (n = 27). Asimilar trend was reported from the correlation foundbetween total and CTC counts in deep sediment samples,which suggests that CTC also stains anaerobic bacteria[34]. In contrast, the lack of universality of CTC waspointed out by Yamaguchi and Nasu [35] for environmen-tal applications. Toxic effects of CTC on bacterial metabo-lism have been reported [36], and this toxicity may explainwhy CTC counts are sometimes lower than active countsdetermined by other metabolic assays (e.g., microautora-diographic counts, or esterase activity) [36, 37].

4.2.2. Esterase activityDetection of esterase activity is measured using lipo-

philic, uncharged and non-fluorescent fluorogenic sub-strates. Once within active cells, the substrate is cleavedby non-specific esterases releasing a polar fluorescentproduct (fluorescein or fluoroscein derivatives) retainedinside cells having an intact membrane. Contrary toeukaryotic cells, Gram-negative bacteria are generallyimpermeable to the lipophilic fluorogenic probes, and apermeabilisation step of the outer membrane is required.

Esterases are present in all living organisms, and theseenzymes can be used to provide information on the meta-bolic state of bacterial cells. Although enzyme synthesisrequires energy, the enzyme–substrate reaction does not,and this assay alone should be considered energy inde-pendent. However, dead or dying cells with damagedmembranes rapidly leak the dye, even if they retain someresidual esterase activity. Consequently, fluorogenic sub-strates for esterases often serve as activity and cell integrityprobes that measure both enzymatic activity, which isrequired to activate their fluorescence, and cell-membraneintegrity, which is required for intracellular retention oftheir fluorescent products.

Fluorescein diacetate (FDA) is known to give weakfluorescence signals, since fluorescein is poorly retainedinside the cells [17]. In contrast, hydrophobic FDA deriva-tives are cleaved into hydrophilic products that are retainedmore efficiently inside cells with an intact membrane.Among these, acetoxymethyl ester (calcein-AM) wasshown ineffective to label different species, with the excep-tion of Staphylococcus aureus [38, 39]. Comparison madeby Jepras et al. [40] of different fluorogenic esters showsthat carboxyfluorescein diacetate (CFDA) is superior toFDA (fluorophore retention problems) and carboxyfluo-rescein diacetate acetoxy methyl ester (CFDA-AM) (solu-bility problems). The ability of a range of fluorogenic estersto stain several bacterial species was investigated by Dia-per and Edwards [38]. They found that ChemChrome B(Chemunex) (a commercial preparation of unknown for-mulation) is superior to FDA, CFDA, and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), as it stains the widest diversityof Gram-negative and Gram-positive species.

The main limitations encountered with the fluorogenicesterases are related to a poor dye uptake, an active dyeextrusion (see section 4.3) [41, 42], and problems of lowlabelling efficiency for some bacterial species in purecultures such as Pseudomonas spp. [41, 43]. Most of these

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limitations were recently overcome by the ChemChromeV6 staining kit (Chemunex) which combines a counter-stain to reduce non-specific fluorescence and a specificbuffer to reduce dye extrusion [44].

Porter et al. [20] found that BCECF-AM and Calcein-AMwere inappropriate dyes to enumerate viable bacteria infreshwater environments. Inversely, these authors observeda clear signal of fluorescence for active cells stained withCFDA and ChemChrome B, and the latter was found moreefficient with river water samples. Universality and highquality of staining was reported for CFDA with both pol-luted and non-polluted river waters [35]. Reynolds andFricker [43] used ChemChrome B to determine the frac-tion of living bacteria in a large number of potable watersand they found that this fluorogenic ester was alwayssuperior to plate counts, and often significantly higherthan CTC counts. The application of ChemChrome V6 todifferent water samples was recently reported by Catala etal. [44].

4.3. Pump activity

Several observations have confirmed that different dyescan be loaded into bacteria and are subsequently activelyremoved by energised cells. This is the case for Rh123[45], BCECF (from BCECF-AM) [41], carboxyfluoroscein(from CFDA) [42], and ethidium bromide [15]. Activeextrusion of these dyes can lead to biased results. To limitpump interference, Nebe-von Caron et al. [15] suggestedthe addition of sodium azide in staining solutions.

When active dye extrusion is not inhibited, probe effluxcan be used as an additional measure of cell activity [15,42]. Actively pumping cells are determined using suitablegrowth conditions and are sometimes detected usingethidium bromide at the single-cell level [15]. However,pump activity assays have only been applied to a fewspecies in culture and are not universal enough to beapplied to environmental samples.

4.4. Membrane integrity

The loss of membrane integrity represents significantdamage for cells due to multiple functions linked to theplasma membrane (permeability barrier, transport, respi-ratory activity, etc.). The maintenance of membrane integ-rity is commonly measured in eukaryotic cells as an indi-cator of cell damage or cell death [46]. Assessment ofmembrane integrity of bacteria is complicated due to theircomplex membrane structure. The envelope of Gram-negative bacteria consists of three interacting layers: theouter membrane, the rigid petidoglycan layer and theinner membrane (plasma membrane). For Gram-positivecells the outer membrane is absent. Additionally, someGram-negative and Gram-positive bacteria have a highlyhydrated polysaccharide layer outside the cell, called thecapsule. Membrane integrity analysis is based on thecapacity of the cells to exclude fluorescent dye com-pounds, which when used at low concentrations do notnormally cross intact membranes. Most of the membraneintegrity assays use nucleic acid stains, due to the highconcentrations of nucleic acids within the cells and thelarge fluorescence enhancement exhibited by nucleic acidstains upon binding, leading to a clear separation between

intact and dead cells. Loss of membrane integrity as mea-sured by uptake of membrane-impermeant dyes is gener-ally considered irreversible. However restoration of themembrane integrity has already been reported by Votya-kova et al. [47] (revealed by the use of the membrane-impermeant fluorescent DNA stain PO-PRO-3) duringresuscitation of starved Micrococcus luteus.

A wide diversity of impermeant nucleic acid stains canbe used; Molecular Probes (Eugene, Oreg., USA) offers alarge variety of dyes with molecular weights ranging from402–1 355, among which propidium iodide (PI) is themost commonly used. In order to simultaneously detectdead and intact cells, Molecular Probes has developed theLive/Dead BacLight kit containing two nucleic acid stains(SYTO-9 and PI) which differ in their spectral characteris-tics and their ability to penetrate intact bacterial mem-branes. SYTO-9 penetrates inside cells with both intact ordamaged membranes, staining the cells green, whereas PIonly penetrates cells with damaged membranes, stainingthe cells red. When the dyes are used in combination, cellswith intact membrane show a green fluorescence whilecells with damaged membranes show a red fluorescence(SYTO-9 emission contributes to the excitation of PI byenergy transfer). According to the manufacturer, SYTO-9should penetrate intact membranes of a large number ofGram-negative and Gram-positive bacteria. However,Langsrud and Sundheim [48] found that 30% of Pseudomo-nas aeruginosa strains tested (n = 18) did not accumulateSYTO-9. To overcome this problem, other membrane-permeable stains have been tested in combination with PI(e.g., SYTO-13 [19]).

The Live/Dead BacLight staining kit was recently usedby different authors to estimate the number of live cells inmarine planktonic environments using epifluorescencemicroscopy [49, 50]. The highly variable percentage ofviable cells reported from these studies may result fromcells exhibiting a continuum of in-between colours. Inorder to obtain a better discrimination between green andred fluorescent cells, Gasol et al. [50] recommended thefilter should be rinsed with isopropanol after staining.Recently, Boulos et al. [30] reported the application of theLive/Dead BacLight kit to drinking water and similar trendswere found between Live/Dead BacLight and CTC in theabsence of stress. In contrast, stress results in a more rapiddecrease of viable counts as determined by CTC com-pared to Live/Dead BacLight kit. Defives et al. [51] usedthe Live/Dead BacLight kit to determine intact and deadcell concentrations in natural mineral water during storageand mechanical bottling. Results suggest that the Live/Dead BacLight kit is a reliable stain for both autochtho-nous and allochthonous bacterial strains introduced bythe bottling system.

Recently, Molecular Probes has introduced a newimpermeant nucleic acid dye (Sytox Green). According tothe manufacturer, Sytox Green can be used to detect bothGram-negative and Gram-positive bacteria with damagedmembranes. However, Lebaron et al. [52] reported limita-tions in the use of Sytox Green alone for death assessmentin the case of treatment leading to DNA degradation (e.g.,long-term starvation). In this case, cells with damagedmembranes and damaged DNA display a low fluores-

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cence signal due to the degradation of target sites and maybe considered cells having intact membrane and intactDNA since these cells are, as living cells, not stained withthe dye. This limitation may exist for all membrane-impermeant DNA stains.

4.5. Nucleic acids

The detection of damaged DNA, such as breaks in theDNA strands, is often used to characterise apoptosis ineukaryotic cells [46]. Application of this method to bacte-ria may be biased by the maintenance of intact DNA forextended periods after cell death has occurred [28, 53].For instance, changes in the topology of the DNA mol-ecule such as those occurring in starved cells may bemisinterpreted as DNA damage [28, 53]. Changes in thefluorescence of DAPI-stained bacteria have been used toestimate the efficiency of drinking water disinfection bysodium hypochlorite [54]. However, death was not adetermining factor in the loss of DAPI fluorescence for alldisinfection treatments, because bacteria killed at highdoses of monochloramine were still fluorescent after DAPIstaining [54]. Recently, evidence has been presented thatbacterial abundance in seawater enumerated by conven-tional DAPI staining and EFM include cells that do notcontain a genome (‘ghost’ cells) due to unspecific bindingof DAPI [14]. Using a destaining procedure with isopro-panol to eliminate non-specific binding of DAPI, Zweifeland Hasgtröm [14] found that a large fraction (68–92%) oftotal counts can be scored as ghost cells. However, doubtspersist about the significance of ghost cells [55].

The cellular rRNA content of bacterial cells can bequantified by fluorescence in situ rRNA hybridization(FISH) of oligonucleotides carrying a fluorochrome.Because rRNA content in many bacterial species variesdepending on their growth rate and decrease rapidly ininactive cells, FISH has been proposed to estimate thephysiological state of cells [56]. Moreover, in the case ofcomplex communities, this assay could be developed todetect activity of specific bacteria using appropriate oligo-nucleotide probes [12]. However, in dynamic environ-ments and when cells are submitted to stress treatment(e.g., cold stress, acetic acid, or ethanol), the rRNA contentis a poor indicator of activity due to the high stability ofrRNA [57]. The recent development of FISH techniquesusing mRNA or pre-rRNAs (precursors in rRNA synthesis)as target molecules and those which determine the expres-sion of specific functional genes may provide more reli-able methods to assess the activity of individual cellswithin complex bacterial communities [58, 59].

5. Heterogeneity of physiological statesat population and community levels

Most investigations at the population level were per-formed in microcosm, to further understand the cellularand molecular responses of targeted species to differentstress conditions. An important part of these studies aimedto identify a definite live versus dead state. Studies at thecommunity level were performed to characterise the physi-ological structure of natural communities with the aim of

discriminating live and dead cells and more recently, toinvestigate if at least one assay could be used to detect andto enumerate growing cells responsible for the productionof bacterial biomass. Both types of studies have given riseto important semantic confusion around the definition ofactivity and viability as already discussed in section 3.

5.1. Heterogeneity at the population level

Probes targeting different cellular functions and thetechnique of flow cytometry have been used to analyse thephysiological heterogeneity of bacterial cells within tar-geted populations in different stress conditions. Many ofthese dyes share common excitation/emission properties,prohibiting simultaneous labelling of samples and there-fore the collection of multiple parameters on a given cell.However, the speed of flow cytometry allows the stainingof identical sub-samples by several dyes and then thecomparison of results. Figure 2 illustrates the succession ofcell changes that occurred at different rates in a Salmo-nella typhimurium population during starvation in artifi-cial seawater. The proportion of culturable cells decreasedrapidly (state 1) followed by a decrease in the proportionof respiring cells without or with nutrient addition (states 2and 3, respectively). After the loss of these functions, cellintegrity is maintained for a few days (state 4) and, after theloss of membrane integrity, cells with intact DNA (state 5)are rapidly subjected to an apparent DNA degradation(state 6) following lysis or formation of non-nucleoid-containing bacteria (state 7). According to the definition ofphysiological states given in section 3, cells in states 2 and3 may be considered ANC, those in state 4 intact cells, instates 5 and 6 dead cells and in state 7 ghost cells.

These results and those from many other studies suggestthat there is a strong heterogeneity of physiological stateswithin bacterial populations subjected to environmentalstress, e.g., [28, 29, 47, 53, 60, 61]. Because each cellularfunction is lost at a different rate, the activity of non-culturable cells is represented by a decreasing number ofactive physiological functions during the time of stress andthe fraction of active cells generally decreases up to theloss of membrane integrity. Today, the significance of eachcellular state in terms of viability remains unclear, mainlybecause of our inability to analyse the recovery of specificcells under favourable growth conditions. In a recent studycombining flow cytometry and cell sorting, it was shownthat S. typhimurium cells with depolarised membranescould be recovered after cell sorting [15]. It suggests thatdepolarisation is a sensitive measure of cell damage but apoor indicator of cell death. The loss of membrane integ-rity as determined by the penetration of impermeantnucleic acid dyes is more often used as an indicator ofdeath, but inversely, the integrity of the membrane cannotbe considered alone as an indicator of viability but iscommonly used as an indicator of cell integrity [62, 63].

Interpretation of results may also vary depending on theconditions affecting the physiological state of the cells. Forinstance, cell death occurs rapidly after exposure to UV orgamma radiation due to DNA damage, but the radiationdoes not affect dehydrogenase activity, membrane poten-tial, membrane integrity and �-D-galactosidase activity[64]. Therefore, this implies a delay following death before

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enzymes are degraded and membranes are damaged. Inthis case, there is no protocol available to rapidly assesscell viability and the use of culture should be also consid-ered with caution, since DNA repair mechanisms mayrestore viability [65]. Some recent and promising investi-gations have been made to improve direct methods bycombining traditional probes targeting enzyme activitiesor membrane integrity with a probe targeting DNA dam-age such as fragmentation [66].

The heterogeneity of cellular physiological statesreported in most microcosm-stressed populations is alwaysobserved by using a large inoculum of cells (> 105 cells permL) because of the low quantitative sensitivity of both flowcytometry and microscopy. As a consequence, high celldensities may introduce a bias in the temporal evolution ofthe physiological states of individual cells due to the lysisof some cells and the use of released organic compoundsby the most metabolically active cells. Consequently, somephysiological states may exist at smaller time scales thanthose found in most laboratory studies. The recent devel-opment of more sensitive instruments such as solid-phasecytometry may help to provide a more accurate descrip-tion of the temporal variations in physiological states, andtherefore to measure the persistence time of active butnon-culturable cells within stressed populations. This infor-mation remains of great importance in the case of patho-

gens and should be determined for each species and for awide variety of stress conditions. If ANC forms are presentand are able to persist in the natural environment, then itwill be important to further evaluate the public healthsignificance of this state.

5.2. Heterogeneity at the community level

In natural waters, culture methods give microorganismcounts that represent less than 1% of the total bacterialcells and overlook an unknown but probably large propor-tion of living cells (that do not grow on the conventionalsubstrates). On the other hand, total counts determined bynucleic acid dyes (such as DAPI or acridine orange) includedead or inactive cells. The detection and enumeration ofactive cells (living bacteria with any detectable metabolicactivity), growing cells (bacteria which participate in theproduction of biomass) and dead cells (representingorganic particles) in natural bacterial assemblages wouldbe of great help and most useful in aquatic microbialecology. It would help to better understand the mecha-nisms behind the regulation of bacterial activity. Differentprobes and methods have been used to estimate the pro-portion of both active and dead cells and only a few recentinvestigations have tried to detect and to enumerate grow-ing cells. However, consensus on what each of the probestested in these studies measures has not yet been reached.

The detection of active cells within complex commu-nities requires the use of universal probes which target thewidest diversity of species. Therefore, only a few probes(formazan salt reduction, esterase activity, and rRNAprobes) have been applied to natural communities todescribe the physiological heterogeneity of bacterial cells.Despite the different drawbacks of CTC reported in section4.2.1, studies suggest that CTC provides an ecologicallymeaningful measure of bacterial activity [26, 67]. Sherr etal. [32] suggested that CTC-positive cells represent thosebacteria characterised by a high level of metabolic activ-ity, and that cells which show no apparent reduction ofCTC have either low or no metabolic activity. This assump-tion is far from clear, and the interpretation of CTC countsremains controversial. The recent and increasing use ofCTC counts in microbial ecology and at the same time thewell-known limitations of the method illustrate the impor-tant need, for microbial ecologists, to identify which cellsare responsible for bacterial community activity. As analternative to CTC, growing cells can also be determinedby flow cytometry as the fraction of cells with a high DNAcontent [68]. The fraction of cells with a high DNA contentis easily discriminated by flow cytometric analysis of SYBRGreen-stained cells, and in a recent study, Gasol et al. [50]indicated that high-DNA bacteria are the dynamic mem-bers of the bacterial assemblage. General acceptance ofthis approach to determine the dynamic members of natu-ral communities will require further investigation to under-stand how it works and what its results mean.

Studies which perform a comparison of methods arescarce and more intercalibration between methods is cur-rently needed. Karner and Fuhrman [37] have applieddifferent methods to assess bacterial activity in sevendifferent marine water samples and they found an averageproportion of 56% of cells containing detectable rRNA,

Figure 2. Evolution of the different cell states in a S. typhimuriumpopulation during starvation survival in artificial seawater. Eachcell state (or lysed/non-nucleoid-containing cells) is expressed as apercentage of the original total cell count. The methods used aredeveloped in the text. Results published in Joux et al. [28].State 1: culturable cells as determined on nutrient agar(bioMérieux, France) plates.State 2: non-culturable cells showing real respiration by the CTC(Polysciences Europe, Germany) method.State 3: non-culturable cells showing no real respiration butpotential respiration as determined by the CTC method afternutrient (1/10 R2A broth) supply.State 4: non-culturable cells showing no respiration but mem-brane integrity maintenance as determined by the Live/DeadBacLight kit (Molecular Probes).State 5: non-culturable cells showing loss of membrane integritywith no DNA change as determined by Hoechst 33342 (Sigma)staining.State 6: non-culturable cells showing cellular integrity withDNA change determined by Hoechst staining.State 7: lysed or non-nucleoid-containing cells.

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49% of cells able to take up added radiolabelled com-pounds, 29% of nucleoid-containing cells and only 0.7%of respiring cells. Parthuisot et al. [69] found that activebacteria determined by esterase activity measurements indifferent water samples are higher than those determinedby CTC and CFU and that CTC counts are sometimeslower than CFU. From these results and other studies [55,70], there is clear evidence that there is an importantheterogeneity of physiological states within natural com-munities. However, each method yields different estimatesof the proportion of bacterial cells that are active. Thephysiological and ecological significance of each cellstate remains unclear and the answer, if it exists, to thisquestion will require further investigations to relate a givencellular physiological state to additional information suchas its growth potential, enzyme activity or taxonomicidentity.

6. Combination of fluorescent probes

The combination of fluorescent probes in single assaysprovides confident tools and some of these are actuallyunder validation for industrial applications such as waterquality assessment in the pharmaceutical industry [43].However, although physiological probes are very usefulfor different industrial and environmental applications,they cannot address the detection of specific active cellssuch as pathogenic microorganisms. Most of these appli-cations are based on the combination of physiological andtaxonomic probes and require instruments which candetect rare events.

6.1. Combination of differentphysiological fluorescent probes

A more accurate evaluation of cell activity should beobtained by combining physiological probes targetingdifferent cellular functions. Assays in which bothmembrane-based and metabolism-based probes are usedsimultaneously provide information on whether the dyeassays accurately reflect cell activity. Probes should beselected with excitation and emission wavelengths whichallow discrimination of each probe in the presence of theother(s). Therefore, dyes with a narrow emission spectrummay be better suited to multicolour applications. Theselection will also be dependent on the instrumentationused for fluorescence analysis (see section 2). One shouldalso keep in mind that the selection of fluorophores inmultiple-staining procedures may take into accountmolecular interactions which can result in quenching, adecrease in the fluorescence signal. This decrease is due tochanges in the excited states of the fluorophores, resultingin energy dissipation by non-radiative transitions. Thesephysico-chemical changes may be caused by the interac-tion between fluorophores.

In multiple-staining assays, PI (dead cells, red fluores-cence) is often combined with CFDA (cells with esteraseactivity, green fluorescence) [35, 42], Rh123 (polarisedcells, green fluorescence) [18, 71] or BOX (depolarisedcells, green fluorescence) [18, 71]. The CTC dye can alsobe combined with the detection of cell depolarisation

(BOX) or cell polarisation (Rh123) [18]. The CV6/CSElabelling kit from Chemunex offers the possibility of simul-taneously detecting cells with an esterase activity (CV6)and membrane integrity (CSE). Application of this stainingprocedure to S. typhimurium cells starved in artificial sea-water allowed a more accurate assessment of active cellcounts by counterstaining cells with permeabilised mem-branes [44]. A nice illustration of multiple-staining assayswas provided by Nebe-von Caron et al. [15] using acombination of three fluorescent dyes to measure mem-brane potential (BOX), pump activity (ethidium bromide)and membrane integrity (PI).

Most of these studies remained methodological studiesand suffer from a lack of validation. Therefore, the signifi-cance of these methods and their possible use for viabilityassessment is unknown. ChemChrome V6 from Chemu-nex is one of the most commonly used methods whichcombines a fluorogenic ester for esterase activity assess-ment with a counterstain which penetrates inside cellswith permeabilised membranes [44, 69]. This method iswidespread in different industrial applications (pharma-ceutical, food and cosmetic industries) and when coupledwith flow cytometry provide real-time estimation of activecell counts. Although active counts are sometimes signifi-cantly higher than viable counts determined by CFU, theyare considered very useful to detect production problemsand perhaps yield a more accurate estimation of viablecounts than do culture methods.

6.2. Combination of physiologicaland taxonomic fluorescent probes

In the case of complex communities, physiologicalfluorescent probes provide useful information on the per-centage of active, inactive or dead cells of the overallcommunity. However, detection of the activity of specificbacteria is of primary importance in different microbio-logical areas. For instance, it could be used to detect activepathogens in food and waters, or to detect active specificpopulations implicated in different transformations (biore-actors, environment). Fluorescent antibodies and fluores-cent oligonucleotide rRNA are taxonomic probes that canbe used at the single-cell level to detect bacteria of interestincluding pathogens. One problem encountered withhybridisation of fluorescent oligonucleotide rRNA con-cerns the permeabilisation of cells and amplification offluorescence signals. The combination of taxonomic andphysiological fluorescent probes is a key challenge formicrobiologists since there is an increasing need to pro-vide numbers of specific cells, depending on their physi-ological state (at least viable or active state).

Whiteley et al. [72] described the combination of fluo-rescent in situ hybridisation (rRNA probes) with the cyto-chrome oxidase assay. Similar combinations with a tetra-zolium salt necessitate a fastidious sequential procedure,inconceivable in routine, in which tetrazolium reductionis evaluated prior to probe hybridisation [72]. Differentauthors have demonstrated the feasibility of combiningimmunostaining with CTC [73], Rh123 [74] and Chem-Chrome staining [75]. The fluorochrome conjugated to themonoclonal antibody must be carefully chosen in order toavoid fluorescence overlap with the physiological dye.

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Recently, Pyle et al. [76] combined an immunomagneticseparation (IMS) with the analysis of respiratory activity(CTC staining) to assess the number of active E. coliO157:H7 present in meat and water. In this procedure,E. coli O157:H7 cells are isolated from the sample byusing a magnetic particle concentrator after addition ofimmunomagnetic beads coated with anti-O157 antibody.Then, cells are incubated with CTC (3 h), stained withFITC-labelled antibody (20 min) and filtered for micro-scopic enumeration or solid-phase laser cytometry. Thisprocedure results in high rates of recovery but cell injury isobserved [76]. However, the combination of taxonomicprobes with the CTC dye has little interest since CTCcounts are well known to underestimate the number ofviable targeted cells (see sections 4.2.1 and 5.2). Further-more, most of these assays were performed by using anartificial mixture of strains or artificial contamination.Such a combination should be treated cautiously for envi-ronmental monitoring, due to additional manipulativesteps that could reduce cell recovery.

One additional limitation to the actual use of thesemethods is that in most cases, cells of interest are highlydiluted within autochthonous populations, and field appli-cations have long been limited because instruments suchas microscopy and flow cytometry do not allow the detec-tion of rare events [77]. Detection of bacterial cells at afrequency of 10–5 to 10–7 is a challenge in various fields ofmicrobiology (industry, medicine, environment, etc.). Therecent development of solid-phase cytometry has coveredthis gap and this technology offers promising perspectives.The ChemScan technology is now increasingly used in theindustry for the rapid and accurate detection of E. coli,Cryptosporidium spp. and Giardia spp. in waters with ahigh sensitivity of detection as well as for the quantifica-tion of active bacteria in process waters (pharmaceuticalindustries). For instance, it is possible to detect a single cellof E. coli or any other species for which specific antibodiesare available in 1 L of tap water and in the presence of106–108 non-targeted cells (Lebaron, unpublished data).This detection limit is similar to that of culture techniqueswhich combine a filtration step. Furthermore, SPC offersthe advantage that it does not require the use of selectivegrowth conditions and thus, allows the detection of injuredcells which are viable cells not detected on selectivemedia. However, although the instrument can detect threefluorescences, applications are still limited because it isequipped with a single source of excitation. Therefore,today the use of at least two probes for physiologicalassessment and an additional probe (taxonomic) for thedetection of specific cells remains difficult if not impos-sible. Many applications will require the use of a secondsource of light.

7. Conclusions and future prospects

There is clear evidence that the application of fluores-cent dyes will provide microbiologists with a set of valu-able tools to complement existing molecular and conven-tional methods by providing information concerningspecific physiological activities at the level of the indi-

vidual bacterial cell. However, despite the continuousdevelopment of new fluorescent dyes, no universal stainor staining procedure that is suitable for all applicationshas been found, and will probably never be found. Theefficiency of dyes and protocols needs to be evaluated foreach application and studies at the population level remainessential to understand how different species react in agiven assay and how the response varies depending on thestress conditions applied to these species.

The development of multiparameter analysis whichcombines at least two physiological probes is very prom-ising but requires further investigations and validationwork to ensure that these methods are reproducible androbust enough for routine applications. Validation shouldbe made in regard to specific applications since no method,as is the case for culturability, can be robust enough to beuniversal and suitable for all applications. For instance,activity assessment after UV or gamma radiation requiresmethods which are not based on membrane integrity orenzyme activities. Inversely, methods based on a combi-nation of probes which target membrane permeability andenzyme activity can be very useful and are already rou-tinely used to quantify active cells in water, food products,drugs and cosmetics, to control the efficiency of freeze-drying procedures in bacterial collections and probablyother applications. Differences between active and cultur-able cell counts do not constitute a limitation to the use ofphysiological probes if active cell counts are obtainedrapidly and allow the detection of any interpretablechanges in terms of quality control. In these cases, limitsthat are based on the standard cultivation techniques,when they exist, should be reconsidered in favour ofalternative methods.

The traditional debate and conflict around viability andactivity as assessed by physiological probes is somehowparadoxical. On the one hand, traditional culture methodsare based on the dividing capacity of a cell which requiresa few hours and sometimes a few days to be detected andon the other hand, physiological probes which have beendeveloped for real-time monitoring of cell viability do notallow the control of cell division. Therefore, viabilityassessment cannot be achieved directly by physiologicalprobes. When assays based on the use of physiologicalprobe(s) are available, they should be evaluated and com-pared to standard methods. However, these probes havenever been applied to the detection of rare events such aspathogens because the instrumentation used for fluores-cence analysis was until recently inappropriate for thedetection of rare events. This last point as well as the priceof these instruments probably explains why alternativemethods have received little field application and valida-tion and why it is difficult if not impossible today toprovide an accurate evaluation of the potential use ofthese probes as alternative methods to the standard culti-vation techniques.

As stated above, the adoption of these new tools willalso require new standards in bacterial quality assessmentsince, in most applications, counts are generally higherthan CFU counts which were used to define the actualstandards. The development of new fluorescent dyes moreclosely related to cellular energy metabolism and assays

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combining different probes remain important to improveand/or to further develop the use of alternative methods ina wide variety of bacterial applications. Furthermore, thedevelopment of new assays, which combine both physi-ological and taxonomic probes, should be encouraged toprovide new tools for the detection of active specific cells.For these combined staining procedures, development oflow-cost instruments equipped with at least two excitationsources is essential.

References

[1] Barer M.R., Harwood C.R., Bacterial viability and cultur-ability, Adv. Microb. Physiol. 41 (1999) 93–137.

[2] Nebe-von Caron G., Badley R.A., Viability assessment ofbacteria in mixed populations using flow cytometry,J. Microscopy 179 (1995) 55–66.

[3] Sidorowicz S.V., Withmore T.N., Prospects for new tech-niques for rapid bacteriological monitoring of drinkingwater, J. IWEN 9 (1995) 92–97.

[4] Roszak D.B., Grimes D.J., Colwell R.R., Viable but non-recoverable stage of Salmonella enteritidis in aquatic system,Can. J. Microbiol. 30 (1984) 334–338.

[5] Ward D., Weller R., Bateson M.M., 16S rRNA sequencesreveal numerous uncultured microorganisms in a naturalcommunity, Nature 345 (1990) 63–65.

[6] Porter J., Deere D., Pickup R., Edwards C., Fluorescentprobes and flow cytometry: new insights into environmen-tal bacteriology, Cytometry 23 (1996) 91–96.

[7] Collier J.L., Campbell L., Flow cytometry in molecularaquatic ecology, Hydrobiologia 401 (1999) 33–53.

[8] Darzynkiewicz Z., Bedner E., Li X., Gorczyca W.,Melamed M.R., Laser-scanning cytometry: a new instru-mentation with many applications, Exp. Cell Res. 249(1999) 1–12.

[9] Reynolds D.T., Slade R.B., Sykes N.J., Jonas A.,Fricker C.R., Detection of Crysporidium oocysts in water:techniques for generating precise recovery data, J. Appl.Microbiol. 87 (1999) 804–813.

[10] Mignon-Godefroy K., Guillet J.C., Butor C., Solid-phasecytometry for detection of rare events, Cytometry 27 (1997)336–344.

[11] Butor C., Duquenne O., Mignon-Godefroy K., Mougin C.,Guillet J.G., Solid-phase cytometry allows rapid in situquantification of human papilloma virus infection in biopsymaterial, Cytometry 29 (1997) 292–297.

[12] Aßmus B., Schloter M., Kirchhof G., Hutzler P., Hart-mann A., Improved in situ tracking of rhizosphere bacteriausing dual staining with fluorescence-labeled antibodiesand rRNA-targeted oligonucleotides, Microb. Ecol. 33(1997) 32–40.

[13] Porter J., Deere D., Hardman M., Edwards C., Pickup R.,Go with the flow –use of flow cytometry in environmentalmicrobiology, FEMS Microbiol. Ecol. 24 (1997) 93–101.

[14] Zweifel U.L., Hagström Å., Total counts of marine bacteriainclude a large fraction of non-nucleoid-containing bacteria(ghosts), Appl. Environ. Microbiol. 61 (1995) 2180–2185.

[15] Nebe-von Caron G., Stephens P., Badley R.A., Assessmentof bacterial viability status by flow cytometry and singlecell sorting, J. Appl. Microbiol. 84 (1998) 988–998.

[16] Kaprelyants A.S., Kell D.B., Dormancy in stationary-phasecultures of Micrococcus luteus: flow cytometric analysis ofstarvation and resuscitation, Appl. Environ. Microbiol. 59(1993) 3187–3196.

[17] Diaper J.P., Tither K., Edwards C., Rapid assessment ofbacterial viability by flow cytometry, Appl. Microbiol.Biotechnol. 38 (1992) 268–272.

[18] López-Amorós R., Castel S., Comas-Riu J., Vives-Rego J.,Assessment of E. coli and Salmonella viability and starvationby confocal laser microscopy and flow cytometry usingrhodamine 123, DiBAC4(3), propidium iodide, and CTC,Cytometry 29 (1997) 298–305.

[19] Comas J., Vives-Rego J., Assessment of the effects of grami-cidin, formaldehyde, and surfactants on Escherichia coli byflow cytometry using nucleic acid and membrane potentialdyes, Cytometry 29 (1997) 58–64.

[20] Porter J., Diaper J., Edwards C., Pickup R., Direct mea-surements of natural planktonic bacterial communityviability by flow cytometry, Appl. Environ. Microbiol. 61(1995) 2783–2786.

[21] Smith J.J., McFeters G.A., Mechanisms of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chlo-ride), and CTC (5-cyano-2,3-ditolyl tetrazolium chloride)reduction in Escherichia coli K-12, J. Microbiol. Methods 29(1997) 161–175.

[22] Bhupathiraju V.K., Hernandez M., Landfear D., Alvarez-Cohen L., Application of a tetrazolium dye as an indicatorof viability in anaerobic bacteria, J. Microbiol. Methods 37(1999) 231–243.

[23] Pyle B.H., Broadaway S.C., McFeters G.A., Factors affect-ing the determination of respiratory activity on the basis ofcyanoditolyl tetrazolium chloride reduction with mem-brane filtration, Appl. Environ. Microbiol. 61 (1995)4304–4309.

[24] Smith J.J., McFeters G.A., Effects of substrates and phos-phate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyltetrazolium chloride) reduction in Escherichia coli, J. Appl.Bacteriol. 80 (1996) 209–215.

[25] Sieracki M.E., Cucci T.L., Nicinski J., Flow cytometricanalysis of 5-cyano-2,3-ditolyl tetrazolium chloride activ-ity of marine bacterioplankton in dilution cultures, Appl.Environ. Microbiol. 65 (1999) 2409–2417.

[26] Lovejoy C., Legendre L., Klein B., Tremblay J.É.,Ingram R.G., Therriault J.C., Bacterial activity duringearly winter mixing (Gulf of St. Lawrence, Canada), Aquat.Microb. Ecol. 10 (1996) 1–13.

[27] del Giorgio P.A., Prairie Y.T., Bird D.F., Coupling betweenrates of bacterial production and the abundance of meta-bolically active bacteria in lakes, enumerated using CTCreduction and flow cytometry, Microb. Ecol. 34 (1997)144–154.

[28] Joux F., Lebaron P., Troussellier M., Succession of cellularstates in a Salmonella typhimurium population during starva-tion in artificial seawater microcosms, FEMS Microbiol.Ecol. 22 (1997) 65–76.

Fluorescent physiological probes for bacteria Review

Microbes and Infection2000, 1523-1535

1533

Page 12: 2000 Use of Fluorescent Probes to Assess Physiological Functions of Bacteria at Single-cell Level

[29] Caro A., Got P., Baleux B., Physiological changes of Salmo-nella typhimurium cells under osmotic and starvation condi-tions by image analysis, FEMS Microbiol. Lett. 179 (1999)265–273.

[30] Boulos L., Prévost M., Barbeau B., Coallier J., Desjar-dins R., Live/Dead Baclight: application of a new rapidstaining method for direct enumeration of viable and totalbacteria in drinking water, J. Microbiol. Methods 37 (1999)77–86.

[31] Huang C.T., Yu F.P., McFeters G.A., Stewart P.S., Nonuni-form spatial patterns of respiratory activity within biofilmsduring disinfection, Appl. Environ. Microbiol. 61 (1995)2252–2256.

[32] Sherr B.F., del Giorgio P.A., Sherr E.B., Estimating abun-dance and single-cell characteristics of respiring bacteriavia the redox dye CTC, Aquat. Microb. Ecol. 18 (1999)117–131.

[33] Yu W., Doods W.K., Banks M.K., Skalsky J., Strauss E.A.,Optimal staining and sample storage time for direct micro-scopic enumeration of total and active bacteria in soil withtwo fluorescent dyes, Appl. Environ. Microbiol. 61 (1995)3367–3372.

[34] Miskin I., Rhodes G., Lawlor K., Saunders J.R.,Pickup R.W., Bacteria in post-glacial freshwater sediments,Microbiology 144 (1998) 2427–2439.

[35] Yamaguchi N., Nasu M., Flow cytometric analysis of bac-terial respiratory and enzymatic activity in the naturalaquatic environment, J. Appl. Microbiol. 83 (1997) 43–52.

[36] Ullrich S., Karrasch B., Hoppe H.G., Jeskulke K., Meh-rens M., Toxic effects on bacterial metabolism of the redoxdye 5-cyano-2,3-ditolyl tetrazolium chloride, Appl. Envi-ron. Microbiol. 62 (1996) 4587–4593.

[37] Karner M., Furhman J.A., Determination of active marinebacterioplankton: a comparison of universal 16S rRNAprobes, autoradiography, and nucleic staining, Appl. Envi-ron. Microbiol. 63 (1997) 1208–1213.

[38] Diaper J.P., Edwards C., The use of fluorogenic esters todetect viable bacteria by flow cytometry, J. Appl. Bacteriol.77 (1994) 221–228.

[39] Comas J., Vives-Rego J., Enumeration, viability and het-erogeneity in Staphylococcus aureus cultures by flow cytom-etry, J. Microbiol. Methods 32 (1998) 45–53.

[40] Jepras R.I., Carter J., Pearson S.C., Paul F.E., Wilkin-son M.J., Development of a robust flow cytometric assay fordetermining numbers of viable bacteria, Appl. Environ.Microbiol. 61 (1995) 2696–2701.

[41] Molenaar D., Bolhuis H., Abee T., Poolman B., Kon-ings W.N., The efflux of a fluorescent probe is catalyzed byan ATP-driven extrusion system in Lactococcus lactis, J. Bac-teriol. 174 (1992) 3118–3124.

[42] Bunthof C., VanDen Braak S., Breeuwer P., Rom-bouts F.M., Abee T., Rapid fluorescence assessment of theviability of stressed Lactococcus lactis, Appl. Environ. Micro-biol. 65 (1999) 3681–3689.

[43] Reynolds D.T., Fricker C.R., Application of laser scanningfor the rapid and automated detection of bacteria in waterssamples, J. Appl. Microbiol. 86 (1999) 785–795.

[44] Catala P., Parthuisot N., Bernard L., Baudart J., Lemarch-and K., Lebaron P., Effectiveness of CSE to counterstainparticles and dead bacterial cells with permeabilised mem-branes: application to viability assessment in waters, FEMSMicrobiol. Lett. 178 (1999) 219–226.

[45] Ueckert J., Breeuwer P., Abee T., Stephens P., Nebe-vonCaron G., TerSteeg P.F., Flow cytometry applications inphysiological study and detection of foodborne microor-ganisms, Int. J. Food Microbiol. 28 (1995) 317–326.

[46] Shapiro H.M., Pratical flow cytometry, 2nd edition, AlanR. Liss, New York, 1988.

[47] Votyakova V., Kaprelyants A.S., Kell D.B., Influence ofviable cells on the resuscitation of dormant cells in Micro-coccus luteus cultures held in an extended stationary phase:the population effect, Appl. Environ. Microbiol. 60 (1994)3284–3291.

[48] Langsrud S., Sundheim G., Flow cytometry for rapid assess-ment of viability after exposure to a quaternary ammoniumcompound, J. Appl. Bacteriol. 81 (1996) 411–418.

[49] Decamp O., Rajendran N., Assessment of bacterioplanktonviability by membrane integrity, Mar. Poll. Bull. 36 (1998)739–741.

[50] Gasol J.M., Zweifel U.L., Peters F., Furhman J.A., Hag-ström Å., Significance of size and nucleic acid contentheterogeneity as measured by flow cytometry in naturalplanktonic bacteria, Appl. Environ. Microbiol. 65 (1999)4475–4483.

[51] Defives C., Guyard S., Oularé M.M., Mary P., Hornez J.P.,Total counts, culturable and viable, and non-culturablemicroflora of a French mineral water: a case study, J. Appl.Microbiol. 86 (1999) 1033–1038.

[52] Lebaron P., Catala P., Parthuisot N., Effectiveness of SYTOXGreen stain for bacterial viability assessment, Appl. Envi-ron. Microbiol. 64 (1998) 2697–2700.

[53] Joux F., Lebaron P., Troussellier M., Changes in cellularstates of the marine bacterium Deleya aquamarina understarvation conditions, Appl. Environ. Microbiol. 63 (1997)2686–2694.

[54] Saby S., Sibille I., Mathieu L., Paquin J.L., Block J.C.,Influence of water chlorination on the counting of bacteriawith DAPI (4',6-diamidino-2-phenylindole), Appl. Envi-ron. Microbiol. 63 (1997) 1564–1569.

[55] Choi J.W., Sherr E.B., Sherr B.F., Relation betweenpresence-absence of a visible nucleoid and metabolic activ-ity in bacterioplankton cells, Limnol. Oceanogr. 41 (1996)1161–1168.

[56] Ruimy R., Breittmayer V., Boivin V., Christen R., Assess-ment of the state of activity of individual bacterial cells byhybridization with a ribosomal RNA targeted fluorescentlylabelled oligonucleotidic probe, FEMS Microbiol. Ecol. 15(1994) 207–214.

[57] Tolker-Nielsen T., Halberg Larsen M., Kyed H., Molin S.,Effect of stress treatments on the detection of Salmonella byin situ hybridization, Int. J. Food Microbiol. 35 (1997)251–258.

[58] Chen F., Gonzáles J.M., Dustman W.A., Moran M.A.,Hodson R.E., In situ reverse transcription, an approach tocharacterize genetic diversity and activities of prokaryotes,Appl. Environ. Microbiol. 63 (1997) 4907–4913.

Review Joux and Lebaron

1534 Microbes and Infection2000, 1523-1535

Page 13: 2000 Use of Fluorescent Probes to Assess Physiological Functions of Bacteria at Single-cell Level

[59] Licht T.R., Tolker-Nielsen T., Holmstrøm K., Krog-felt K.A., Molin S., Inhibition of Escherichia coli precursor-16S rRNA processing by mouse intestinal contents, Envi-ron. Microbiol. 1 (1999) 23–32.

[60] Caro A., Got P., Lesne J., Binard S., Baleux B., Viability andvirulence of experimentally stressed nonculturable Salmo-nella typhimurium, Appl. Environ. Microbiol. 65 (1999)3229–3232.

[61] Lisle J.T., Pyle B.H., McFeters G.A., The use of multipleindices of physiological activity to access viability in chlo-rine disinfected Escherichia coli O157:H7, Lett. Appl. Micro-biol. 29 (1999) 42–47.

[62] Novo D.J., Perlmutter N.G., Hunt R.H., Shapiro H.M.,Multiparameter flow cytometric analysis of antibiotic effectson membrane potential, membrane permeability, and bac-terial counts of Staphylococcus aureus and Micrococcus luteus,Antimicrob. Agents Chemother. 44 (2000) 827–834.

[63] Weichart D., McDougald D., Jacobs D., Kjelleberg S., Insitu analysis of nucleic acids in cold-induced nonculturableVibrio vulnificus, Appl. Environ. Microbiol. 63 (1997)2754–2758.

[64] Fiksdal L., Tryland I., Effect of UV light irradiation, star-vation and heat on Escherichia coli �-D-galactosidase activ-ity and other potential viability parameters, J. Appl. Micro-biol. 87 (1999) 62–71.

[65] Joux F., Jeffrey W.H., Lebaron P., Mitchell D.L., Marinebacteria isolates display diverse responses to ultraviolet-Bradiation, Appl. Environ. Microbiol. 65 (1999) 3820–3827.

[66] Rohwer F., Azam F., Detection of DNA damage in prokary-otes by terminal deoxyribonucleotide transferase-mediateddUTP nick end labeling, Appl. Environ. Microbiol. 66(2000) 1001–1006.

[67] Smith E.M., Coherence of microbial respiration rate andcell-specific bacterial activity in a coastal planktonic com-munity, Aquat. Microb. Ecol. 16 (1998) 27–35.

[68] Jellet J.F., Li W.K.W., Dickie P.M., Boraie A., Kepkay P.E.,Metabolic activity of bacterioplankton communitiesassessed by flow cytometry and single carbon substrateutilization, Mar. Ecol. Prog. Ser. 136 (1996) 213–225.

[69] Parthuisot N., Catala P., Lemarchand K., Baudart J., Leb-aron P., Evaluation of ChemChrome V6 for bacterial viabil-ity assessment in waters, J. Appl. Microbiol. (2000)370–380.

[70] Joux F., Lebaron P., Ecological implication of an improveddirect viable count method for aquatic bacteria, Appl.Environ. Microbiol. 63 (1997) 3643–3647.

[71] López-Amorós R., Comas J., Vives-Rego J., Flow cytomet-ric assessment of Escherichia coli and Salmonella typhimuriumstarvation–survival in seawater using rhodamine 123, pro-pidium iodide, and oxonol, Appl. Environ. Microbiol. 61(1995) 2521–2526.

[72] Whiteley A.S., O’Donnell A.G., Macnaughton S.J.,Barer M.B., Cytochemical colocalization and quantitationof phenotypic and genotypic characteristics in individualbacterial cells, Appl. Environ. Microbiol. 62 (1996)1873–1879.

[73] Pyle B.H., Broadaway S.C., McFeters G.A., A rapid, directmethod for enumerating respiring enterohemorragicEscherichia coli O157:H7 in water, Appl. Environ. Micro-biol. 61 (1995) 2614–2619.

[74] Clarke R.G., Pinder A.C., Improved detection of bacteriaby flow cytometry using a combination of antibody andviability markers, J. Appl. Microbiol. 84 (1998) 577–584.

[75] Hennington E.W., Krocova Z., Sandström G., Forsman M.,Flow cytometric assessment of the survival ratio of Fran-cisella tularensis in areobiological samples, FEMS Microbiol.Ecol. 25 (1998) 241–249.

[76] Pyle B.H., Broadaway S.C., McFeters G.A., Sensitive detec-tion of Escherichia coli O157:H7 in food and water byimmunomagnetic separation and solid-phase laser cytom-etry, Appl. Environ. Microbiol. 65 (1999) 1966–1972.

[77] Pinder A.C., Purdy P.W., Poulter S.A.G., Clark D.C.,Validation of flow cytometry for rapid enumeration ofbacterial concentrations in pure cultures, J. Appl. Bacte-riol. 69 (1990) 92–100.

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