APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1992, p. 1801-18080099-2240/92/061801-08$02.00/0Copyright © 1992, American Society for Microbiology

Use of a Fluorescent Redox Probe for Direct Visualizationof Actively Respiring Bacteria

G. G. RODRIGUEZ, D. PHIPPS, K. ISHIGURO,t AND H. F. RIDGWAY*

Biotechnology Research Department, Orange County Water District,10500 Ellis Avenue, Fountain Valley, California 92728-8300

Received 14 January 1992/Accepted 14 March 1992

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was employed for direct epifluorescentmicroscopic enumeration of respiring bacteria in environmental samples. Oxidized CTC is nearly colorless andis nonfluorescent; however, the compound is readily reduced via electron transport activity to fluorescent,insoluble CTC-formazan, which accumulates intracellularly. Bacteria containing CTC-formazan were visual-ized by epifluorescence microscopy in wet-mount preparations, on polycarbonate membrane filter surfaces, or

in biofilms associated with optically opaque surfaces. Counterstaining of CTC-treated samples with theDNA-specific fluorochrome 4',6-diamidino-2-phenylindole allowed enumeration of active and total bacterialsubpopulations within the same preparation. Municipal wastewater, groundwater, and seawater samplessupplied with exogenous nutrients yielded CTC counts that were generally lower than total 4',6-diamidino-2-phenylindole counts but typically equal to or greater than standard heterotrophic (aerobic) plate counts. Inunsupplemented water samples, CTC counts were typically lower than those obtained with the heterotrophicplate count method. Reduction of CTC by planktonic or biofilm-associated bacteria was suppressed byformaldehyde, presumably because of inhibition of electron transport activity and other metabolic processes.

Because of their bright red fluorescence (emission maximum, 602 nm), actively respiring bacteria were readilydistinguishable from abiotic particles and other background substances, which typically fluoresced at shorterwavelengths. The use of CTC greatly facilitated microscopic detection and enumeration of metabolically active(i.e., respiring) bacteria in environmental samples.

Enumeration of metabolically active bacteria in environ-mental samples is frequently required to estimate systemproductivity, biomass turnover, or substrate utilizationpotentials. However, the detection of active microorganismsis often problematic, since no single analytical methodidentifies all physiological types of bacteria. Plate countmethods are often employed for this purpose, but they are

time consuming (because of required lengthy incubations)and typically do not provide useful information concerningmicrobial activity (or viability) in the absence of exogenousnutrient supplementation. Moreover, the bacteria are gener-

ally physically removed (by filtration or dilution) from thenative sample and are therefore no longer subject to possibleinhibitory substances or conditions that may limit theirmetabolic activity in situ. Plate count procedures cannot beused to directly observe active cells in situ, especially whenthe cells are attached to suspended particulate matter or

other solid surfaces.Iturriaga (5) employed the redox dye 2-(p-iodophenyl)-3-

(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) to di-rectly observe respirometrically active bacteria in aquaticenvironmental samples. This vital stain directly competeswith molecular oxygen as an artificial electron acceptor (1, 3,5, 9, 10, 17, 18). The reducing power generated by theelectron transport system converts INT into insoluble INT-formazan crystals, which accumulate in metabolically activebacteria (1, 5, 18). The INT-formazan may be observedmicroscopically with bright-field optics as opaque red intra-cellular deposits (1), or the formazan may be extracted with

* Corresponding author.t Present address: Kurita Water Industries, Ltd., 4-7, Nishi-

Shinjuku 3-chome, Shinjuku-ku, Tokyo 160, Japan.

ethanol or other organic solvents from filtered cells andmeasured spectrophotometrically as a quantitative assay

(13). Zimmermann et al. (18) first developed a technique forsimultaneously determining total and respiring bacteria byusing acridine orange for total bacterial counts and INT toenumerate active cells. Although the INT procedure hasproved extremely useful in many environmental studies, it isdifficult or impossible to visualize actively respiring cells onmembrane filters and other surfaces that are opticallyopaque. King and Parker (6) adopted the filter-transfer-freeze technique of Hewes and Holm-Hansen (4) to transfergroundwater bacteria captured on membrane filter surfacesto microscope slides. However, this approach adds a time-consuming step, and the efficiency of cellular transfer fromfilter to slide is uncertain. In addition, small bacteria con-

taining INT or bacteria that reduce INT at low rates may beeasily mistaken for abiotic debris, resulting in an underesti-mation of metabolic activity.Another direct technique for enumerating metabolically

active bacteria, the Kogure method, involves incubation of a

water sample amended with an assimilable carbon source inthe presence of nalidixic acid, a specific DNA gyrase inhib-itor that interferes with cell division in many gram-negativebacteria (6). Growing cells that respond to the antibioticbecome elongated or swollen and can be directly enumeratedmicroscopically to determine the actively growing subpop-ulation. However, the presence of filamentous or pleomor-phic cells in certain water samples may complicate interpre-tation of results obtained with the Kogure procedure.Furthermore, in the absence of an exogenously suppliedcarbon source, cell elongation may occur too slowly or maybe insufficient to measure accurately, precluding the appli-cation of this procedure in studies involving unamendedsamples.

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Lundgren (8) proposed a fluorescent method for directlydetermining the viability of soil bacteria based on the enzy-matic hydrolysis of the fluorogenic ester fluorescein diace-tate. However, many bacteria are evidently unable to trans-port fluorescein diacetate into the cell; the fluorescenceemission tends to be weak, and backgrounds tend to be highbecause of autofluorescence (at similar emission wave-lengths) of abiotic particles in many samples. Furthermore,the hydrolysis of fluorescein diacetate is primarily a functionof cellular esterase activity and, as such, does not necessar-ily provide useful information concerning the respiratorystatus or growth potential of a bacterium.

In recent years, novel ditetrazolium redox dyes that aresimilar to INT in molecular structure and function have beendeveloped. However, unlike INT, these new compoundsproduce fluorescent formazans when they are chemically orbiologically reduced. One such compound that has beensuccessfully applied in cytochemical and histochemical stud-ies is 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) (14, 15).Because of the fluorescent nature of its reduced formazan,which enhances detection and sensitivity, CTC has provensuperior to INT. The reduction of CTC is inhibited bycyanide and other electron transport inhibitors (14, 15). Thecompound is now commercially available in relatively highpurity, which diminishes the likelihood of side reactions thatmay lead to misinterpretation of fluorescence data or cellulartoxicity (14, 15). So far, CTC has only been employed as acellular redox indicator of respiratory (i.e., electron trans-port) activity in cytochemical experiments with Ehrlichascites tumor cells and has not been applied in bacteriolog-ical studies (14-16).

In this paper we describe the first application of CTC fordirect microscopic visualization of actively respiring bacte-ria in native and nutrient-amended environmental samples.This compound acts similarly to INT as a vital redox dye butis more easily detected intracellularly because of its brightred fluorescence when illuminated by longwave UV light(>350 nm). Moreover, the fluorescent nature of the com-pound greatly facilitates its use in studying actively respiringbacteria captured on dark membrane filters or bacteria inbiofilms associated with optically nontransparent surfaces.

MATERIALS AND METHODS

Chemicals and reagents. CTC was purchased from Poly-sciences, Inc. (Warrington, Pa.), and INT was purchasedfrom Sigma Chemical Co. (St. Louis, Mo.).

Collection of water samples. Secondary treated (activatedsludge) effluent was obtained from Orange County WaterDistrict Water Factory 21, an advanced municipal wastewa-ter reclamation facility located in Fountain Valley, Calif.Groundwater was obtained from Orange County WaterDistrict Deep Well 2, a blending well extracting from asemiconfined aquifer approximately 400 m beneath the landsurface. Coastal seawater was collected at the mouth ofUpper Newport Back Bay, Newport Beach, Calif. All sam-ples were collected aseptically in sterile 1-liter plastic bottlesand processed within 30 min.

Heterotrophic plate count. The numbers of viable bacteriapresent in water samples were determined by a standardheterotrophic plate count (HPC) procedure with R2A me-dium. This medium was developed by Reasoner and Gel-dreich (11) for the isolation and enumeration of bacteria in awide variety of nonmarine aquatic environments. Marinebacteria were enumerated on sterile filtered seawater sup-

plemented with agar (15 g/liter) and R2A medium (1:10dilution). All HPC plates were incubated aerobically.CTC reduction assay. Environmental samples were incu-

bated in triplicate with CTC at a concentration of 2.0 or 5.0mM in sterile test tubes. Two sets of samples were prepared;one was amended with a 1:2 dilution of R2A broth (desig-nated CTC+R2A), and the other was unamended (CTC).Killed controls were prepared by incubating identical cellsuspensions with formaldehyde (3.7%, wt/vol) for 5 minbefore CTC was added. Experimental and control prepara-tions were incubated for 4 h at 28°C with agitation (200 rpm).After incubation, samples were sometimes counterstainedwith 1.0 ,ug of the DNA-binding fluorochrome 4',6-dia-midino-2-phenylindole (DAPI) per ml by a modification ofthe procedure of Coleman (2). Counterstaining with DAPIallowed concurrent determination of total (i.e., viable plusnonviable) and respiring (i.e., cells exhibiting CTC-formazanfluorescence) cell counts in a single preparation. Stainedbacteria were captured by microfiltration through a 0.2-,um-pore-size black polycarbonate membrane filters (NucleporeCorp., Pleasenton, Calif.). Filters were air dried andmounted with low-fluorescence immersion oil (Resolve;Stephens Scientific, Division of Cornwell Corp., Debville,N.J.) on glass microscope slides. It was also possible toperform all CTC and DAPI staining operations after cellswere filtered onto the Nuclepore membrane surface. Prepa-rations were examined with the x 100 oil immersion fluores-cence objective of an Olympus Vanox model AHBT3 micro-scope equipped with a 200-W mercury burner. The filtercombination found to be most effective for viewing CTC-treated preparations consisted of a blue (420-nm) excitationfilter (Olympus model BP490) used in combination with a590-nm-barrier (cutoff) filter (Olympus model 0590). CTC-and DAPI-stained bacteria in the same preparation could beviewed simultaneously with a 365-nm-excitation filter(Olympus model UG1), an emission filter (Olympus modelY455), and a 400-nm-cutoff filter, although this filter combi-nation resulted in significantly reduced detection of CTC-stained cells and a corresponding underestimation of respir-ing bacteria (data not shown).

Effect of CTC and INT concentrations on redox activity.Experiments were performed to determine the optimumconcentration of CTC with a pure culture of Pseudomonasputida 54g, a hydrocarbon-degrading isolate previously iso-lated from groundwater in a shallow aquifer in Seal Beach,Calif. (10). Several concentrations of CTC, ranging from 0 to10.0 mM, were evaluated; all cell suspensions were am-mended with a 1:5 dilution of R2A broth. The starting celldensity for all preparations was 108 bacteria per ml (mea-sured by epifluorescence direct counts with DAPI [2]).Samples were incubated for 90 min at 28°C with agitation(200 rpm). After incubation, the CTC- or INT-formazan wasextracted by filtering 1.0 ml of sample through a 0.45-,um-pore-size membrane filter and then extracting with 2.5 ml ofabsolute ethanol. The relative amount of CTC-formazan inethanol extracts was determined by A450 with a Sequoia-Turner model 690 spectrophotometer. Relative INT-for-mazan in cells was determined at A480. The presence of theCTC-formazan was qualitatively confirmed by epifluores-cence microscopy.

Reduction kinetics of CTC and INT. The reduction kineticsof equimolar (5.0 mM) concentrations of INT and CTC werecompared with P. putida 54g as the test organism. Both theINT and CTC preparations were amended with a 1:5 dilutionof R2A broth. Two 125-ml Erlenmeyer flasks contained themagnetically stirred cell suspensions (108 cells per ml)

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FIG. 1. Epifluorescence photomicrograph of CTC-treated P. putida cells captured on 0.2-p.m-pore-size black Nuclepore membrane filtersurface (5.0 mM CTC+R2A; 30-min incubation). Bacteria are 1 to 2 pLm long.

amended with R2A medium and the respective redox dye. Atintervals, 1.0-ml aliquots were removed from each flask andfiltered through 0.45-,um-pore-size membrane filters. Therelative amounts of CTC-formazan and INT-formazan weredetermined as described above.

Effect of formaldehyde on CTC reduction. Glass micro-scope slides previously coated with a thin (50- to 100-nm)layer of polysulfone were submerged in coastal seawater(Upper Newport Back Bay, Newport Beach, Calif.) con-tained in a Plexiglass flow cell through which seawater wasrecirculated. After 3 days of continuous recirculation, theslides were removed and exposed to 2.0% (wt/vol) formal-dehyde prepared in filter-sterilized seawater for periodsranging from 15 s to 5 min. Residual formaldehyde wasremoved by three sequential changes of filtered seawater (5min each) and then incubated for 2 h in 5.0 mM CTCprepared in filtered seawater amended with 10% (vol/vol)R2A broth. After the incubation, the slides were rinsed twicewith filtered seawater and counterstained with DAPI for 30min (2). The slides were rinsed, air dried, and then observedby epifluorescence microscopy as described above.

RESULTS

Effect of CTC concentration. An environmental P. putidaisolate, strain 54g, previously recovered from a gasoline-contaminated aquifer (12) was used to determine the effect ofCTC concentration on the ability of the microorganism toreduce the compound. Actively respiring bacteria containing

the fluorescent CTC-formazan were clearly visible with UVoptics and epi-illumination (Fig. 1). A concentration of 2 to 6mM CTC resulted in optimal CTC-formazan deposition forthis particular microorganism and set of experimental con-ditions; higher concentrations resulted in lower CTC-for-mazan deposition within the cells (Fig. 2). The respirometricreduction of nonfluorescent INT, like that of CTC, exhibiteda concentration optimum (Fig. 2); however, the INT opti-mum occurred at a lower concentration (approximately 2.0mM [about 0.1%, wt/vol]). Formazan deposition was re-duced by approximately 50% at an INT concentration of 4.0mM, suggesting partial inhibition of electron transport activ-ity. Elucidation of the mechanism of inhibition was notwithin the scope of this investigation. The addition of 3.7%(wt/vol) formaldehyde to cell suspensions of P. putida 54gbefore CTC addition resulted in a time-dependent decreasein the number of red-fluorescing bacteria, presumably be-cause of inhibition of respiratory activity and other cellularprocesses (see below). Since CTC can be reduced by exog-enous chemical reductants (e.g., sodium dithionite) in theabsence of biological electron transport, the addition offormaldehyde provided a convenient negative control toaccount for any abiotic CTC reduction in experimentalpreparations. Similar inhibition of CTC reduction has beennoted after exposure of Pseudomonas diminuta to the elec-tron transport inhibitor sodium azide (13a).

Reduction kinetics of CTC and INT. At equimolar concen-trations of 5.0 mM, CTC and INT displayed similar kineticsof reduction by P. putida 54g (Fig. 3). The reduction kinetics

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0.8 -

001

CD

00O.

'tl1-04

CL0

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0 1 2 3 4 5 6 7 8

Concentration (mM)FIG. 2. Formation of CTC-formazan and INT-formazan by P. putida as a function of the initial concentrations of oxidized CTC and INT,

respectively. Ethanolic extracts of CTC-formazan and INT-formazan were determined by optical density at 450 and 480 nm, respectively (seeMaterials and Methods for details).

exhibited by each compound were curvilinear in nature andbiphasic, with an early period of rapid respirometric reduc-tion lasting for about 1 h. The initial rapid reduction phasewas followed by slower, more linear reduction (approxi-mately one-half the initial rate). At an INT concentration of

1.2-

1.0.-

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0.4 - #

0.2 /

0.0-

0.39 mM (0.02%, wt/vol), the concentration at which INT isnormally applied in most environmental studies (4-6, 17, 18),somewhat more linear reduction kinetics was observed (datanot presented). However, lowering the concentration ofCTC from 5.0 mM to 1.0 mM did not appreciably change its

0 50 100 150 200 250 300Minutes

FIG. 3. Kinetics of reduction of CTC and INT to their respective formazans by P. putida (see Materials and Methods for details). The CTCand INT data are from separate experiments that have been normalized to the fractional amount of compound reduction (ordinate) over thecourse of the experiment. An ordinate value of 0.5 corresponds to 50% of the maximum observed reduction. The initial concentrations of CTCand INT were 5.0 mM.

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DIRECT VISUALIZATION OF ACTIVELY RESPIRING BACTERIA

7.0

6.0-1.E 5.0

j 4.0

3 3.0

2.0

1.0

w ste-water

'total' 'respiring 'respiring' HPC+ substrate'

FIG. 4. Enumeration of bacteria in groundwater, seawater, and municipal wastewater by different methods. Bars: total, microscopicenumeration after DAPI staining; respiring + substrate, microscopic enermeration after treatment with CTC in the presence of 50% (wt/vol)R2A broth; respiring, microscopic enumeration after CTC treatment without substrate; and HPC, HPC on R2A medium (see Materials andMethods). The error bars are standard deviations of mean values based on triplicate samples.

reduction kinetics (data not shown). The curvilinear natureof CTC and INT reduction kinetics suggests partial inhibi-tion of respiration by the oxidized compounds or theirrespective formazans, although other explanations (e.g.,inhibition by impurities) are possible.CTC reduction with native water samples. A primary

objective of this study was to determine whether CTCreduction could be used to rapidly estimate the number ofactively respiring bacteria from different aquatic environ-ments. Water samples selected for study included municipalactivated-sludge effluent (wastewater), groundwater, andcoastal seawater (see Materials and Methods). Since therewas no apparent adverse effect of 5.0 mM CTC on thereduction kinetics of P. putida 54g (see above), this level ofreagent was selected for examination of native microfloraassociated with the three environmental samples.

Supplementation of water samples with R2A medium(50%, vol/vol) typically resulted in a substantial increase inthe number of red-fluorescent bacteria enumerated relativeto that of identical control preparations lacking nutrientsupplementation (Fig. 4). This observation suggests that thelack of oxidizable carbon substrates or other required nutri-ents in native water samples limited the in situ respiratoryactivity of resident bacteria. A similar stimulatory effect ofglucose (0.1%, wt/vol) supplementation on CTC reductionwas also observed (data not shown). It should be noted thatlittle or no background (i.e., abiotic) CTC reduction wasobserved in any of the water samples examined in thisinvestigation.

In the R2A-supplemented wastewater and seawater sub-samples, the CTC microscopic counts approximated theR2A plate (HPC) counts. In the deep-well aquifer sample,the CTC microscopic counts (with R2A supplementation)exceeded the R2A plate count by nearly 4 orders of magni-tude. These data suggest that the CTC-reduction methodmight provide a more sensitive indicator of viable (i.e.,actively respiring) bacteria in environmental samples thanthe standard HPC.

Significantly lower counts of actively respiring (i.e., red-

fluorescing) bacteria were obtained when CTC was em-ployed at a concentration of 2.0 mM instead of 5.0 mM (datanot shown). This suggested a possible dose-response rela-tionship between the CTC concentration and the number ofrespiring bacteria detected; however, this effect was notexplored further in this study. The elevated bacterial countsobserved with 5.0 mM CTC tended to substantiate selectionof this CTC concentration for the examination of the watersamples described above.

Visualization of respiring bacteria in biofilms. A distinctadvantage of CTC over other nonfluorescent tetrazoliumdyes such as INT is the direct visualization of respiringbacteria in biofilms associated with optically nontransparentsurfaces (e.g., wood, metal, plastic, etc.). An example offluorescent CTC-stained marine bacteria composing a bio-film on the surface of a thin polysulfone membrane substra-tum is shown in Fig. 5. The red-fluorescing (CTC-stained)bacteria were readily observed at a 365-nm excitation wave-length, even against the blue background autofluorescenceattributable to the polysulfone substratum. The biofilm bac-teria could also be readily counterstained with DAPI afterCTC treatment to allow enumeration of total and respiringbacteria within the same preparation (Fig. 5). Soaking ofbiofilm-coated coupons in 2.0% (wt/vol) formaldehyde be-fore CTC treatment strongly inhibited CTC reduction, pre-sumably by inactivation of electron transport and othermetabolic processes (Fig. 6). Thus, CTC appears to be auseful reagent for directly and rapidly evaluating the abilityof certain biocides to inactivate bacteria associated withattached biofilms.

DISCUSSION

Previous work with CTC has been limited to cytochemicalinvestigations of the electron transport activity of Ehrlichascites tumor cells (14-16). In those earlier studies it wasdemonstrated that CTC reduction was suppressed by spe-cific electron transport inhibitors such as cyanide. The dataprovided herein indicate that CTC can also be biologically

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FIG. 5. Epifluorescence photomicrograph of a marine biofilm formed on thin polysulfone substratum after treatment with CTC and DAPI(see Materials and Methods). The excitation wavelength was 365 nm, and a 455-nm barrier filter was used. The larger red cells areapproximately 2 ,um in size.

reduceiformazcroscolchemicpresen(primariclear

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d in respiring bacteria to a water-insoluble fluorescent tants in aerobic systems independently of biological electronan that is readily detectable by epifluorescence mi- transport. However, this possibility seems unlikely in thepy. It should be noted that CTC and INT can be studies reported here, since formaldehyde and azide bothally reduced in low-redox environments (e.g., in the suppressed CTC reduction and reduction tended to increasece of sodium dithionite); hence, their use is restricted strongly upon the addition of oxidizable carbon substratesily to aerobic or microaerophilic systems. It is not such as glucose or R2A broth.vhether CTC can be reduced by endogenous reduc- Intracellular deposition of the CTC-formazan appears to

be similar or identical to the deposition of INT-formazan;however, CTC-formazan is more stable and, because of itsred fluorescence, is more easily detected than INT-formazan(emission wavelength, approximately 602 nm). BecauseCTC-formazan fluoresces primarily in the red region of thevisible spectrum, it is readily distinguishable from mostbackground fluorescence and autofluorescing abiotic parti-cles, which typically emit in the blue or blue-green regions ofthe visible spectrum in most natural water samples. The

±1+ increased detectability of the fluorescent CTC-formazanfacilitated enumeration of very small bacteria and bacteriaexhibiting low electron transport activity with minimal dep-osition of the vital dye. In many bacteria, only a small(<0.2-pLm) region of CTC-formazan deposition could beobserved. In other instances, it was possible to differentiate

0 50 100 150 200 250 300 specific areas within a single bacterium where CTC-for-mazan deposition occurred. It is possible that the multiple

Contact Time (Seconds) sites of CTC-formazan deposition represent areas in cells

6. Inhibition of CTC reduction in marine biofilm by pre- where electron transport activity is occurring, although thisnt with 2.0% (wt/vol) formaldehyde. was not proven.

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Standard INT-formazan is recognized as opaque red orpink intracellular deposits under bright-field optics (1). How-ever, visual detection of these deposits may be difficult insamples containing abiotic particulate or colloidal materialresembling INT-stained bacteria. In bacteria <0.4 ,um insize, INT-formazan deposits have not been observed (18),and INT-formazan has been reported to be unstable and todissolve in immersion oil (6). Hewes and Holm-Hansen (4)and King and Parker (6) attempted to avoid the latterproblem by transferring stained bacteria from a filter surfaceto a microscope slide and employing glycerol instead ofimmersion oil as a mounting medium. The fluorescent CTC-formazan used in this investigation was not subject to theabove limitations, and fluorescence was stable in immersionoil preparations even after several days of storage at roomtemperature. It is noteworthy that CTC yielded good resultswith groundwater, which has been reported to be proble-matic in studies employing INT (6).Carbon supplementation (with R2A medium or glucose) of

most environmental samples was required to maximize thenumber of bacteria that could actively reduce CTC in theselected time frame. A similar stimulatory effect of glucosesupplementation on CTC reduction has been noted in coli-form bacteria introduced into a marine environment (14a).The enhancement effect of R2A or glucose supplementationmay reflect nutrient-limiting conditions associated with nat-ural aquatic environments, although other explanations arepossible.The observed suppression of CTC reduction activity in P.

putida at elevated reagent concentrations (>6.0 mM) mayreflect a toxic effect of the compound or possibly an impurityassociated with the commercial product. The apparent sup-pression of CTC reduction activity at high reagent concen-trations and the increased CTC counts observed with 5.0mM CTC relative to those with 2.0 mM CTC suggest thatthere may be an optimum level of CTC for enumeratingbacteria in environmental samples. The ability to detectgreater numbers of respiring bacteria at higher CTC concen-trations may be offset at some point by an inhibitory effect ofthe reduced or oxidized form of the compound on bacterialrespiration. Additional experiments will be needed to delin-eate more carefully the optimal reagent concentrations andto identify physiological and environmental factors thatinfluence compound reduction.The proportion of actively respiring bacteria detected with

CIC plus a substrate relative to the total cell count deter-mined by DAPI staining ranged from about 6 to 88%; thehighest proportion was observed in the groundwater sample,and the lowest proportion was observed in seawater. Inprevious studies in which INT reduction was used as ameasure of metabolic activity, the proportions of active (i.e.,respiring) bacteria ranged from 4 to 61% in the marineenvironment (17), 5 to 36% in freshwater systems (18), and 9to 25% in populations from lakes with the malachite green-INT method of Dutton et al. (3). The fraction of respiringbacteria seems to be variable and unpredictable, dependingon the particular sampling environment.The fluorescent nature of CTC-formazan permits for the

first time direct microscopic visualization of metabolicallyactive bacteria on dark membrane filter surfaces (e.g., blackNuclepore membranes). This unique feature allows directenumeration of metabolically active bacteria in filtered watersamples. Similarly, it is possible to observe actively respir-ing bacteria in biofilms associated with optically nontrans-parent solid surfaces. It may be possible with CTC tomonitor fluorometrically the electron transport activity of a

single immobilized bacterium. Other potential applicationsof this new fluorescent redox-sensitive probe include (i)rapid enumeration of actively respiring (i.e., viable) plank-tonic or attached bacteria in environmental samples, (ii)fluorometrically based toxicity determination, (iii) determi-nation of substrate utilization potentials and bacterial star-vation responses, (iv) rapid evaluation of biocide activity,and (v) determination of assimilable organic carbon. Most ofthese applications could be automated with a suitable fluo-rometer.The CTC reduction assay method described in this paper

proved to be a rapid and convenient procedure for determin-ing metabolically active bacteria by direct epifluorescencemicroscopy. It is not known whether all bacteria transportand reduce CTC equally well, or whether they exhibit similarsensitivities to the compound. A variety of coliform bacteria,including Escherichia coli, Salmonella typhimunum, andYersinia enterocolitica, have been observed to activelyreduce CTC under arctic marine conditions (14a) and torespond to carbon (glucose) supplementation. Experimentscurrently under way are designed to define more carefullythe limitations and applications of CTC for enumeratingmetabolically active microorganisms in environmental sam-ples.

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