Vol. 47, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1984, p. 160-1660099-2240/84/010160-07$02.00/0Copyright © 1984, American Society for Microbiology

Bacteriological Analysis of Water by Potentiometric Measurement ofLipoic Acid Reduction: Preliminary Assays for Selective Detection

of Indicator OrganismsGUY CHARRIERE,1 THIERRY JOUENNE,1 JEAN-FRANCOIS LEMELAND,2 ERIC SELEGNY,3 AND GUY-ALAIN

JUNTER3*Laboratoire Municipal d'Hygiene, 76600 Le Havrel; Laboratoire de Microbiologie, U.E.R. de Medecine-Pharmacie, 76800Saint-Etienne-du-Rouvray2; and Laboratoire de Chimie Macromolculaire, E.R.A. 471, Faculte des Sciences de Rouen,

76130 Mont-Saint-Aignan3, France

Received 6 July 1983/Accepted 3 October 1983

The practical task of adapting an original potentiometric technique to the bacteriological analysis of wateris discussed. Various laboratory strains of organisms belonging to the usual aquatic flora were inoculatedone by one in a minimal lactose broth supplied with lipoic (thioctic) acid. The time evolution of the redoxpotential of the cultures was followed during incubation by combined gold versus reference electrodes.When the incubation temperature was regulated at 36°C, most organisms were able to grow and to reducethe coenzyme, generating changes in the redox potential of the culture. However, very few organismsdeveloped significant reductive activity when the temperature was increased to 41°C and when the brothwas provided with sodium deoxycholate. Among the fecal coliform organisms, only Escherichia coli andKlebsiella pneumoniae exhibited early but reproducible potential-time responses. Positive potentiometricresponses were also recorded with Acinetobacter calcoaceticus. E. coli showed rapid potentiometric signalsas compared with K. pneumoniae. The time required for a 100-mV shift of potential to be detected wasrelated to the logarithm of the initial concentration of E. coli or K. pneumoniae in the culture broth.Experiments on natural surface water samples showed that the potentiometric method, associated with theselective incubation conditions, mainly detected E. coli among the bacterial flora of the tested environmen-tal water. The calibration curve relating the time required for a 100-mV shift of potential to be detected tothe number of fecal coliforms, as determined by control fecal coliform-selective plate counts, was consistentwith the composite standard curve of detection times obtained with six different laboratory strains of E. coli.No false-positive reading due to the aquatic bacterium A. calcoaceticus could be elicited.

In previous papers (4-7), we have described the theoreti-cal bases and the experimental standardization of an electro-chemical method suitable for detection and enumeration ofbacteria (4) or for monitoring their responses to drugs (5).Redox potential as a function of time was measured withcombined gold versus reference electrodes in a minimalculture broth supplied with exogenic lipoic (thioctic) acid.During bacterial growth, lipoic acid is reduced into dihydro-lipoic acid, and the lasting appearance of the reduced form inthe medium makes the zero-current potential of the goldelectrode fall rapidly. A potential "wave" attaining severalhundred millivolts is observed. For more sophisticated anal-yses of antibacterial effects of drugs, the full time-course ofpotential had to be recorded (5). However, for routinedetection and counting of organisms, it was sufficient tomeasure the time that elapsed between inoculation and theappearance of a preselected potential shift chosen on thewave: an arbitrary but sufficient 100-mV potential shift waschosen as the practical lag time [t(100 mV)] (4). A theoreticallogarithmic relationship between the lag time and the inocu-lum size was obtained from the law expressing dissolvedoxygen consumption by bacteria (4). This relationship hasbeen standardized with a single laboratory strain of Esche-richia coli (4).Under the standard culture conditions used previously,

many bacterial species were able to reduce exogenic lipoicacid and produce potential-time signals without any specific-ity; the method was useful for fundamental investigations

* Corresponding author.

with single bacterial strains or for sterility control, but notfor selective analysis. In this paper, we present new experi-mental results that allow the further application of thepotentiometric method to practical measurements of bacteri-al contamination of water. To promote selective growth ofspecific bacterial indicators offecal contamination (i.e., fecalcoliforms), we modified the incubation conditions during thepotentiometric measurements. Such a procedure has beenfollowed recently by different authors who proposed newtechniques for detecting and counting pollution indicators inwater, e.g., the potentiometric detection of hydrogen gasproduction (18), the measurement of changes in electricalimpedance (14), the gas chromatographic analysis of meta-bolically produced ethanol (11), or the colorimetric measure-ment of o-nitrophenyl-,3-D-galactopyranoside hydrolysis(16).

Respecting these more selective conditions of incubation,we successively (i) investigated the "potentiometric behav-ior" of laboratory strains of bacterial species representativeof the environmental aquatic flora, (ii) calibrated the rela-tionships between lag times t(100 mV) and fecal coliformindicator concentrations, and finally (iii) tested the techniqueon natural samples collected from environmental water.

MATERIALS AND METHODSOrganisms. Bacteria representative of the natural flora of

water (Table 1) were obtained from the American TypeCulture Collection, Rockville, Md., the Town Hygiene Lab-oratory Collection, Le Havre, France, the Microbial Eco-

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TABLE 1. Potentiometric detection times t(100 mV) for variousbacterial species growing under different incubation conditions

Incubation conditions'Organism Origina 410C 41°C + sodium

deoxycholate

Acinetobacter calcoace- THLC +++ +++ + ++ticus

Acinetobacter sp. strain MEUC + --B5W

Aeromonas dourgesi MEUC - --Aeromonas hydrophila MEUC - --Bacillus pumilus PIC +++ +++ +Bacillus subtilis A-TCC + + + + + + AButtiauxella agrestis MEUC ++++Citrobacter freundii THLC + + + +Enterobacter amnigena MEUC +++ - -Enterobacter cloacae ATCC +++ ++ ++Enterobacter hafniae THLC +++ + +Enterobacter intermedium MEUC ++++Escherichia coli THLC +++ +++ + ++Flavobacterium sp. MEUC ++ - -Klebsiella oxytoca THLC +++ ++ + + +Klebsiella pneumoniae THLC +++ +++ +++Klebsiella terrigena MEUC +++ - -Levinea amalonatica THLC +++ + + +Levinea malonatica THLC +++ +Micrococcus sp. PICPseudomonas aeruginosa MEUC + +Pseudomonas fluorescens MEUCPseudomonas putida MEUC + - -Pseudomonas putrefa- PIC +

ciensRahnella aquatilis MEUC ++ - -Serratia fonticola MEUC +++ - -Serratia marcescens MEUC +++ - -Staphylococcus aureus PIC +++ +Staphylococcus capitis MEUC + +Staphylococcus saprophy- MEUC + +

ticusStaphylococcus sp. strain PIC ++ +Oxford

Streptococcus faecalis ATCC +++ +++ +Yersinia enterocolitica THLC + + --Yersinia frederiksenii THLC + + + + +

a Abbreviations: THLC, Town Hygiene Laboratory Collection,Le Havre, France; ATCC, American Type Culture Collection,Rockville, Md.; MEUC, Microbial Ecotoxicology Unit Collection,Lille-, France; PIC, Pasteur Institute Collection, Lille, France.

b +++, 5 h < t(100mV) < 10h; ++, 10h < t(100mV) < 15 h;+,15 h < t(100 mV) < 20 h; -, t(00 mV) > 20 h.

toxicology Unit Collection, Institut National de la Santd etde la Recherche Mddicale unit no. 146, Lille, France, and thePasteur Institute Collection, Lille, France.Inoculum preparation and calibration. (i) Pure laboratory

cultures. All organisms tested were cultured at 36°C onordinary agar plates (nutrient agar; Difco Laboratories,Detroit, Mich.). Inocula for the potential-time measurements(0.2-ml volume) were prepared by suspending the bacteria insterile distilled water after incubation for 24 h.

Populations of the inocula were estimated by plating out 1-ml portions of appropriate 10-fold dilutions, incubating for 24h at 36°C, and counting the colonies pn Difco Plate CountAgar. Each viable cell count was the average of threereplicates of the dilution yielding 30 to 300 colonies per plate.

In preliminary experiments, initial cell concentrationsranging from 5 x i07 to 5 x fo8 organisms per liter were usedto test the potentiometric responses of organisms repre-

sentative of the natural aquatic flora. At least six experi-ments were carried out for each species.The calibration curves converting potentiometric lag times

to initial E. coli or Klebsiella pneumoniae concentrationswere constructed by using a wider range of inocula obtainedby successive 10-fold dilutions of the mother cell suspen-sion.

(ii) Natural samples. Natural samples were collected at thesurface of the Saint-Laurent, a river in the suburbs of LeHavre, France, receiving untreated sewage effluents. Thenumber of fecal coliforms in each sample was determined byplate counts on a standard coliform-selective, deoxycholate-lactose agar (Difco) and by spreading 1-ml portions ofappropriate dilutions and incubating for 24 h at 44°C. Thecell concentration in each sample was given by the averageof three experiments. Successive 10-fold dilutions of thenatural water samples in sterile distilled water were used asinocula.Most experiments were carried out by using 0.2-ml inocu-

la. To gain higher cell concentrations in the flasks, weinoculated larger volumes (i.e., 30 ml) in some experiments.These larger inocula were pre-equilibrated with sterilized airat 41°C before inoculation into a more concentrated medium.

Substrates and growth conditions. The potential-time mea-surements were carried out during bacterial growth in aselective broth modified from the minimal medium describedpreviously (4). The minimal medium was enriched by yeastextract (Difco) to provide a concentration of 2 g/liter,sterilized by autoclaving (20 min at 120°C), and supplement-ed with sterile solutions of lactose (4 g/liter) and lipoic acid (2mg/liter; E. Merck AG, Darmstadt, West Germany). Aspecific inhibitor of gram-positive bacteria, i.e., sodiumdeoxycholate (0.5 g/liter; Merck) was also added in mostexperiments as specified in the text.

Cultures were incubated at either 36 or 41°C in the 50-mlErlenmeyer-type flasks already described (4). For experi-ments on laboratory strains, the flasks contained 35 ml ofsingle-strength medium. For experiments on environmentalwater, the flasks contained either 35 ml of single-strengthmedium (0.2-ml inocula) or 5 ml of a septuple-strengthmedium (corresponding to the maximal possible concentra-tion of salts, substrates, and reagents in water) to which 30-ml inocula were added. The flasks were placed in a recipro-cal water bath shaker (type S.B.K.25, Salvis) (agitationspeed, 85 rpm; stroke, 15 mm).

Before inoculation, the initial oxygen content of the medi-um was standardized by equilibration with sterilized air atthe temperature of the experiments, providing oxygen con-centrations of 2.2 x 10-4 M (36°C) or 2.0 x 10-4 M (410C).The experimental conditions controlling oxygen transferfrom residual air in the flasks to the culture medium (4, 7)were maintained constant during all potential-time measure-ments.

Isolation and identification. All flasks supplied with naturalwater inocula and exhibiting bacterial growth were kept forisolation and identification of the responsible gram-negativebacilli by means of the standard methods used routinely inthe Le Havre water control laboratory.The flasks were streaked onto bromocresol purple lactose

agar (Pasteur Institute Production, Paris), and plates wereincubated for 24 h at 37°C. Isolates were picked off (up tofour on every plate), and both were transferred to Kliger ironagar slants (Pasteur Institute Production) and streaked ontonutrient agar (Difco).

Isolates obtained on Kliger iron agar after 24 h of incuba-tion at 37°C were further differentiated by indole, methyl

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162 CHARRIERE ET AL.

red, Voges-Proskauer, citrate biochemical tests, by screen-ing through the API-20 Enteric System (Analytab Products,Plainview, N.Y.), and by the ability of the organisms toproduce gas in brilliant green lactose bile broth (Difco) andto produce indole in peptone water after 24 h of incubation at44°C (Mackenzie test).The colonies which were isolated on nutrient agar after 24

h of incubation at 37°C were subjected to the standardoxidase test (filter paper disks impregnated with a 1%aqueous solution of N,N,N',N'-tetramethyl-p-phenylenedia-mine dihydrochloride) to confirm the absence of oxidase-positive organisms.

Potentiometric measurements. The experimental setup forpotential-time measurements has already been described (4).

RESULTSPotentiometric responses of different organisms repre-

sentative of the aquatic flora. As a first step, we inoculatedthe minimal lactose-yeast extract medium with various labo-ratory cultures of organisms belonging to the natural flora ofwater and investigated the potential-time responses in differ-ent incubation conditions, i.e., at 36, 41, or 41°C, withsodium deoxycholate added. As reported previously (4), thepresence of yeast extract caused the potential waves toflatten, but the waves kept their reproducibility and suffi-cient amplitude, and the t(100 mV) lag time values weresufficient to distinguish the positive from the negative re-sponses. The potentiometric responses of organisms willalways be expressed by means of these lag times.The results obtained are summarized in Table 1. The

choice of the selective incubation conditions for furtherexperiments was based on these results.

(i) Nonpsychrophilic coliforms. The organisms usually des-ignated as nonpsychrophilic coliforms exhibited dissimilarpotentiometric behaviors. All of them gave significant poten-tial-time responses at 36°C, but the development of hostilegrowth conditions led to variable and extended t(100 mV)responses. Only E. coli and K. pneumoniae remained practi-cally unaffected by the increase in incubation temperatureand the presence of sodium deoxycholate.

(ii) Psychrophilic coliforms. These nonfecal speciesshowed homogenous potentiometric responses. An incuba-tion temperature of 41°C caused the wave of potential todisappear. The only exception was Yersinia frederiksenii,giving late signals at 41°C that were further delayed bysodium deoxycholate.

(iii) Gram-negative bacteria other than members of thefamily Enterobacteriaceae. Such organisms commonly belongto the autochthonous aquatic flora. Since they are psychro-philic or nonglucidolytic species, they gave late signals at36°C and no potentiometric response at 41°C with or withoutsodium deoxycholate. A noticeable exception to this behav-ior was Acinetobacter calcoaceticus which exhibited poten-tiometric responses in the range of the t(100 mV) timesrecorded with E. coli and K. pneumoniae.

(iv) Gram-positive bacteria. Various types of potentiomet-ric behavior were observed at 36 and 41°C, according to thethermoresistance and the glucidolytic activity of organisms,but all bacteria tested gave negative potential-time responseswhen incubated in the presence of sodium deoxycholate.

Calibration curves for pure E. coli and K. pneumoniaecultures. We then studied, as a function of the inoculum size,the potential-time responses of E. coli and K. pneumoniaeunder the most hostile incubation conditions (41°C in thepresence of sodium deoxycholate). The calibration curves,

relating t(100 mV) to initial cell concentrations in the culturemedium, are shown in Fig. 1 to 3.

(i) E. coli. The results of 85 potential-time measurementswith a single strain of E. coli are depicted in Fig. 1. Theregression line, correlating the t(100 mV) value to thelogarithm of the initial concentration of organisms, followedthe equation:

t(100 mV) = -85.7 log x0 + 968 (1)

where t(100 mV) is given in minutes, and x0 represents theinitial cell concentration in the culture broth (cells per liter).The value of r was 0.989.We investigated the influence of the strain on the potentio-

metric responses of E. coli. The results obtained with sixdifferent strains of E. coli are given in Fig. 2. For each straintested, a linear relationship between log x0 and detectiontimes t(100 mV) existed. Values of r ranged from 0.954 to0.999, but the slope of the straight line was dependent on thestrain used. The least-squares method, applied to the totalityof the experimental values (about 200 data points) withoutreference to the strain tested, led to a composite standardcurve of detection time for E. coli which corresponded to theequation:

t(100 mV) = -83.0 log x0 + 930 (2)

where r is 0.967.Such a composite straight line was representative of

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0 1 2 3 4 5 6 7 8 9 10 11FIG. 1. Standard curve of detection times t(100 mV) for a single

laboratory strain of E. coli. Symbols: 0, experimental values;least-squares line.

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POTENTIOMETRIC DETECTION OF INDICATOR ORGANISMS 163

LOG INITIAL CELL10

NUMBER/ LITER

0 l 2 3 4 5 6 7 8 9 l0 1 12FIG. 2. Calibration curves of detection times t(100 mV) for

different laboratory strains of E. coli. Slopes (in minutes perlogarithmic unit) and values of r for the lines fitted by the least-squares method are as follows: 1, -60.5, 0.954; 2, -68.2, 0.992; 3,-78.8, 0.999; 4, -74.2, 0.987; 5, -93.7, 0.999; 6, -90.9, 0.981.Symbol: ----, composite calibration curve of detection times for E.coli.

"average behavior" of the bacterial species, E. coli. Thecorresponding average generation time was obtained fromthe slope as follows.A theoretical relationship between t(100 mV) and x0 has

been proposed previously (4):

t(100 mV) = C +og2 log 1 + [02]0ln 2 (3)

where p is the generation time of bacteria, [0210 iS the initialdissolved oxygen concentration in the culture broth, Qm isthe maximum specific oxygen uptake rate, and C is a

constant depending on the growth conditions.At x0 values inferior to 109 cells per liter, [02]0 ln 21Q,,pxo

>> 1, and equation 3 is reducible to:

t(100 mV) = C' - lP log xOlog 2

the potentiometric method to the detection and counting ofbacteria in natural water, it appeared advisable to determinethe potential-time responses of some mixed cultures inselective growth conditions to detect possible interferences.

(i) Mixed cultures of E. coli and K. pneumoniae. Detectiontimes t(100 mV) recorded in mixed cultures of E. coli and K.pneumoniae are given in Table 2. When the initial cellnumber in the culture medium was much higher for K.pneumoniae than for E. coli (e.g., 100 K. pneumoniae for 1E. coli), the potentiometric signal was clearly attributable toK. pneumoniae. For lower proportions of K. pneumoniae inthe mixed inoculum, E. coli dominated, and its potentiomet-ric responses were not significantly perturbed by the pres-ence of K. pneumoniae.

(ii) Influence of gram-positive bacteria. Gram-positive or-ganisms had no noticeable influence on the potential-timeresponses of E. coli, whatever their relative proportion in themixed inoculum (Fig. 4).

Potentiometric responses elicited by natural water samples.To test the practicality of the method under actual fieldconditions, we carried out potential-time measurements inselective medium inoculated with samples of natural surfacewater. A total of 75 potentiometric measurements corre-sponding to 33 natural samples were performed, and thet(100 mV) values were related to the logarithm of the initialconcentration of fecal coliforms as determined by a controlplating method. The experimental data and the correspond-

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where C' is a constant.Multiplying the slope of the correlation line by log 2 gave

the generation time (p) of 25.0 min, i.e., 5 min more than inthe previously used (more stimulative) growth conditions(4).

(ii) K. pneumoniae. The calibration curves correspondingto two different strains of K. pneumoniae are shown in Fig.3. In agreement with the results relative to E. coli, the slopeof the correlation line changed from one strain to another.Other potential-time measurements were carried out withdifferent strains of K. pneumoniae; the greatest part of thecorresponding t(100 mV) fell as a function of log x0 betweenthe two straight lines of Fig. 3.

Potential-time measurements in mixed cultures (laboratorystrains). The preceding results were obtained with purelaboratory cultures of organisms belonging to the flora ofenvironmental water. Considering a further application of

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experimental values; -, least-squares lines. Slopes (in minutes perlogarithmic unit) and values of r are -88.4 and 0.967 (0), and-117.5 and 0.987 (-).

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164 CHARRIERE ET AL.

TABLE 2. Detection times t(100 mV) for mixed cultures of E.coli and K. pneumoniae (the same strains were used for all

experiments)Log xo (cells per liter)

t(100 mV) (min)E. coli K. pneumoniae

7.32 3209.38 160

7.32 9.38 1657.23 315

8.46 3307.23 8.46 3307.32 300

7.45 4257.32 7.45 3157.26 310

6.31 4957.26 6.31 325

ing least-squares line are depicted in Fig. 5. The correlationline had the following equation:

t(100 mV) = -84.2 log x0 + 852 (5)

where r is 0.955.The average concentration offecal coliforms in the natural

samples tested was 1.66 x 105 cells per liter. Diluting andinoculating appropriate volumes of diluted surface watergave initial cell concentrations in the flasks ranging fromabout 30 organisms per liter to 5 x 105 organisms per liter, ascalculated from the cell concentration of the mother suspen-sion of organisms in natural water (Fig. 5).

All contaminated flasks exhibited positive bacterialgrowth. A total of 183 bacterial strains were isolated fromthese flasks and identified as E. coli (172 strains), K.pneumoniae (10 strains), and A. calcoaceticus (1 strain). Theisolated strains of K. pneumoniae and A. calcoaceticus werealways accompanied by E. coli.

DISCUSSIONSince exogenously supplied lipoic acid can be reduced by

various species of microorganisms, the only means for ourpotentiometric method to gain any specificity was to makethe incubation conditions more selective than during theprevious standardization experiments (4).

In the present work, we directed our efforts toward theadaptation of the growth conditions to the selection of thebacterial indicators of fecal pollution. Glucose was replacedby lactose as the only sugar in the medium, and the growthmedium was supplied with sodium deoxycholate, a bile saltinhibiting most gram-positive bacteria capable of fermentinglactose. Less classical was the choice of 41°C for theincubation temperature. Manual detection of fecal coliformdensities by traditional pour plate techniques or most-proba-ble-number procedures is usually carried out at a higherincubation temperature, e.g., 44 or 44.5°C. In the same way,the recently developed nonconventional techniques unani-mously enumerate coliform bacteria of fecal origin at 44.5°C(11, 14, 16, 18). Our choice of 41°C was based on resultsreported by Gavini et al. (2), who showed by biophotometricmeasurements that members of the family Enterobac-teriaceae of human or fecal origin exhibited a higher growthrate at 41 than at 44°C, whereas autochthonous Enterobac-teriaceae of water, which are psychrophilic, were unable togrow at 41°C. Moreover, Reasoner et al. (12) noticed that

stressed fecal coliforms unable to grow when incubated at44.5°C were recovered by lowering the incubation tempera-ture to 41.5°C. Warren et al. (16) also reported that thegrowth and o-nitrophenyl-p-D-galactopyranoside hydrolysisexerted by different strains of E. coli were increased bylowering the classical temperature of 44.5°C by 1 or 2°C.The results obtained with these new selective growth

conditions divided the organisms tested into two maingroups: (i) metabolically inactive bacteria, giving infinitet(100 mV) (no potentiometric signal); (ii) organisms able todevelop a metabolic activity even in the most hostile incuba-tion conditions, exhibiting early and reproducible signals.

(i). The first group is obviously larger. All gram-positivebacteria were inhibited, essentially on account of the pres-ence of sodium deoxycholate. All thermosensitive (psychro-philic) species were also inhibited: lactose-positive Entero-bacteriaceae of aquatic origin (i.e., nonfecal coliforms) andgram-negative bacteria other than Enterobacteriaceae (i.e.,the main autochthonous organisms of water).The potentiometric behavior of Pseudomonas aeruginosa

is of particular interest. The role of P. aeruginosa as anopportunistic pathogen is well known, and its use as an indexof fecal pollution has been suggested (3, 8). This organismusually offers a quite good resistance to heat; however,under our growth conditions, it produced late signals at 36°Cand no potentiometric response at 41°C.

(ii). Although very limited, the second group of organisms,exhibiting positive potential-time responses, is of the highestimportance with regard to the significance of the potentio-metric technique. Only three species were found to belong tothis group: E. coli, K. pneumoniae, and A. calcoaceticus.

E. coli exhibited its classical high resistance to a highincubation temperature in the presence of bile salts. Thepotentiometric responses of this organism were stronglydependent on the strain. The slope of the regression linerelating t(100 mV) to log x0 varied from -60.5 min perlogarithmic unit to -93.7 min per logarithmic unit, whichcorresponded to generation times (p) ranging from 18 to 28min. Such growth rate differences between strains of fecalcoliforms in environmental water have also been elicited by

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FIG. 4. Effect of gram-positive bacteria on the potentiometricresponses of E. coli (strain corresponding to curve 1 in Fig. 2).Constant initial concentration of each gram-positive organism in theculture broth was 3.6 x 107 cells per liter. Symbols: ----, calibrationcurve for E. coli alone; A, experimental t(100 mV) recorded inmixed cultures of E. coli, Bacillus pumilus, Bacillus subtilis, andStreptococcus faecalis as a function of the initial E. coli concentra-tion.

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POTENTIOMETRIC DETECTION OF INDICATOR ORGANISMS 165

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FIG. 5. Potential-time responses of inocula obtained from envi-ronmental surface water, detection times t(100 mV) as a function ofthe initial concentration of fecal coliform organisms. Symbols: 0,

experimental values; -, least-squares line.

different techniques, e.g., impedance measurements (14).The least-squares analysis of the whole t(100 mV) values ledto a straight line with a reasonably good value for r (0.967)that substantiated a statistically good distribution of thepotentiometric responses for strains chosen at random.Among the other fecal coliforms, only K. pneumoniae

could be compared with E. coli with regard to its potential-time signals. This organism exhibited positive potentiomet-ric responses, although these were delayed in comparisonwith E. coli t(100 mV). The calibration curves had strain-dependent slopes with r values close to 1. The correspondinggeneration times, ranging from 27 to 35 min, were clearlylonger than for E. coli. This difference could be attributed toa greater sensitivity of K. pneumoniae to sodium deoxycho-late, as the generation times of the two species looked to becloser without the inhibitor.The potentiometric responses of mixed cultures of E. coli

and K. pneumoniae were clearly attributable to E. coli whenboth organisms had the same initial concentration in thebroth but attributable to K. pneumoniae when the initial cellproportion was of, e.g., 1 E. coli for 100 K. pneumoniae. It isunlikely to find such relative proportions of E. coli and K.pneumoniae in most environmental waters. Thus, E. coli isthree or four logarithmic units more numerous than K.pneumoniae in human feces (10), and their relative propor-tions are usually unchanged in recently contaminated water.However, K. pneumoniae may be found in high numbers inwaters with high organic nutrient status, e.g., in paper milleffluents (1, 9). Under such circumstances, the possibledetection of K. pneumoniae alone (without E. coli) would beof interest for the health of the public because of thepotential pathogenicity of this organism. It would give

evidence of a particular ecological state, i.e., high organicpollution, where the fecal origin would be more doubtful.

All other fecal coliform species tested gave nonreproduci-ble potential-time responses that were delayed as comparedwith E. coli or K. pneumoniae. Potentiometric responses ofthese organisms were intermediate between the responses ofE. coli and K. pneumoniae and the group of psychrophiliccoliforms. These organisms occasionally belong to the hu-man fecal flora, but they occur at low numbers, i.e., closer tothe number of K. pneumoniae than to E. coli (10), and arelikely to be concealed by E. coli or eventually K. pneumo-niae during the potential-time measurements in natural wa-ter.A. calcoaceticus is an aquatic bacterium, the potential

pathogenicity of which is well known. This organism gavepotentiometric responses surprisingly similar to those of E.coli or K. pneumoniae. Trinel and Leclerc (15) have alreadyreported such a noninhibited growth of A. calcoaceticus insemiselective culture conditions (buffered lactose broth at41°C) used for the specific enumeration of E. coli in water.From the data in the literature, A. calcoaceticus does notbelong to the fecal flora (10) and is not considered a pollutionindicator organism. Its specific detection in environmentalwater would induce false-positive information on the fecalcontamination of water, even if, as in the case of K.pneumoniae, it would be significant for public health accord-ing to its potential pathogenicity.

Results obtained from analysis of natural water samplesfor fecal coliforms by the lipoic acid potentiometric methodwere consistent with results from laboratory experimentswith pure and mixed bacterial cultures. Only E. coli, K.pneumoniae, and A. calcoaceticus present in the surfacewater samples gave positive responses in the potentiometrictest. Of 183 bacterial strains isolated from flasks afterpotentiometric measurements, 172 (94%) were E. coli, 10(5.5%) were K. pneumoniae, and 1 (0.5%) was A. calcoace-ticus. E. coli was isolated from all samples that containedeither K. pneumoniae or A. calcoaceticus. Thus, the newincubation conditions allowed a satisfactory selective isola-tion and enumeration of E. coli, whereas there was no false-positive reading due to the aquatic bacterium, A. calcoace-ticus, alone. It must be noted that if A. calcoaceticus alonewere detected during experiments on environmental water(which is unlikely), it would be possible to increase theincubation temperature by 3 or 4°C. Potential-time measure-ments carried out in cultures of E. coli or A. calcoaceticushave shown (unpublished data) that a temperature of 44°Cdelayed appreciably the potentiometric responses of A.calcoaceticus without greatly lengthening the t(100 mV)detection times for E. coli.The different calibration curves presented in this paper

were obtained by least-squares analysis of experimental datawhich did not include low cell concentrations. The totalvolume of the incubation broth was 35 ml per measurement,and consequently, the minimal cell concentration tested wasabout 30 organisms per liter. This is clearly much higher thanthe concentrations found in disinfected water suitable fordrinking, for example, where the maximum E. coli concen-tration permitted is 1 bacterium per 100 ml of water (19).Preliminary ultrafiltration procedures or inoculum volumesincreased to 100 ml or more would be necessary to obtainstatistically significant results with lower cell concentra-tions. However, by extrapolating the least-squares line ofFig. 5 to low cell concentrations, we can estimate that 1 E.coli per 100 ml of surface water would be detected within 15h. Such a value is in the range of the detection times needed

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166 CHARRIERE ET AL.

by other recently developed enumeration techniques (13, 14,17). This estimation is now submitted for experimentalproof.

In conclusion, the selective incubation conditions adoptedensure sufficient predominance of the potential-time re-sponses of E. coli and adequately short detection times forthis organism. The potentiometric measurement of lipoicacid reduction can therefore be used for the selective detec-tion and enumeration of E. coli in environmental watersamples.Our present interest, in addition to further measurements

on surface water, is focused on deep or disinfected waterwith very low bacterial contaminations. The results of thesecomplementary investigations will be reported in a laterpaper.

ACKNOWLEDGMENTSThis work was supported by the French National Institute of

Health and Medical Research (Institut National de la Santd et de laRecherche Medicale) grant no. 810151.We thank the technical staff of Le Havre Town Hygiene Labora-

tory, more especially L. Bertin, for helpful assistance.

LITERATURE CITED1. Caplenas, N. R., M. S. Kanarek, and A. P. Dufour. 1981.

Source and extent of Klebsiella pneumoniae in the paperindustry. Appl. Environ. Microbiol. 42:779-785.

2. Gavini, F., B. Marais, G. Leroy, D. Izard, and H. Leclerc. 1980.Essai de differentiation des souches d'Enterobacteriaceae enfonction de leur origine, Zentralbl. Bakteriol. Parasitenkd.Infektionskr. Hyg. Abt. 1 Orig. Reihe C 1:188-192.

3. Highsmith, A. K., and R. L. Abshire. 1975. Evaluation of amost-probable-number technique for the enumeration of Pseu-domonas aeruginosa. Appl. Microbiol. 30:596-601.

4. Junter, G.-A., J.-F. Lemeland, and E. Selegny. 1980. Electro-chemical detection and counting of Escherichia coli in thepresence of a reducible coenzyme, lipoic acid. Appi. Environ.Microbiol. 39:307-316.

5. Junter, G.-A., J.-F. Lemeland, and E. Selegny. 1982. Evolutionwith time of the zero-current potential of a gold electrode inEscherichia coli cultures supplied with lipoic acid. Part II.Properties of the model and their application to investigations

on drug effects on bacterial activity. Bioelectrochem. Bioenerg.9:699-709.

6. Junter, G.-A., E. Selegny, and J.-F. Lemeland. 1979. Analysetheorique des variations de potentiel dans les cultures deEscherichia coli K 12 en presence d'un transporteur d'elec-trons. Ann. Microbiol. (Paris) 130A:295-313.

7. Junter, G.-A., E. Selegny, and J.-F. Lemeland. 1982. Evolutionwith time of the zero-current potential of a gold electrode inEscherichia coli cultures supplied with lipoic acid. Part I.Mathematical modeling and computer simulation. Bioelectro-chem. Bioenerg. 9:679-697.

8. Kenner, B. A., and H. P. Clark. 1974. Detection and enumera-tion of Salmonella and Pseudomonas aeruginosa. J. WaterPollut. Control Fed. 46:2163-2171.

9. Knittel, M. D., R. J. Seidler, C. Eby, and L. M. Cabe. 1977.Colonization of the botanical environment by Klebsiella isolatesof pathogenic origin. Appl. Environ. Microbiol. 34:557-563.

10. Leclerc, H. 1977. Sur les methodes d'etude de la contaminationbacterienne de la mer. Rev. Int. Oceanogr. Med. 47:113-122.

11. Newman, J. S., and R. T. O'Brien. 1975. Gas chromatographicpresumptive test for coliform bacteria in water. Appl. Microbi-ol. 30:584-588.

12. Reasoner, D. J., J. C. Blannon, and E. E. Geldreich. 1979.Rapid seven-hour fecal coliform test. Appl. Environ. Microbiol.38:229-236.

13. Sakato, K., H. Tanaka, H. Samejima, I. Karube, and S. Suzuki.1981. Electrochemical measurements of cell populations. Ann.N.Y. Acad. Sci. 369:321-334.

14. Silverman, M. P., and E. F. Munoz. 1979. Automated electricalimpedance technique for rapid enumeration of fecal coliforms ineffluents from sewage treatment plants. Appl. Environ. Micro-biol. 37:521-526.

15. Trinel, P. A., and H. Leclerc. 1977. Automatisation de l'analysebacteriologique de l'eau: description d'une nouvelle methode decolimetrie. Ann. Microbiol. (Paris) 128A:419-432.

16. Warren, L. S., R. E. Benoit, and J. A. Jessee. 1978. Rapidenumeration of fecal coliforms in water by a colorimetric -galactosidase assay. Appl. Environ. Microbiol. 35:136-141.

17. Wilkins, J. R. 1978. Use of platinum electrodes for the electro-chemical detection of bacteria. Appl. Environ. Microbiol.36:683-687.

18. Wilkins, J. R., and E. H. Boykin. 1976. Electrochemical methodfor early detection and monitoring of coliforms. J. Am. WaterWorks Assoc. 68:257-263.

19. World Health Organization. 1971. International standards fordrinking water, 3rd ed. World Health Organization, Geneva.

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