In the present study, water was circulated in asimulated dental unit water line with electrifying asmall current. The morphology of the biofilmdeveloped on inner surface of the water line andthe number of heterotrophic bacteria were investi-gated to elucidate the effect of a low level electriccurrent on the biofilms formation associated withbacteria reproduction.

Destruction and malconformation of biofilms byelectrification was observed using SEM, in additionto deformation and hypertrophy of the bacteria. Bynaked eye observation, small pieces, which werepossibly exfoliated biofilms, were detected inelectrified water. While an adherent, yellow gelwas demonstrated on the inner surface of thewater line without electrification.

With electrification, the number of bacteriadecreased during the first week, however the bac-teria increased gradually after that. The number ofbacteria without electrification was consistentlygreater than that with electrification and the differ-ence was statistically significant (P<0.05). Thepredominant bacteria were identified asSphingomonas paucimobilis. The excess chlorinelevels decreased to a minimum value within oneweek.

The small current appeared to have effects onbiofilm formation of heterotrophic bacteria thatresulted in enhanced chlorine sterilization of dentalunit water. Thus, electrification has considerablepotential for the extermination of bacterialbiofilms in dental unit water lines.

Key words: dental unit water line, electrifying,sterilization, biofilm, excess chlorinelevel

Introduction

Bacterial contamination of dental unit water lines hasbeen a problem since air turbines began to spread inthe 1960’s. At that time “suck-back” of intraoral bacteriawas recognized as the main cause of contamination.This contamination was eventually eliminated by use ofan anti-retraction valve in the handpiece. However, in2000, Araki et al.1 reported that contamination with het-erotrophic bacteria, such as Sphingomonas paucimo-bilis, Methylobacterium mesophilicum, Pseudomonasstutuzeri, was found to predominate in dental unitwater lines. While pathogenic bacteria, such asLegionella, Streptcoccus, Pseudomonas andColibacillus were not evident. Similar findings havesince been reported2-6. Thus, it was recently revealedthat bacterial contamination of dental unit water lineswas caused not by bacteria from the oral cavity but byheterotrophic bacteria from the water supply systems.On the other hand, the United States Environmental

Original Article

Effects of a small electric current on sterilization of a dental unit water line

Takayuki Tanahashi1, Ken-ichi Tonami2, Kouji Araki3 and Norimasa Kurosaki1

1) General Dentistry, Department of Comprehensive Oral Health Care, Division of Comprehensive PatientCare, Graduate School, Tokyo Medical and Dental University2) Oral Diagnosis and General Dentistry, Dental Hospital, Tokyo Medical and Dental University 3) Center for Education Research in Medicine and Dentistry, Tokyo Medical and Dental University

J Med Dent Sci 2006; 53: 111–118

Corresponding Author: Takayuki TanahashiGeneral Dentistry, Department of Comprehensive Oral HealthCare, Division of Comprehensive Patient Care, Graduate School,Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku,Tokyo, Japan, 113-8549Tel: +81-3-5803-5765, Fax: +81-3-5803-5765E-mail: takayuki.gend@tmd.ac.jpReceived January 23; Accepted March 17, 2006

Protection Agency (EPA) has determined that thenumber of heterotrophic bacteria should be less than500 CFU/ml in drinking water7. The American DentalAssociation has also recommended that water fordental treatment should have bacteria levels less than200-CFU/ml8. In Japan, chlorine has been used forsterilization of tap water, including dental water lines.However, water used in dental treatment is not suffi-ciently controlled, nor does it meet with these interna-tional standards1. Therefore, additional countermea-sures are necessary to supplement treatment for den-tal water lines.

In order to resolve this issue, a new sterilization sys-tem has been developed in which water lines areelectrified with a small electric current. It has beenreported that the number of heterotrophic bacteria indental unit water lines is decreased and kept less than102 CFU/ml with this procedure9. Heterotrophic bacteriapropagate inside biofilms that protect bacteria from dis-infectants, such as chlorine6. The electrification ofsmall current cause electrolysis of water which resultsin a specific oxidation reduction potential (ORP) and pHof water. The ORP and pH relates to metabolism toform biofilms and reproduce bacteria10. Therefore, itcould be hypothesized that the small amount of electriccurrent could affect biofilm formation as well as bacte-ria reproduction.

In the present study, water sampled from a dentalunit was circulated in a simulated water line with a smallamount of electric current. The morphology of thebiofilm and the number of heterotrophic bacteria wereinvestigated in an effort to elucidate the effect of a lowlevel electric current on the biofilms formation associ-ated with bacteria reproduction.

Materials and Methods

A 1000-ml water sample was obtained from a dentalunit in General Dentistry II of the Dental Hospital ofTokyo Medical and Dental University, and was pooled ina beaker. The water was then circulated in a seven-meter long tube at a flow rate of 20 ml/min using anelectromagnetic metering pump (EH-BI5VC-100PRI,IWAKI Co.LTD, Tokyo, Japan).

The water line was electrified with a direct currentvoltage of 20V, limited current of 10mA, using an elec-tric current generator (Bioprotector, Tamagawa,Tokyo, Japan) one meter downstream from the pump(Fig. 1). The electrification was conducted continuous-ly throughout the experiment. The experimental

schedule and conditions were, as follows: Circulationwith electrification for five weeks (hereafter, Exp. 1); cir-culation for five weeks without electrification afterthorough exchange of the waterline tube (hereafter,Exp. 2 control); finally, circulation with electrification foreight weeks immediately after Exp. 2 using the sametube (hereafter, Exp. 3) (Fig. 2). In all conditions, thewater was circulated from 9:00 to 17:00 from Monday toFriday and temperature was set at 23 °C. And the fol-lowing parameters were investigated for each condition.

(1) Observation of Biofilm Configuration A section of tube, 5 mm in length, that was located

five meters downstream from the electric current gen-erator was cut. The piece was fixed and stained using2.5% glutaraldehyde and 1% osmium, and was dehy-drated with 50-100% ethanol. After critical point dryingwith a critical point dryer (HCP-2, HITACHI, Tokyo,Japan), the inner surface of tube was coated with pal-ladium and platinum using an ion-spattering machine(E102, HITACHI, Tokyo, Japan). The inner surface oftube was then examined using scanning electronmicroscope (S-4500, HITACHI, Tokyo, Japan) with

T. TANAHASHI et al. J Med Dent Sci112

Fig. 1. Schema of the simulated dental unit water line: Pooled waterfrom a dental unit was circulated in a seven-meter long tube at a flowrate of 20 ml/min using an electromagnetic metering pump. The cir-culation time was from 9:00 to 17:00 from Monday to Friday; the cir-culation was stopped on Saturday and Sunday.

Fig. 2. Experimental Schedule

acceleration voltage 15 kV. Biofilm formation inside thebeaker and tube was also observed with naked eyes.These observations were done on the first day ofevery week in Exp. 1, Exp. 2 and Exp. 3.

(2) Number measurement of heterotrophic bacteria Water was sampled from locations one meter and

five meters downstream from the electric current gen-erator and the number of bacteria was measuredusing R2A agar medium. Two sets of counts were aver-aged and the number of bacteria was determined foreach location and condition. These measurementswere carried out on the first day of every week for Exp.1 and Exp. 2. The numbers of bacteria were analyzedby paired-t test using statistical software (SPSS 11.0 Jfor Windows, SPSS Inc.), comparing between thenumbers of bacteria in Exp. 1 and Exp.2 of every timeand local condition.

(3) Identification of predominant bacteriaFive colonies were randomly sampled from the 5-

weeks medium in Exp.2 and a predominant bacteriumwas decided with examining shape, Gram stainabilityand affinity for oxygen. Finally the predominant bac-terium was identified with a Gram-negative rod identifi-cation kit (API20NE, bioMerieux Japan, Tokyo,Japan).

(4) Measurement of ORP, pH and excess chlorinelevel

The ORP and pH of water in the beaker were mea-sured hourly using an ORP meter (PH1500, EutechInstruments Pte. Ltd, Singapore). Excess chlorine lev-els were also measured at the beginning of circulation,and after 1 hour, 8 hours, 2, 5, and 7 days of circulationusing a digital excess chlorine meter (Kuroru II,TACMINA, Osaka, Japan). Each measurement wasconducted for samples in Exp. 1, Exp. 2 and Exp. 3.

Results

1. SEM observationIn Exp. 1, after 1-week of electrification, bacilli and

cylindrical objects considered to be the product ofdeformation of bacteria were observed on the inner sur-face of the tube (Fig. 3). Under high magnification,granular structures were apparent on the surface of thebacteria including cylinder shaped objects (Fig. 4). After5-weeks of electrification, no network-connecting bac-teria were apparent (Fig. 5). In Exp. 2, colonies

formed on the inner surface of the tube after the firstweek (Fig. 6); the predominant bacteria were bacilli(Fig. 7). Filamentous objects that connected thecolonies were apparent after 3 weeks and formed anetwork (Fig. 8). After 5 weeks, no bacteria wereobserved on the surface of the film but were buriedinside the film (Fig. 9, 10). The number of bacteriaexposed on the film surface tended to decrease grad-ually in number throughout Exp. 2. In Exp. 3, after elec-trification for 1week, the bacteria observed on the filmsurface increased in number (Fig. 11). After 6-weeks of

113ELECTRONIC CURRENT STERILIZATION OF DENTAL UNIT WATER

Fig. 3. Exp. 1, 1st week: In Exp. 1 after 1-week electrification, cylin-drical objects were observed on the inner surface of the tube.

Fig. 4. Exp. 1, 1st week, high magnification: Granular structuresappeared on the surface of bacteria bodies and the cylindricalobjects.

electrification, spiral fibrous structures were recog-nized on bacterial cell surface that had not beenobserved before electrification (Fig. 12). After electrifi-cation for 8 weeks, the shape of bacteria on the filmcould be observed more clearly and the number wasgreater than that after electrification for 1week in Exp. 3.The network was not as significant as that in Exp. 2(Fig. 13).

2. Naked-eye observationIn Exp. 1 and Exp. 3, the transparent, small and yel-

low particulates that would likely be regarded as disin-tegrated biofilm were observed in the stream.Adherence of a biofilm was not recognized on the innersurface of the beaker, although a sediment of trans-parent yellow particulates was found in the bottom of abeaker (Fig. 14 -a). On the other hand, in Exp. 2, we didnot observe the small particulates such as observed inExp. 1 and Exp. 3 in the stream or in the beaker. In itsplace, a yellow transparent gel that appeared to be abiofilm, was attached to the inner surface of thebeaker (Fig. 14 -b).

T. TANAHASHI et al. J Med Dent Sci114

Fig. 5. Exp. 1, 5th week: No network connecting bacteria were rec-ognized.

Fig. 6. Exp. 2, 1st week: Colonies were formed on the inner surfaceof the tube by the end of the first week.

Fig. 7. Exp. 2, 1st week, high magnification: The predominant bac-teria were bacilli.

Fig. 8. Exp. 2, 3rd week: Filamentous objects which connected thecolonies appeared to form a network.

3. Counting the number of bacteria (CFU)In Exp. 1, the bacteria decreased in number after the

first week, and then increased gradually to 4×103

CFU/ml after 5-weeks of circulation. In Exp. 2, the num-ber of bacteria increased immediately after the start ofcirculation; the number of the bacteria increased to2.7×106 CFU/ml at 5 weeks. The number of bacteria inExp. 2 was 102 times greater at maximum than that ofExp. 1 (Fig. 15). A significant difference was observedbetween the number of bacteria in Exp. 1 and Exp. 2 bypaired t-test (P<0.05).

4. Identification of predominant bacteriaThe predominant bacterium was identified as

Sphingomonas paucimobilis.

5. ORP, pH and excess chlorine levelIn Exp. 1 and Exp. 3, ORP ranged from -100 to

+200mV and varied throughout the day, correspondingto the circulation program (Fig. 16). The pH also variedwith in the range of 7.4-7.7. The pH tended to changealong with ORP. The ORP gradually increased in Exp.2 and the daily periodicity was not apparent (Fig. 16).

115ELECTRONIC CURRENT STERILIZATION OF DENTAL UNIT WATER

Fig. 9. Exp. 2, 5th week

Fig. 10. Exp. 2, 5th week, high magnification: Bacteria were notobserved on the surface of the film but were buried inside the film.

Fig. 11. Exp. 3, 1st week: After electrification for 1 week, the bacte-ria on the film surface increased in number.

Fig. 12. Exp. 3, 6th week: Spiral fiber structures that had not beenobserved before electrification were apparent.

The excess chlorine in the beaker was 0.3 ppm imme-diately before the start of the experiment; however,chlorine levels decreased afterward to a minimumvalue within about 1 week (Fig. 17).

Discussion

The characteristics of electrolyzed water depend onpH and ORP. For medical purposes, strongly acidicwater can be used to sterilize devices or tissues; thelow pH, 2.0 to 3.0, and high ORP, 1000 to 1150mV, are

T. TANAHASHI et al. J Med Dent Sci116

Fig. 13. Exp. 3, 8th week: The network was not remarkable. Theshape of bacteria on the film could be observed more clearly and thenumber was greater than that after electrification for 1week in Exp. 3.

Fig. 14. Reserved water in the beaker: a: Exp.1, No biofilm wasapparent on the inner surface of the beaker by naked eye, althougha sediment of small, yellow particulates was found in the bottom ofthe beaker after circulation of five weeks with electrifying. b: Exp. 2,The small particulates were not observed in the beaker after circula-tion of five weeks without electrifying. Instead, a yellow transparentgel, seemingly a biofilm, was attached to the inner surface of thebeaker.

Fig. 15. Numbers of bacteria: ◇: Exp1, ◆: Exp2, bold line: Onemeter downstream from the electric current generator, dotted line:Five meters downstream from the electric current generator. ForExp.1, the bacteria decreased in number during the first week, butincreased gradually to 4×103 CFU/ml after 5-weeks of circulation.The number of bacteria in Exp.2 was 102 times greater than that ofExp.1.

Fig. 16. Change of ORP over one week: For Exp.1 and 3, the ORPranged from -100 to +200mV and changed periodically during the daycorresponding to the circulation. The ORP gradually rose in Exp.2and the daily periodicity was not clear.

Fig. 17. Change of excess chlorine level during the first week of cir-culation: The excess chlorine level in the beaker was 0.3ppm imme-diately before the experiment started; however, it subsequentlydecreased to a minimum value within about 1 week.

considered to directly damage bacteria. There is alsoreported that neutral electrolyzed water which was gen-erated by electrifying water containing 25% NaCl11. Thewater indicated pH 8 and ORP more than 700mV usingelectric current of about 32A. The sterilization effectdepended on biocide produced by electrolysis such asHOCl, ClO- and HO. In the present study, the water cir-culated with electrification using much less electric cur-rent of 10mA without NaCl. Therefore, the amount andthe kinds of generated biocide is not the same as neu-tral electrolyzed water. The water in this study exhibitedan ORP of -100 to +300mV and pH from 7.4 to 7.7; i.e.,the ORP was lower than with strongly acidic water andthe neutral electric water. Thus, no significant directkilling of bacteria was expected; a different mechanismmight account for the decrease in number of het-erotrophic bacteria in this study. Through observationwith SEM, we found that there was a prominent differ-ence in biofilm morphology between circulation withand without electrification. The bacteria were buriedwithin the matrix after 5-weeks of circulation withoutelectrification. On the other hand, remarkable exposureof bacteria on the inner surface was observed with elec-trification. In addition, suspended yellow matter wasnoted within the beaker and the tube by naked-eyeobservation. Sphingomonas paucimobilis was detectedas the predominant bacteria in this study and isknown to generate a yellow pigment as one of itsmetabolites12. Therefore, it was possible that the sus-pended yellow matter could be a biofilm that wasremoved from the fixed surfaces. Thus, the biofilm wasdestroyed or at least showed malconformation byelectrification that resulted in exposure of bacteria onthe inner surface. Deformation and hypertrophy ofbacteria itself was also observed; therefore, electrifica-tion may also exhibit direct effects on the bacteria itself.Furthermore, the number of bacteria with electrificationwas consistently less than that of without electrification.The ORP and pH are known to be closely related tometabolism of the bacteria10,13. In the present study,ORP was controlled at relatively low level for propaga-tion of bacteria and changed repeatedly with a dailyperiodicity. Such an environment might interfere withthe metabolism and disturb the reproduction of thebiofilm and bacteria.

For sterilization of water supply systems, chlorine isgenerally used at levels of 1 ppm or less in tap water inJapan14. However, the sterilizing effect of chlorine hasbeen reported to be inadequate to eradicate bacteriafrom water lines15-17. The primary reason for this is thatbacteria reside inside a biofilm. There have been

reports that biofilm cells were more resistant to halogenbiocides than planktonic cells.18,19. In other words,chlorine would be more effective at sterilizing if thebiofilm was removed from the bacteria. In this study, aremarkable decrease of bacteria number wasobserved after the first week of electrification. Wealso confirmed that excess chlorine remained in thesimulated water line throughout the first week.Therefore, the decrease of bacteria number at the firstweek was likely caused by an interaction between chlo-rine and electrification. Recover of excess chlorine lev-els by electrification has also been demonstrated20. Onthe other hand, the amount of chlorine in water hasbeen shown to decrease following exposure to light21.The water in this study was exposed to room light suchthat excess chlorine disappeared after the first week.By contrast, water lines in original dental units wouldgenerally be in dark; thus the level of chlorine would notdecrease by light as much as that of the simulatedwater line. Therefore, sterilization using electrificationwould be more effective in a real dental unit comparedwith the simulation used in this study. This assumptionis consistent with the report of Araki et al.9, which indi-cated that the number of bacteria from the end of awater line decreased to 8-11x103 CFU/ml after twoweeks, 3-10x102 CFU/ml after one month and almostzero after two month when the same electric generatorwas connected directly to dental units in clinics.

Thus, electrification of the water line exhibited aneffect on both biofilm formation and bacteria reproduc-tion. Combination of electrification and chlorine wasseemingly more efficient than sterilization with chlorinealone. Consequently, electrification has considerablepotential for extermination of bacteria in dental unitwater lines.

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