establishment of gold standard for electrolyzed sodium ... · 7.4) containing 0.05% (w/v) ruthenium...

Bulletin of the Osaka Medical College 53（1）：11-19, 2007 11

Address correspondence to:Kouichi Sano, Department of Preventive and Social Medicine, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki-city, Osaka 569-8686, JapanPhone: +81-72-684-6417 Fax: +81-72-684-6517 E-mail: [email protected]

〈Original Article〉

Establishment of Gold Standard for Electrolyzed

Sodium Chloride Solution in Disinfection

Isao YOKOYAMA, Takashi NAKANO, Chizuko MORITA, Yasuhiro ARAI,

Takanori HIRAYAMA, Hiroaki AOKI, Jun HIROSE and Kouichi SANO

Department of Preventive and Social Medicine, Osaka Medical College

Takatsuki-city, Osaka 569-8686, Japan

Key Words：disinfection, electrolysis, electron microscopy, gold standard.

ABSTRACT

Electrolyzed saline as a recommended gold standard for the bactericidal activity of electrolyzed

products was examined in this study. The electrolyzed saline showed stronger bactericidal activity

against a Gram-positive bacterium (Staphylococcus aureus) than against a Gram-negative one

(Pseudomonas aeruginosa). This difference in bactericidal activity was analyzed by measuring the

cytoplasmic nitrate reductase activity and morphological changes in both bacteria. The difference in

the manner of inactivation of nitrate reductase activity between the Gram-positive and Gram-

negative bacteria indicates that the destruction of the cell wall differs between these bacteria.

Morphological analysis showed that the outer layer of the Gram-positive bacterium degenerated with

swelling and the periplasmic layer of the Gram-negative bacterium degenerated first. These findings

indicate that the penetration of free chlorine differs between Gram-positive and Gram-negative

bacteria, resulting in different susceptibilities to electrolyzed saline. Furthermore, the cause of the

previously reported bleb formation in the Gram-negative bacterium, which was treated with

electrolyzed products containing a low sodium chloride concentration, was shown to result from the

osmotic pressure of the product. The usefulness of electrolyzed saline as gold standard for the

evaluation of various electrolyzed products is discussed in this paper.

1. Introduction

The electrolyzed products of NaCl solutionhave recently been used as disinfectant (1), andpermitted as food additives by the Japanese FoodHygiene Law (Act No. 370 from the Ministry ofHealth and Welfare). Because these products areeasily inactivated in the environment, their toxicload is considered to be low compared with that ofother disinfectants.

Various electrolyzed products of NaCl solutionare available in terms of their pH and NaClconcentration. Electrolyzed solutions containingNaCl that show bactericidal activity are classifiedinto three groups: strong, weak and slight acidwaters (2). Strong acid water has been applied tothe disinfection of gastric endoscopes (3) on thebasis of results of basic studies (4-7).

In sodium hypochlorite solution, almost all Clexists as HClO at pH 5; more Cl2 is generated from

HClO at a lower pH, whereas more OCl- isgenerated from HClO at a higher pH. In variouselectrolyzed NaCl solutions, however, bactericidalfree chlorine such as HClO, Cl2 and OCl- exist inan unknown distribution, and therefore freechlorine concentration has been mentioned asindex of bactericidal activity.

According to the Japanese Food Hygiene law,more than 10 ppm free chlorine is required forreliable disinfection; however, approximately 5-10ppm free chlorine is confirmed effective fordisinfection with acidic electrolyzed NaCl solution(3-7) and tap water (8). Various electrolyzedNaCl solutions can be produced by changing theconcentrations of NaCl, free chlorine and otheradditives to adjust pH. pH adjustment to obtainacidic electrolyzed NaCl solution is difficult innormal situations. The electrolysis of saline indistilled water without pH adjustment is one of thepossible methods of producing standardizedelectrolyzed NaCl solution as disinfectant.Currently, no gold standard for evaluating thebactericidal activity of electrolyzed NaCl solutionis available. Because the most standardized NaClsolution used in biological and medical fields issaline, electrolyzed saline (ES) should be used asgold standard for the evaluation of electrolyzedproducts. However, the effectiveness ofelectrolyzed saline in disinfection has not yet beenwell clarified.

To establish a gold standard for the evaluationof the effectiveness of electrolyzed NaCl solutionin disinfection, we require knowledge of thecharacteristics of electrolyzed saline. In thisstudy, we evaluate the bactericidal activity of ESfor Gram-positive and Gram-negative bacteria.

2. Materials and Methods

2.1. Electrolysis, and measurement of free

chlorine and pH

Electrolysis was performed with a high-voltagepower supplier (PMC35-3A; Kikusui ElectronicsCo., Tokyo, Japan) and platinum-iridiumelectrodes were placed in a 500-ml plastic bottle.Saline was electrolyzed at 200 mA and a currentdensity of 0.446 A dm-2 for a designated time atroom temperature.

Free chlorine content was measured with achlorine meter (RC-2Z, Kasahara ChemicalInstruments Co., Saitama, Japan), and pH wasmeasured with a pH meter (Checker, HannaInstruments, Woonsocker, RI).

Bulletin of the Osaka Medical College 53（1）：11-19, 2007

Isao YOKOYAMA et al.12

2.2. Bacteria and culture

Pseudomonas aeruginosa ATCC10145 asGram-negative bacterium that commonly lives inwater-rich environments and Staphylococcus

aureus FDA 209P as Gram-positive bacterium,were used in this experiment. These bacteriawere cultured on heart infusion agar (HIA; BectonDickinson Co., Sparks, MD) overnight at 37ºC andfurther cultured in heart infusion broth (HIB;Becton Dickinson) for more than 3 h at 37ºC withagitation.

The bacterial cells were suspended in saline,centrifuged and washed. The final pellet wasresuspended in ES, which was sterilized with a0.45-µm-pore-size membrane filter. After placingthe bacterial cells in contact with ES, free chlorinewas neutralized with 1% bovine serum albumin(BSA) in saline, and the resulting mixtures wereused as samples. Surviving bacteria were countedby an endpoint dilution method that wascalibrated with a colony forming unit assay.

2.3. Morphological analysis

The bacterial cells were placed in contact withES at room temperature, and free chlorine in thesuspension was neutralized with BSA solution.The bacterial suspension was placed on a glassslide and Gram-stained.

For transmission electron microscopy, thebacterial cells were pelleted and fixed with 2%glutaraldehyde in cacodylate buffer (0.05 M, pH7.4) containing 0.05% (w/v) ruthenium red. Thepellets were washed with cacodylate buffer andpostfixed with 1% osmium tetroxide in the samebuffer containing 0.05% ruthenium red. Theywere then embedded in Epoxy resin and cut intoultrathin sections. The sections were doublystained with 4% uranyl acetate and lead citrate,and observed under an electron microscope (H-7100, Hitachi, Tokyo, Japan).

For scanning electron microscopy, a drop ofbacterial emulsion on a cover glass was fixed with2% glutaraldehyde in 0.15 M PBS (pH 7.2) at 4ºCfor 2 h. After washing three times in PBS, thebacterial cells were fixed with 1% osmiumtetroxide in 0.15 M PBS. The sample wasdehydrated in a graded ethanol series, coveredwith a platinum-palladium layer about 5 nm thickin an ion coater (E-102, Hitachi), and examinedunder a scanning electron microscope (S-5000,Hitachi).


Gold standard for electrolyzed NaCl solution 13

2.4. Inactivation of bacterial nitrate

reductase

The activity of bacterial nitrate reductase wasdetected using an Api 20 NE system (bioMerieux,sa Marcy-l’Etoile, France) by a modified method.In brief, after placing the cells in contact with ES,the suspension was neutralized, and then P. aeruginosa and S. aureus cells were cen-trifuged and resuspended at McFarland #2 in theAUX medium supplied with the kit and applied toan Api 20 NE plate. The plate was incubated for 1h at 37ºC. The activity of nitrate reductase wasdetected from the change in the color of thesolution in the “NO3” well of the plate. Thereaction mixture was centrifuged, and thesupernatant was transferred to a microplate. Anappropriate wavelength for measuring the level ofreaction was determined, and optical density(OD) at that wavelength was measured with areference wavelength of 680 nm.

3. Results

In subsequent experiments, a stable amount offree chlorine in ES is required. To examine theproduction of free chlorine and to estimate thenumber of generated molecules in the electrolysisof saline, free chlorine concentration and pH weremeasured in ES. Electrolysis seemed to generate

free chlorine in a linearly time-dependent mannerand pH increased up to 8.4 (Fig. 1). To obtainfree chlorine concentrations of 5, 10 and 20 ppm,electrolysis was required for 39, 77 and 155 sec,respectively. Because these values are repro-ducible, we used electrolyzed saline generatedunder such conditions in subsequent experiments.

To determine the bactericidal activity ofelectrolyzed saline, Gram-positive and Gram-negative bacteria were examined for viability afterthey were placed in contact with ES of variousfree chlorine concentrations. ES containing 5ppm free chlorine (ES5ppm) did not show thecomplete disinfection of S. aureus or P. aeruginosa (Fig. 2a). Increasing the freechlorine concentration to 10 ppm (ES10ppm)showed the complete disinfection of S. aureus

within 3 min, but not that of P. aeruginosa (Fig.2b). To confirm its bactericidal activity against P. aeruginosa, the free chlorine concentrationwas increased to 20 ppm and its bactericidalactivity was examined. ES20ppm disinfected P. aeruginosa within 5 min and S. aureus within1 min (Fig. 2c). These data indicate that ESshows a difference in bactericidal activity betweenS. aureus and P. aeruginosa. This difference inbactericidal activity may be due to a difference inthe inactivation of free chlorine between thebacteria.

Fig. 1 Production of free chlorine in and change in pH of electrolyzed salineAn increase in free chlorine and a slight increase in pH were observed in the electrolysis ofsaline. n (number of experiment) = 3. Bar = SD.


Isao YOKOYAMA et al.14

To examine the difference in the inactivation offree chlorine by the bacteria, free chlorineconcentration was measured in a bacterialsuspension in ES10ppm. The free chlorineconcentration in the suspension similarlydecreased in a time-dependent manner aftermixing with each bacterium (Fig. 3), indicatingthat P. aeruginosa does not inactivate free

chlorine more rapidly than S. aureus. In thisexperiment, free chlorine concentration of ESwithout putting bacteria did not change within 5min.

One enzyme that explains the disinfectionmechanism is nitrate reductase (8). To determinehow nitrate reductase is inactivated when mixingwith ES, its activity was measured. For such

Fig. 2 Bactericidal activity of electrolyzed salinesS. aureus or P. aeruginosa cells were placed in contact with electrolyzed saline containing 5, 10and 20 ppm free chlorine. Different numbers of viable bacteria were detected by placing thebacterial cells in contact with electrolyzed saline containing >10 ppm free chlorine. n (number ofexperiment) = 3. Bar = SD.



measurement, the reacted fluid in the “NO3” wellof the API 20 NE plate was examined for OD atvarious wavelengths (Fig. 4a). The maximum ODwas recorded at a wavelength of 540 nm. Thereacted fluids for S. aureus and P. aeruginosa

were transferred to a microplate and OD wasmeasured at a wavelength of 540 nm (reference at680 nm) in subsequent experiments. Thepercentage reduction in OD540-680 decreased in atime-dependent manner for S. aureus (Fig. 4b).

For P. aeruginosa, OD increased 1 min after itwas placed in contact with ES and rapidlydecreased as it did for S. aureus (Fig. 4b). Thesedata indicate that the manner of destruction of thebacterial cell wall differs between S. aureus andP. aeruginosa at the early stage of disinfection byES.

To determine the manner of destruction of thebacterial cell wall, morphological analysis usinglight and electron microscopies was performed.

Fig. 3 Inactivation of free chlorine by bacteriaS. aureus or P. aeruginosa cells were placed in contact with electrolyzed saline containing 10ppm free chlorine, and the decrease in free chlorine concentration was measured. P. aeruginosa

did not inactivate free chlorine more rapidly than S. aureus. n (number of experiment) = 3. Bar =SD. “n. s.” = not significant (t-analysis, p<0.05)

Fig. 4 Optical density of nitrate reductase assay (a) and inactivation of nitrate reductase activity inbacteria (b)a) The optical density peak was recorded at approximately 540 nm.b) Nitrate reductase activity of S. aureus decreased rapidly, but that of P. aeruginosa increased

in the first 1 min and then decreased. (* p<0.05, t-analysis)

Gram staining showed a loss of stainability in bothbacteria in a time-dependent manner (Fig. 5),indicating that ES destroyed their cell wall. Toconfirm the changes in the cell wall, its surfacestructure was observed by scanning electronmicroscopy. S. aureus cells after treatment withES were observed as slightly large (Figs. 6a and

6b) compared with untreated cells (Fig. 6c). Thesurface of the S. aureus cells was rough (Fig. 6d),particularly after 2 min of treatment with ES (Fig.6e) compared with that of untreated ones (Fig.6f). Small knob-like structures were observed onthe surface of the ES-treated S. aureus cells (Fig.6e). P. aeruginosa cells were also observed with

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Isao YOKOYAMA et al.

Fig. 5 Light microscopic images of bacteria treated with electrolyzed salineS. aureus and P. aeruginosa treated and untreated with electrolyzed saline (10 ppm) werestained with Gram staining and observed by light microscope. The loss of stainability in bothbacteria in a time-dependent manner is shown.

Fig. 6 Scanning electron microscopic images of bacteria treated with electrolyzed salinea), b) and c) are low-magnification, and d), e) and f) are high-magnification images of treated anduntreated S. aureus with electrolyzed saline (10 ppm) for 1 or 2 min. g), h) and i) are low-magnification, and j), k) and l) are high-magnification images of treated and untreated P. aeruginosa with electrolyzed saline (10 ppm) for 1 or 2 min.

Treated with electrolyzed saline for

Treated with electrolyzed saline for



(Figs. 6g and 6h) and without (Fig. 6i) EStreatment. The surface of the ES-treated P. aeruginosa cells was smooth (Figs. 6j and 6k)compared with that of the untreated ones, whichshowed a wavy surface (Fig. 6l). Those differ-ences were considered to be a result of structuralchanges in the cell wall.

To determine structural changes in the cellwall, transmission electron microscopy wasperformed. For the ES-treated S. aureus cells,the swollen cell wall was observed as not so sharpand the cytoplasm had an electron-dense lesion(Fig. 7a), which was not observed for theuntreated cells (Fig. 7b). In the observation of S. aureus cells at high magnification, thepeptidoglycan layer of the cell wall of the ES-treated cells was swollen and its surface wasrough and fuzzy (Fig. 7c) compared with that ofthe untreated ones, whose surface revealed a tightbrushlike structure (Fig. 7d). For the ES-treatedP. aeruginosa cells, the cell wall was thin andelectron-dense, and the cytoplasm had electron-dense speckled aggregates (Fig. 7e). For theuntreated P. aeruginosa cells, the cell wall wasthick and less electron-dense, ribosomal granules

were distributed throughout the cytoplasmhomogenously, and few large granules wereobserved (Fig. 7f). At high magnification, thepeptidoglycan layer of the ES-treated P. aeruginosa cells was highly electron-dense(Fig. 7g) compared with that of the untreatedones (Fig. 7h).

4. Discussion

Electrolyzed products of sodium chloridesolution disinfected both Gram-positive and Gram-negative bacteria as previously reported (3-7,9-13). Because the concentration of sodiumchloride is not described in some previous reports(11), it is difficult to compare the disinfectivepotential of and to analyze the mechanism ofdisinfection by such products.

In this study, we observed a reproduciblegeneration of free chlorine and a change in pHwhen saline was electrolyzed. Thus, werecommend ES as a gold standard for disinfection.We also suggest that some of the characteristics ofES be determined.

A difference between the bactericidal activities

Fig. 7 Transmission electron microscopic images of bacteria treated with electrolyzed salinea) and b) are images of treated and untreated S. aureus with electrolyzed saline (10 ppm) for 1min, respectively. c) and d) are respective images of the same bacterium at high magnification. e)and f) are images of treated or untreated P. aeruginosa with electrolyzed saline (10 ppm) for 1min, respectively. g) and h) are respective images of the same bacterium at high magnification.

of ES for Gram-positive and Gram-negativebacteria was found. We will attempt to explainthis difference. Because the consumption of freechlorine were almost the same between S. aureus

and P. aeruginosa, the relative resistance againstfree chlorine of P. aeruginosa may not be due tothe inactivation of free chlorine by this bacterium.

Kiura et al. (6) and Nakajima et al. (8) showedthat nitrate reductase from P. aeruginosa isinactivated by products of an electrolyzed solutioncontaining sodium chloride at a low concentrationand of an electrolyzed tap water containing a lowchlorine concentration, respectively. In this study,we confirmed that the cytoplasmic enzyme wasinactivated in S. aureus and P. aeruginosa. Theenzyme’s activity transiently increased in P. aeruginosa, which can be explained as follows:electrolyzed products break the outer membraneof the bacterium, the substrate reaches theenzyme rapidly through the inner membrane (6,8), and finally free chlorine reaches the enzymeafter breaking the periplasm and inner membrane.This time lag may be the cause of the transientincrease in the activity of nitrate reductase. Incontrast, in S. aureus, which has no outermembrane, no transient increase in the enzyme’sactivity was observed.

The stainability of both bacteria by Gramstaining decreased in a time-dependent manner,indicating the destruction of cell wall structures.The findings of scanning and transmissionelectron microscopies supported this indication.Because the cell wall structures of Gram-positiveand Gram-negative bacteria are different, thestructural changes owing to ES are different. Theouter membrane of Gram-negative bacteria mayplay a protective role against the bactericidaleffect of ES. This difference in structuraldestruction between Gram-positive and Gram-negative bacteria probably is the reason for thedifference in the degree of inactivation ofcytoplasmic enzymes and the difference insusceptibility between these bacteria against ES.

Kiura et al. (6) and Nakajima et al. (8) showedthat P. aeruginosa exhibits bleb formation bycontact with electrolyzed products of sodiumchloride solution at a low concentration and of tapwater containing a very low chloride concentra-tion; no such bleb formation, however, wasobserved when P. aeruginosa was placed incontact with ES. In both studies, the originalsolutions had low osmotic pressures different fromthat of our saline solution. The bleb probablyformed owing to the electrolyzed products and

low osmotic pressure of the solution, suggestingthat its formation is not a specific change resultingfrom contact with an electrolyzed solution.

Although there is an infinite number ofelectrolyzed products of sodium chloride solutiondepending on the sodium chloride concentrationin the original solution, no standard is available forevaluating such products at present. Because theosmotic pressure of ES has no effect on bacteria,ES directly reveals the effect of electrolyzedproducts. Therefore, ES may be useful as a goldstandard for bactericidal activity regardless ofother factors.

Durante et al. (14) reported on the acutetoxicity of glutaraldehyde in a clinical accidentcaused by a disinfectant, and Russell et al. (15)showed the need for caution in handlingglutaraldehyde. Because electrolyzed sodiumchloride solution has been focused on assubstitute for glutaraldehyde in recent years (3-5,7, 10), the evaluation of various electrolyzedproducts is urgent. We recommend electrolyzedsaline as an established gold standard for theevaluation of electrolyzed sodium chloridesolution. We also suggest that some characteris-tics of electrolyzed saline and its disinfectivepotential be determined.

5. Acknowledgement

We thank Mr. Y. Fujioka for technical assistancewith electron microscopy.

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Received October 2, 2006Accepted October 30, 2006

establishment of gold standard for electrolyzed sodium ... · 7.4) containing 0.05% (w/v) ruthenium...

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