Evaluation of pH, available chlorine content, and antibacterialactivity of endodontic irrigants and their combinationsagainst Enterococcus faecalis

Juliane M. Guerreiro-Tanomaru, DDS, PhD,a Renata D. Morgental, DDS, MSc,a

Danilo L. Flumignan, DDS, MSc,b Fabricia Gasparini, DDS, MSc,b

José E. Oliveira, DDS, PhD,b and Mário Tanomaru-Filho, DDS, PhD,a Araraquara, SP, BrazilUNIV ESTADUAL PAULISTA

Objectives. The objectives of this study were to evaluate pH, available chlorine content, and antibacterial activity ofendodontic irrigants and their combinations.Study design. The pH and chlorine content of sodium hypochlorite (NaOCl) were analyzed pure and in combinationwith 10% citric acid (CA) and apple vinegar (AV). The antibacterial effect of the following solutions was measured bydirect contact test against Enterococcus faecalis: 2.5% NaOCl, 2.5% NaOCl �10% CA (7:3), 2.5% NaOCl � AV (5:5),10% CA, and AV. Sterile saline was used as control. The colony-forming units were determined by serial decimaldilutions.Results. The combination of 2.5% NaOCl with CA or AV lowered the pH and the chlorine content. NaOCl, alone orin combination was able to eliminate E. faecalis in 30 seconds, and CA, after 10 minutes. AV promoted reduction(32.2%) after 10 minutes.Conclusions. NaOCl with acidic solutions lowered the pH and the chlorine content, but did not alter its antibacterial

effect. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;xx:xxx)

Endodontic infection plays an essential role in the eti-ology of pulpal and periapical disease.1,2 Thus, one ofthe main objectives of endodontic therapy of teeth withperiapical lesions is the control of infection.3 The per-sistence of microorganisms in the root canal systemafter endodontic therapy is determinant to treatmentfailure.4,5

Instrumentation and irrigation of the root canals areextremely important, but these procedures alone areunable to eliminate endodontic infection.6,7 This is at-tributable to microbial propagation into the root canalsystem and apical cementum,8,9 which are challengingareas to reach during the root canal preparation.

Sodium hypochlorite is the most widely used irrigat-ing solution in endodontics for its antimicrobial activ-ity, tissue-dissolving property, detergent action, and theability to neutralize toxic products.10,11 Some factorsmay affect the efficacy of NaOCl, such as solutionconcentration,12 temperature,13 and pH.14,15 The 2.5%

aAraraquara Dental School, UNESP, Univ Estadual Paulista, Depart-ment of Restorative Dentistry, Araraquara, SP, Brazil.bAraraquara Chemical School, UNESP, Univ Estadual Paulista, De-partment of Organic Chemistry, Araraquara, SP, Brazil.Received for publication Sep 28, 2010; returned for revision Dec 5,2010; accepted for publication Jan 6, 2011.1079-2104/$ - see front matter© 2011 Published by Mosby, Inc.

doi:10.1016/j.tripleo.2011.01.009

sodium hypochlorite solution has been used because ofits antimicrobial and solvent activity, with better bio-compatibility than more concentrated solutions.10-13

At lower pH, the chlorine in the NaOCl solution ispredominantly available as hypochlorous acid (HOCl),more active than the hypochlorite anion (OCl–) preva-lent at more alkaline pH.16 HOCl is able to betterpenetrate the bacterial cell membrane, because of itslack of electrical charge and molecular structure similarto water. Once within the cell, HOCl presents bacterio-static effect, reacting with the DNA, RNA, and othernucleotides. Additionally, this substance acts as a bac-teriostatic agent, reacting with amino acids and produc-ing chloramines.17 Therefore, the antimicrobial effectof NaOCl can potentially be enhanced by lowering thepH of the solution.14,15

Citric acid was first suggested as an irrigant byWayman et al.18 This substance is generally recom-mended as a final irrigant because of its ability toremove the smear layer19,20 and it can be associatedwith NaOCl solutions to lower their pH. Irrigation withNaOCl followed by citric acid may reduce the micro-biota in root canals.21 Apple vinegar, containing mainlyacetic acid and malic acid, also has the ability to re-move the smear layer22 and is active against endodonticmicroorganisms,23 thus being another alternative foruse in combination with NaOCl.

The goal of this study was to evaluate the pH and

available chlorine content in 2.5% NaOCl solutions,

1

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pure and in combination with 10% citric acid or applevinegar in different ratios, and to investigate the in vitroantibacterial action of these substances and their com-binations.

MATERIAL AND METHODSpH level and available chlorine assessment

The pH and the chlorine content of 2.5% NaOClsolution (Araquímica, Araraquara, SP, Brazil) weremeasured pure and in combination with 10% citric acid(Dinâmica, Diadema, SP, Brazil) or apple vinegar (Cas-telo, Jundiaí, SP, Brazil) in different ratios (by volume):8:2, 7:3, 6:4, 5:5, 4:6, 3:7, and 2:8.

The pH was assessed using a DMPH-2 pH meter(Digimed, São Paulo, SP, Brazil), calibrated at 25°C.The chlorine content was evaluated by iodometry, us-ing an AT500-N2 automatic potentiometric titratorAT500-N2 (KEM—Kyoto Electronics Manufacturing,Tokyo, Japan). All analyses were conducted in tripli-cate.

Antibacterial activity against Enterococcusfaecalis

The microbiological assays were carried out in alaminar flow chamber (VecoFlow Ltda, Campinas, SP,Brazil). The following solutions and combinations wereinvestigated: 2.5% NaOCl, 2.5% NaOCl � 10% citricacid at 7:3 ratio, 2.5% NaOCl � apple vinegar at 5:5ratio, 10% citric acid, and apple vinegar. Because so-dium hypochlorite solutions show better activity at pHof approximately 5,24 when the chlorine is availablemostly as hypochlorous acid,16 the solutions with this

Table I. Mean and standard deviation of pH and avacombination with 10% citric acid and apple vinegar

Sample Ratio

2.5% NaOCl —10% Citric acid —Apple vinegar —2.5% NaOCl � 10% citric acid 8:22.5% NaOCl � 10% citric acid 7:32.5% NaOCl � 10% citric acid 6:42.5% NaOCl � 10% citric acid 5:52.5% NaOCl � 10% citric acid 4:62.5% NaOCl � 10% citric acid 3:72.5% NaOCl � 10% citric acid 2:82.5% NaOCl � apple vinegar 8:22.5% NaOCl � apple vinegar 7:32.5% NaOCl � apple vinegar 6:42.5% NaOCl � apple vinegar 5:52.5% NaOCl � apple vinegar 4:62.5% NaOCl � apple vinegar 3:72.5% NaOCl � apple vinegar 2:8

pH (Table I) were selected for these tests. Sterile saline

was used as negative control and it allowed the deter-mination of the initial number of colony-forming units(CFU).

The antibacterial activity was evaluated by directcontact test, using a standard strain of E. faecalis(ATCC 29,212). The bacteria were plated onto trypticsoy agar (Difco, Detroit, MI), 12 hours before each test.Purity of the inoculum was confirmed by Gram stain.

The inoculum was suspended in sterile saline and theoptical density was adjusted using a spectrophotometer(model 600 Plus, Femto, São Paulo, SP, Brazil). Thebacterial suspension was adjusted to a concentration of3 � 107 CFU/mL and used within 60 minutes.

Automated pipettes were used to carry 1.45 mL ofeach solution or combination into 2-mL Eppendorf testtubes. Following that, a 50-�L aliquot of E. faecalissuspension was added to the tube, and the mixture wasagitated for 30 seconds (Vortex AP 56, Phoenix, Ara-raquara, SP, Brazil). Contact times were 30 seconds,and 1, 3, and 10 minutes.

After the contact period, serial decimal dilutions upto 10�5 were made. Then, 100-�L aliquots of themixture were transferred to another test tube containing0.9 mL of neutralizing agent. For the pure NaOClsolution, the neutralizer was 1% sodium thiosulfate,whereas for the other groups, 1% sodium thiosulfate �1% Tween 80 solution was used. The contents from thefirst dilution were homogenized and 100 �L was trans-ferred to a third test tube containing 0.9 mL of thecorresponding neutralizing solution. The fourth, fifth,and sixth tubes contained 0.9 mL sterile saline.

Finally, 20-�L aliquots from each dilution were

chlorine content in 2.5% NaOCl solution, pure or in

pH Chlorine, %

11.75 � 0 2.52 � 0.021.51 � 0.01 0 � 02.76 � 0.01 0 � 0

6.415 � 0.035 2.085 � 0.0054.82 � 0.04 1.815 � 0.0053.79 � 0.08 1.555 � 0.0053.08 � 0.02 1.30 � 0.01

2.685 � 0.005 1.045 � 0.0052.40 � 0.01 0.785 � 0.005

2.125 � 0.045 0.53 � 07.365 � 0.045 2.07 � 06.565 � 0.055 1.805 � 0.0055.43 � 0.02 1.545 � 0.0054.69 � 0.01 1.285 � 0.005

4.345 � 0.035 1.035 � 0.0054.03 � 0.02 0.775 � 0.0053.76 � 0.04 0.52 � 0

ilable

s

seeded in triplicate on the surface of agar plates, then

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incubated at 37°C for 48 hours under aerobic condi-tions. Readings were carried out on the dilution sam-ples with 5 to 50 colonies in each area of bacterialgrowth. The mean values were used to calculate thenumber of CFU/mL at each period of contact betweenthe irrigant and the bacterial suspension.

These experiments were repeated 3 times. Mean val-ues obtained were expressed as mean percentages ofviable colonies after contact with the irrigants at eachexperimental period.

RESULTSpH and chlorine content

The combination of 2.5% NaOCl with 10% citricacid lowered both the pH and the available chlorinecontent. The higher the amount of acid in the solution,the lower the values found for pH and chlorine avail-ability. A similar effect was observed for the combina-tion of 2.5% NaOCl with apple vinegar (Table I).

Antibacterial activityPlain 2.5% NaOCl solution, as well as in combina-

tion with 10% citric acid or apple vinegar fully elimi-nated E. faecalis in 30 seconds of contact. Plain 10%citric acid eliminated E. faecalis in 10 minutes. Applevinegar promoted only a reduction in the number ofviable cells by 32.20% at the end of the experiment, asshown in Table II.

DISCUSSIONInvestigations on the influence of pH values on the

antibacterial activity of NaOCl have been de-scribed.14,15,25 E. faecalis was used because of its highprevalence in endodontic treatment failures,26 which islikely related to its ability to invade dentin tubules andbind to collagen.27

The use of chloridric acid and acetic acid to adjustthe pH of NaOCl solutions has been previously as-sessed.14,15 In this study, citric acid and apple vinegar

Table II. Percentage of viable Enterococcus faecaliscells after direct contact with the different solutions andcombinations

Sample

Percentage of viable cells

30 s 1 min 3 min 10 min

2.5% NaOCl 0 0 0 02.5% NaOCl � 10% citric acid (7:3) 0 0 0 02.5% NaOCl � apple vinegar (5:5) 0 0 0 010% Citric acid 4 4 2.3 0Apple vinegar 97.8 93.6 88.0 67.8Negative control 100 100 100 100

were used with the same purpose. Citric acid has been

used in endodontics as a final irrigant,19,21 and applevinegar has shown activity against endodontic micro-biota.23

In a preliminary study, a remarkable reduction on theavailable chlorine content in combinations of NaOClwith acidic solutions was observed after 10 minutes.For this reason, the substances were mixed only imme-diately before the physical-chemical and microbiolog-ical analyses took place. Combination of 2.5% NaOClwith 10% citric acid gradually lowered the pH and theavailable chlorine content as the ratios progressed from8:2 to 2:8. Zehnder et al.28 studied the combination of1% NaOCl with 10% citric acid in different ratios andobserved absence of available chlorine after the firstminute. A possible explanation for this fact would beevaporation of chlorine in the form of gas during in-teraction of the substances, as reported by Baumgartnerand Ibay.29 Similar results were observed by mixingNaOCl with apple vinegar.

According to Hugo and Russel,24 NaOCl solutionsshow stronger antimicrobial activity when the pH isclose to 5, which corresponded to the ratio of 7:3 for thecombination of NaOCl with citric acid and 5:5 forNaOCl with apple vinegar. These were the selectedratios for the direct contact test. However, in thesesolutions, the chlorine contents were 1.815 and 1.285,respectively. It is important to point out that the anti-bacterial effect of NaOCl is directly related to theamount of HOCl available, besides the total chlorinepresent in the solution.

NaOCl solution in high concentration presents moreaggressive action to the periapical tissues.11 In thisstudy, the combinations of irrigating solutions weretested with the aim of improving the antibacterial ac-tivity of 2.5% NaOCl by lowering the pH.

Camps et al.14 and Mercade et al.15 demonstrated, inextracted human teeth, that NaOCl solutions with neu-tral pH are more active against E. faecalis than con-ventional solutions with pH 12. In the present study,NaOCl was mixed with acidic solutions, resulting in apH of approximately 5, showing antibacterial activitycomparable with that of the pure solution with alkalinepH. All the solutions analyzed were able to eliminate E.faecalis in the minimum period (30 seconds). In vitro,2.5% NaOCl was capable of eliminating E. faecalis andadjustment of the pH proved unnecessary. In addition,plain citric acid solution and apple vinegar were lessefficient.

The direct contact test allows comparisons betweenthe substances without the influence of extrinsic factorsthat could decrease or increase their antimicrobial ef-fect. So, it should be the first step in the evaluation ofantimicrobial agents. In this methodology, the first and

the second serial dilution tubes contained a neutralizing

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solution to prevent trace amounts of irrigant from beingtransferred into the culture medium.30

In clinical situations, the results of this study suggestthat when NaOCl solution is combined with acidicsolution, these substances should be placed directly intothe root canal instead of being mixed in a syringe, toprevent the potential loss of chlorine that would occurby previously mixing the solutions. Thus, it would beinteresting to study the efficacy of an initial irrigation ofthe canal with the acidic solution, followed by irriga-tion with NaOCl.

Considering the methodology used and the resultsobtained, the present study allows the conclusion thatthe combination of 2.5% NaOCl with 10% citric acid orapple vinegar lowered the pH and the chlorine content,but did not affect the antibacterial activity, comparedwith pure NaOCl solution. Plain citric acid and applevinegar showed weak activity against E. faecalis. Ad-ditional studies are necessary to clarify the physical andchemical interactions between NaOCl and different ac-ids.

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Reprint requests:

Mário Tanomaru-Filho, DDS, PhDDisciplina de EndodontiaFaculty de Odontologia de Araraquara, UNESPRua Humaitá, 1680CEP 14801-903 Araraquara, SP, Brazil

tanomaru@uol.com.br