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 Table of Contents  
Year : 2022  |  Volume : 34  |  Issue : 4  |  Page : 254-258

Antibacterial efficacy of sodium hypochlorite versus apple cider vinegar against Enterococcus faecalis in contracted endodontic cavity: An in vitro study

Department of Conservative Dentistry and Endodontics, K M Shah Dental College and Hospital, Sumandeep Vidyapeeth, Vadodara, Gujarat, India

Date of Submission07-Apr-2022
Date of Decision28-May-2022
Date of Acceptance31-May-2022
Date of Web Publication28-Dec-2022

Correspondence Address:
Dr. Karkala Venkappa Kishan
Department of Conservative Dentistry and Endodontics, K. M. Shah Dental College and Hospital, Sumandeep Vidyapeeth, Piparia, Vadodara, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/endo.endo_93_22

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Aim: The aim of this study is to evaluate and compare the antibacterial efficacy of 3% sodium hypochlorite with 5% apple cider vinegar using passive ultrasonic irrigation (PUI) against Enterococcus faecalis in contracted endodontic access cavity on mandibular molars.
Methods: Contracted access cavity was prepared in 30 extracted human permanent mandibular molar teeth. The root canals were incubated with strain of E. faecalis MTCC 9845 (Strain designation – HBL/BAC– 002/08–09) using 15 K-type manual file for 3 weeks. At this point, the S1 sample was collected using 15 paper points. The extracted teeth were randomly divided into two groups: sodium hypochlorite group and apple cider vinegar group. After mechanical instrumentation up to 25/04 rotary files HyFlex CM (Coltene Whaledent, Altstätten, Switzerland) and use of respective irrigants (3% sodium hypochlorite and 5% apple cider vinegar) using PUI, S2 sample was collected using 25 size paper point. The cultivated samples were incubated at 37°C for 24 h, and the number of bacterial colonies was counted as colony-forming units (CFUs)/mL. Data obtained from S1 and S2 were tabulated and subjected to Wilcoxon signed-ranks test and Mann–Whitney test.
Results: There was a significant reduction in microbial count in both groups (P < 0.05) when preoperative CFUs were compared with postoperative. There was slightly higher reduction in the apple cider vinegar group in S2 sample, as compared to the sodium hypochlorite group, but it was not statistically significant (P > 0.05).
Conclusion: Antimicrobial activity of 5% apple cider vinegar is similar to 3% sodium hypochlorite.

Keywords: Apple cider vinegar, bacterial reduction, contracted access cavity, passive ultrasonic irrigation, sodium hypochlorite

How to cite this article:
Supreet K, Kishan KV, Shah NC. Antibacterial efficacy of sodium hypochlorite versus apple cider vinegar against Enterococcus faecalis in contracted endodontic cavity: An in vitro study. Endodontology 2022;34:254-8

How to cite this URL:
Supreet K, Kishan KV, Shah NC. Antibacterial efficacy of sodium hypochlorite versus apple cider vinegar against Enterococcus faecalis in contracted endodontic cavity: An in vitro study. Endodontology [serial online] 2022 [cited 2023 Jan 28];34:254-8. Available from: https://www.endodontologyonweb.org/text.asp?2022/34/4/254/365819

  Introduction Top

The goal of endodontic therapy is to disinfect the root canal system completely. As a result, root canal disinfection requires a combination of mechanical and chemical cleaning.[1]

Although sodium hypochlorite is a routinely used endodontic irrigant, its extrusion beyond the apical foramen may result in tissue necrosis.[2] However, when herbal irrigants such as neem and cinnamon were used as endodontic irrigants, they were found to be safer but with less antimicrobial efficacy.[3] Therefore, research should be focused on finding newer irrigants which are safer and as antimicrobial as sodium hypochlorite.

Apple cider vinegar is a natural product formed as a result of the fermentation reaction of apple, sugar, and yeast.[4] In the field of medicine, it has been used for the management of diabetes, sore throat, and candidiasis, but in dentistry, its known use is as a chelating agent in endodontics and for the management of denture psoriasis.[5]

The notion of minimally invasive endodontics, or Contracted Endodontic Cavity (CEC), was recently presented to retain pericervical dentin to improve the prognosis of endodontically treated teeth, although Viera et al. claimed that CEC has hampered disinfection.[6],[7],[8] To enhance the antibacterial efficacy of the irrigant in a contracted access cavity, side-vented needles and passive ultrasonic irrigation (PUI) can be used.[9]

Since there is limited literature available comparing both irrigants in minimally invasive access cavity, this study was aimed to compare and evaluate the antibacterial efficacy of 3% sodium hypochlorite with 5% apple cider vinegar in CEC against Enterococcus faecalis.

The null hypothesis of the present study stated that there will not be any difference in antibacterial efficacy of 3% sodium hypochlorite with 5% apple cider vinegar using PUI against E. faecalis in CEC on extracted mandibular molars.

  Materials and Methods Top

“The manuscript of this laboratory study has been written according to the PRILE 2021 guidelines.[10]

Sample size calculation

Approval for this study was obtained from the Institutional Ethical Committee (SVIEC/ON/Dent/SRP/20130). G*Power 3.1 (Heinrich Heine University, Düsseldorf, Germany) was used to calculate the sample size, and it had a power of 90% and an alpha type error of 0.05.[11] The calculated sample size was 15 per group.

Sample selection

This study employed 30 extracted adult mandibular first molar teeth. A stereomicroscope (Olympus BX43; Olympus Co, Tokyo, Japan) was used to examine the mesial and distal roots of all the teeth at a ×20 to search for root fractures, fissures, or fusion. The study excluded extracted teeth with a history of root canal therapy, internal or external root resorption, or pulpal calcification. To maintain standardization, teeth with similar lengths (20 mm) were selected. Ultrasonic scaler (Woodpecker, Japan) was used to remove the debris. Periapical radiographs were taken from various angles (mesiodistal and buccolingual angles), scanned, and stored electronically. Schneider's approach was used to determine the curvature of the root canal.[12] Only extracted teeth with a curvature angle ranging from 10° to 20° were chosen.

Preparation of teeth

The extracted teeth were disinfected for 1 week in a 10% formalin solution before being kept in normal saline at 4°C. The samples were handled according to the Centers for Disease Control and Prevention protocols.[13]

Primary investigator prepared the contracted access cavity using small round bur (Dentsply/Maillefer bur size no #1), SS White STF 10932, FG-169L (SS White Burs Inc., Lakewood, NJ, USA), diamond-coated ultrasonic tip E5D (Woodpecker, Japan), and dental loupes – ×4. A 10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) was used to determine apical patencies, which was then validated using radiography.

The canals were first shaped up to size 20 with stainless steel hand files (Dentsply Maillefer, Ballaigues, Switzerland), then with a HyFlex CM 20/04 rotary files (Coltene Whaledent, Altstätten, Switzerland). All of the canals were irrigated with a 5% sodium hypochlorite (NaOCl, Vishal, Ahmedabad, Gujarat, India) solution to wipe out the debris. The effects of sodium hypochlorite were neutralized for 4 h with a 10% sodium thiosulfate (Na2S203) solution before the samples were stored in sterile saline solution for 20 h. The apical foramen was sealed with nail varnish. The samples were autoclaved for 15 min at 121°C and 15 lbs pressure in an Eppendorf tube containing 1.5 ml sterile brain–heart infusion (BHI) agar (HiMedia, Mumbai). To ensure that the teeth were free of bacterial contamination, they were sterilized and placed in an incubator at 37°C for 48 h.

Contamination of the root canals with Enterococcus faecalis

The study used a pure culture of the E. faecalis test strain MTCC 9845 (Microbial Type Culture Collection and Gene Bank, Chandigarh). To make the McFarland suspension, 1 mL pure E. faecalis was added to BHI and then diluted 30-fold to generate a bacterial solution with 1 × 107 colony-forming units (CFUs) per milliliter. The samples were inoculated with 10 μl E. faecalis suspension using a sterile pipette under a streamline flow hood. The suspension was inserted into the canal using a sterile #15 K-type file and subsequently transported throughout the working length (WL). The roots were incubated for 3 weeks at a temperature of 35°C ± 2°C and a humidity of 95%. Before instrumentation, the canals were washed with 1 mL of sterile saline solution to remove any remaining free cells.

Two sterile #15 paper points (Dentsply Maillefer) were placed at the WL in the mesiobuccal canals for 1 min each to obtain the primary sample (S1). The paper points should contact the MB canal walls on the periphery. CFUs were then determined by placing the paper points in tubes containing sterile saline solution.

Irrigation groups

After the S1 sample, the extracted teeth were randomly divided using the flip coin method into two groups for the irrigation protocol.

  • Group A: (n = 15) 10 mL of 3% sodium hypochlorite (Vishal, Ahmedabad, Gujarat, India)
  • Group B: (n = 15) 10 mL of 5% apple cider vinegar (American Garden, New York, USA).

After first sampling (S1), mesiobuccal root canals of the samples were enlarged using 25/04 rotary files HyFlex CM (Coltene Whaledent, Altstätten, Switzerland). In both groups, the irrigants – 3% sodium hypochlorite and 5% apple cider vinegar were injected into the canal using the side-vented 27-gauge needle C-K Endo (CK Dental Ind. Co. Ltd., Korea) and ultrasonically activated for 2 min using 20 U-file (Mani Inc. Tochigi, Japan), placed to a depth 1 mm short of WL. The irrigating solution was left in contact with the tooth surface for 20 min. The effect of the test irrigants was neutralized with the same protocol as mentioned above. Then, the second samples (S2) from the mesiobuccal canals were collected using #25 sterile paper points by placing it into the canal throughout the WL for 1 min, which were transferred to tubes with sterile saline solution.

Quantification of the bacterial load

The samples were taken from paper points that were placed in tubes containing 1 mL 0.85% phosphate-buffered solution and vortexed for 1 min. A 0.1 mL sample was cultured on Columbia Agar-Sheep Blood after 10X serial dilutions in sterile saline solution. The numbers of bacterial colonies were quantified as CFUs/mL after the cultivated samples were incubated at 121°C for 24 h.

Statistical analysis

Statistical significance was set at P < 0.05 in SPSS software (Version 20.0.1; IBM Corp, Armonk, NY, USA). For intragroup analysis, the Wilcoxon signed-ranks test was used, and the Mann–Whitney test was used for intergroup analysis.

  Results Top

In [Table 1], the mean of CFU values at S1 and S2 in the root canals are presented. The microbial count observed before instrumentation and irrigation (S1) was nearly identical in both groups. After using the respective irrigant, from S1 to S2, there was a significant reduction in microbial count in both groups (P < 0.05). There was slightly higher reduction in the apple cider vinegar group as compared to the sodium hypochlorite group, but it was not statistically significant (P > 0.05) [Table 2].
Table 1: Comparison of colony-forming units values between sodium hypochlorite and apple cider vinegar irrigant

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Table 2: Intergroup comparison of colony-forming units values between sodium hypochlorite irrigant and apple cider vinegar irrigant

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  Discussion Top

The main objective of cleaning and shaping is to eradicate microorganisms from the root canal system which can be accomplished by mechanical instrumentation supported by various irrigating solutions.[14] To achieve complete disinfection, we must choose an irrigant with better efficacy and less toxicity. NaOCl has proven to be an effective root canal irrigant but its toxicity is always a matter of concern.[15] As seen in previous studies, its efficacy in a contracted endodontic access has shown a reduced antimicrobial efficacy.[8] The use of herbal alternatives, especially Apple cider vinegar (ACV) has shown promising results in the previous studies in conventional access.[15],[16]

The results of the present study proved that the antimicrobial activity of 5% apple cider vinegar is similar to 3% sodium hypochlorite in CEC in mandibular molars. Despite the fact that there was no statistically significant difference between the two irrigating agents, ACV performed better in terms of lowering E. faecalis load. Sodium hypochlorite's pH disrupts cytoplasmic integrity, resulting enzyme inhibition, biosynthetic changes in cellular metabolism, and phospholipid breakdown, all of which contribute to bacterial lysis. Whereas in the case of ACV when it combines with endodontic biofilm, there's stimulatory action on the release of ATP synthase. With the release of this enzyme into the extracellular environment, there will be a rupture of cytoplasmic membrane, due to proteolysis reaction. Furthermore, the terminal enzymes of glycolysis, enolase, and phosphoenolpyruvate, are released into the extracellular environment. As a result, no pyruvate will be formed, which will result in inhibition of glycolysis, causing programmed cell death of bacteria.[17]

This is in accordance with the study done by Mohanty et al. to assess the antimicrobial efficacy of apple cider vinegar and 5% sodium hypochlorite against E. faecalis, in which the authors concluded that ACV is as antibacterial as 5% sodium hypochlorite.[17] Like sodium hypochlorite, apple cider vinegar enhances the release of antioxidant enzymes – catalase, dismutase, and glutathione peroxidase, causing release of reactive oxygen species. As a result, superoxide anion is also converted into H2O2 by spontaneous dismutation, further reduced to largely reactive hydroxyl radical, causing point mutation of bacterial DNA, which will result in free radical injury of bacteria.[18],[19] Similarly, Sayed et al. found no statistically significant difference between ACV and 5% sodium hypochlorite in another study utilizing EndoVac to investigate antibacterial activity of ACV and sodium hypochlorite against E. faecalis.[20]

Following the technique of Clark and Khademi, a constricted endodontic access cavity was created on the occlusal surface of the mandibular molar.[7] In the present study, initial access involving penetration into dentin was done using small round bur (Dentsply/Maillefer bur size no #1) in the center of the tooth. Enlargement and deroofing of the pulp chamber were done using SS White STF 10932, FG-169L (SS White Burs Inc., Lakewood, NJ, USA), and diamond-coated ultrasonic tip E5D (Woodpecker, Japan), to achieve a conservative access [Figure 1]. Maximum care was taken for standardization of all the procedures in both the groups, which include: initial apical diameter, total length, mechanical preparation using HyFlex CM, amount of irrigant, and gauge of the irrigating needle and its penetration. To negate the effect of apical vapor lock, the use of ultrasonic agitation and side-vented needle were done as the samples had contracted access cavities.[20]

The study's test organism was E. faecalis, a facultative anaerobe that easily enters dentinal tubules and survives by converting to a viable but noncultivable state during periods of adverse environmental conditions, does not require symbiotic support from other bacteria such as primary infections, and is relatively easy to culture and manipulate.[21] ACV not only kills E. faecalis but it also removes the smear layer, allowing its components to penetrate deeper into the dentinal tubules. It is a mixture of acetic, citric, formic, lactic, succinic, and tartaric acids with a small amount of alcohol that aids in lowering the solution's surface tension. Acetic (5%) and maleic (0.35%) acids, on the other hand, have the highest acid concentrations in vinegar.[22]

Due to the presence of maleic acid, apple cider vinegar is biocompatible. The ethyl alcohol produced is transformed and oxidized into acetic acid in this process, which takes place in the presence of particular bacteria. This process is known as acetification.[23] The presence of H+ ions causes the total amount of calcium ions detected in apple vinegar solution to increase. The more H+ ions present, the more effective the acid attack will be.

Based on a study conducted by Mittal et al., it was concluded that ACV is associated with greater root dentin demineralization.[24] Similarly, in another study conducted by Ali LA et al., it was concluded that ACV is more effective in the removal of smear layer than sodium hypochlorite.[25] Showkat et al. investigated the antibacterial efficacy of chitosan, chlorhexidine, apple cider vinegar, and sodium hypochlorite on E. faecalis biofilm in primary teeth and concluded that ACV was ineffective.[26]


Although endodontic biofilm is multispecies, we have assessed the antibacterial efficacy of ACV against single organism only. Although it has been utilized as a mouth wash for denture stomatitis and diabetic patients, no studies have reported its interaction with periapical tissues and other irrigants. Thus, future investigations should be planned in this direction.

  Conclusion Top

Within the study's limits, it can be inferred that:

  1. Antibacterial irrigants such as 3% sodium hypochlorite and 5% apple cider vinegar are effective against E. faecalis
  2. Despite the fact that the difference is not statistically significant, 5% apple cider vinegar is slightly more antimicrobial than 3% sodium hypochlorite
  3. Apple cider vinegar is a promising alternative to sodium hypochlorite.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Siqueira JF Jr, Alves FR, Versiani MA, Rôças IN, Almeida BM, Neves MA, et al. Correlative bacteriologic and micro-computed tomographic analysis of mandibular molar mesial canals prepared by self-adjusting file, reciproc, and twisted file systems. J Endod 2013;39:1044-50.  Back to cited text no. 1
Mohammadi Z. Sodium hypochlorite in endodontics: An update review. Int Dent J 2008;58:329-41.  Back to cited text no. 2
Ghonmode WN, Balsaraf OD, Tambe VH, Saujanya KP, Patil AK, Kakde DD. Comparison of the antibacterial efficiency of neem leaf extracts, grape seed extracts and 3% sodium hypochlorite against E. feacalis – An in vitro study. J Int Oral Health 2013;5:61-6.  Back to cited text no. 3
Yagnik D, Serafin V, Shah AJ. Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression. Sci Rep 2018;8:1732.  Back to cited text no. 4
Mota AC, de Castro RD, de Araújo Oliveira J, de Oliveira Lima E. Antifungal activity of apple cider vinegar on Candida species involved in denture stomatitis. J Prosthodont 2015;24:296-302.  Back to cited text no. 5
Boveda C, Kishen A. Contracted endodontic cavities: The foundation for less invasive alternatives in the management of apical periodontitis. Endod Topics 2015;33:169-86.  Back to cited text no. 6
Clark D, Khademi J. Modern endodontic access and dentin conservation, Part I. Dent Today 2009;28:86, 88, 90 passim.  Back to cited text no. 7
Vieira GC, Pérez AR, Alves FR, Provenzano JC, Mdala I, Siqueira JF Jr., et al. Impact of contracted endodontic cavities on root canal disinfection and shaping. J Endod 2020;46:655-61.  Back to cited text no. 8
Goel S, Tewari S. Smear layer removal with passive ultrasonic irrigation and the NaviTip FX: A scanning electron microscopic study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:465-70.  Back to cited text no. 9
Nagendrababu V, Murray PE, Ordinola-Zapata R, Peters OA, Rôças IN, Siqueira JF Jr, et al. PRILE 2021 guidelines for reporting laboratory studies in endodontology: Explanation and elaboration. Int Endod J 2021;54:1491-515.  Back to cited text no. 10
Bedier MM, Hashem AA, Hassan YM. Improved dentin disinfection by combining different-geometry rotary nickel-titanium files in preparing root canals. Restor Dent Endod 2018;43:e46.  Back to cited text no. 11
Ingle JI. Endodontic cavity preparation. In: Ingle J, Tamber J, editors. Endodontics. 3rd ed. Philadelphia: Lea & Febiger; 1985. p. 102-67.  Back to cited text no. 12
Kohn WG, Collins AS, Cleveland JL, Harte JA, Eklund KJ, Malvitz DM, et al. Guidelines for infection control in dental health-care settings–2003. MMWR Recomm Rep 2003;52:1-61.  Back to cited text no. 13
Parikh M, Kishan KV, Solanki NP, Parikh M, Savaliya K, Bindu VH, et al. Efficacy of removal of calcium hydroxide medicament from root canals by endoactivator and endovac irrigation techniques: A systematic review of In vitro studies. Contemp Clin Dent 2019;10:135-42.  Back to cited text no. 14
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Hülsmann M, Hahn W. Complications during root canal irrigation – Literature review and case reports. Int Endod J 2000;33:186-93.  Back to cited text no. 15
Kishan KV, Shah NC, Das DT, Parikh M. Herbal medicaments in endodontics – Current guidelines for in vivo studies in India. J Conserv Dent 2019;22:411-4.  Back to cited text no. 16
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Mohanty S, Ramesh S, Muralidharan NP. Antimicrobial efficacy of apple cider vinegar against Enterococcus faecalis and Candida albicans: An in vitro study. J Adv Pharm Edu Res 2017;7:137-41.  Back to cited text no. 17
Dong Y, Hu J, Fan L, Chen Q. RNA-Seq-based transcriptomic and metabolomic analysis reveal stress responses and programmed cell death induced by acetic acid in Saccharomyces cerevisiae. Sci Rep 2017;7:42659.  Back to cited text no. 18
Nakanishi-Matsui M, Sekiya M, Futai M. ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols. Biochim Biophys Acta 2016;1857:129-40.  Back to cited text no. 19
El-Sayed T, Nour El-Deen M, Rokaya M, Sherif M. Evaluation of the antibacterial effect of apple vinegar as a root canal irrigant using endovac irrigation system. Al-Azhar Dental Journal for Girls 2019;6:53-9.  Back to cited text no. 20
Townsend C, Maki J. An in vitro comparison of new irrigation and agitation techniques to ultrasonic agitation in removing bacteria from a simulated root canal. J Endod 2009;35:1040-3.  Back to cited text no. 21
Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of root-filled teeth with periapical lesions. Int Endod J 2003;36:1-11.  Back to cited text no. 22
Caligiani A, Acquotti D, Palla G, Bocchi V. Identification and quantification of the main organic components of vinegars by high resolution 1H NMR spectroscopy. Anal Chim Acta 2007;585:110-9.  Back to cited text no. 23
Mittal A, Dadu S, Yendrembam B, Abraham A, Singh NS, Garg P. Comparison of new irrigating solutions on smear layer removal and calcium ions chelation from the root canal: An in vitro study. Endodontology 2018;30:55-61.  Back to cited text no. 24
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Ali LA, Toma IS, Saeed RK. Compartive evalution of a new endodontic irrigation solution – apple vinegar, ginger oil and sodium hypochlorite to remove the smear layer by scanning electron microscope study. Journal of Dohuk University 2020;22:30-7.  Back to cited text no. 25
Showkat I, Chaudhary S, Sinha A, Ghaus M, Priya N. Evaluation the antibacterial efficacy of chitosan, chlorhexdine, apple cider vinegar and sodium hypochorite on E. fecalis biofilm in primary teeth: An in vitro study. Eur J Pharm Med Res 2020;7:667-73.  Back to cited text no. 26


  [Table 1], [Table 2]


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