May 19, 2026
Antibacterial Efficacy of Triton All-in-One Solution versus Sodium Hypochlorite against Enterococcus faecalis in Extracted Mandibular Premolars: An In Vitro Study
- Nayera Hamdy Mohamed Hassan1,
- Shaimaa Ismail Gawdat2,
- Laila Zakaria3
- 1BDS New Giza University - 2021, Master's degree candidate, Department of Endodontics - Faculty of Dentistry, Cairo University;
- 2Professor of Endodontics, Faculty of Dentistry, Cairo University;
- 3Lecturer of Endodontics, Faculty of Dentistry, Cairo University
- Nayera Hamdy Mohamed Hassan: Researcher and principal investigator;
- Shaimaa Ismail Gawdat: Chief supervisor;
- Laila Zakaria: Co-supervisor
- Faculty of Dentistry, Cairo University
Protocol Citation: Nayera Hamdy Mohamed Hassan, Shaimaa Ismail Gawdat, Laila Zakaria 2026. Antibacterial Efficacy of Triton All-in-One Solution versus Sodium Hypochlorite against Enterococcus faecalis in Extracted Mandibular Premolars: An In Vitro Study. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jqo5vqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: May 11, 2026
Last Modified: May 19, 2026
Protocol Integer ID: 316796
Keywords: antibacterial efficacy of triton all, enterococcus faecalis in extracted mandibular premolar, antibacterial efficacy, reproducible assessment of antimicrobial efficacy, bacterial sample, antimicrobial efficacy, enterococcus faecali, standardized root canal specimen, biofilm formation, bacterial reduction, extracted mandibular premolar, in vitro study this protocol, irrigation solution, solution versus sodium hypochlorite, in vitro study, triton solution, irrigation protocol, using sterile paper point, mandibular premolar, sodium hypochlorite
Abstract
This protocol describes an in vitro method to evaluate the antibacterial efficacy of Triton All-in-One irrigation solution compared with sodium hypochlorite (NaOCl) against Enterococcus faecalis in extracted mandibular premolars. Fifty-one standardized root canal specimens are prepared, sterilized, and inoculated with E. faecalis and incubated for 21 days to allow biofilm formation. Pre-irrigation bacterial samples (S1) are collected using sterile paper points and cultured in Brain Heart Infusion broth for colony-forming unit (CFU) quantification. Specimens are randomly assigned to three irrigation protocols: Triton solution, NaOCl + EDTA, and NaOCl alone. Post-irrigation samples (S2) are collected and analyzed to determine bacterial reduction. The protocol enables reproducible assessment of antimicrobial efficacy using CFU analysis.
Title
Antibacterial Efficacy of Triton All-in-One Solution versus Sodium Hypochlorite against Enterococcus faecalis in Extracted Mandibular Premolars: An In Vitro Study
Introduction
Root canal treatment aims to eliminate microorganisms harbored within the complex anatomy of the root canal system, thereby creating a bacteria free environment prior to obturation to ensure long term treatment success and prevent reinfection. However, mechanical preparation alone is insufficient to effectively eradicate intracanal pathogens, particularly Enterococcus faecalis (E. faecalis), a Gram-positive facultative anaerobe capable of surviving under extreme environmental conditions. Consequently, E. faecalis has been reported in approximately 4 to 40% of primary endodontic infections and 24 to 77% of persistent infections.[1]
Despite advances in endodontic instrumentation and the availability of files in diverse shapes, sizes, and tapers, complete contact with all root canal walls remains unattainable. Accordingly, chemical irrigants are employed adjunctively with mechanical instrumentation to address anatomical complexities beyond canal ramifications, including isthmuses, curved canals, oval canals, and irregular canal morphologies, particularly within the apical third which may harbor resistant microorganisms.[2,3]
Sodium hypochlorite is widely regarded as the gold standard root canal irrigant owing to its broad-spectrum antibacterial activity and its ability to dissolve organic tissue. It is highly effective against both Gram-positive and Gram-negative microorganisms, including E. faecalis, the major cause of persistent and refractory endodontic infections. In addition to its potent antimicrobial action, sodium hypochlorite is the only commonly used irrigant capable of dissolving necrotic pulp tissue and organic components of the smear layer, thereby enhancing canal debridement and disinfection.[4]
Despite these advantages, its clinical use is associated with several well recognized drawbacks. Sodium hypochlorite exhibits cytotoxic effects on vital tissues, accidental extrusion beyond the apical foramen can result in severe irritation and inflammation of the periapical tissues, accompanied by pain, swelling, and tissue damage.[5]
In addition, sodium hypochlorite is unable to remove the inorganic component of the smear layer produced during root canal preparation, which necessitates the use of 17% ethylene diamine tetra acetic acid (EDTA) as a final flush. EDTA, a chelating agent, reacts with calcium ions in hydroxyapatite crystals, leading to demineralization and removal of the inorganic smear layer. This process opens the dentinal tubules and facilitates deeper and more effective cleaning of the root canal system.[6]
Root canal treatment aims to eliminate microorganisms harbored within the complex anatomy of the root canal system, thereby creating a bacteria free environment prior to obturation to ensure long term treatment success and prevent reinfection. However, mechanical preparation alone is insufficient to effectively eradicate intracanal pathogens, particularly Enterococcus faecalis (E. faecalis), a Gram-positive facultative anaerobe capable of surviving under extreme environmental conditions. Consequently, E. faecalis has been reported in approximately 4 to 40% of primary endodontic infections and 24 to 77% of persistent infections.[1]
Despite advances in endodontic instrumentation and the availability of files in diverse shapes, sizes, and tapers, complete contact with all root canal walls remains unattainable. Accordingly, chemical irrigants are employed adjunctively with mechanical instrumentation to address anatomical complexities beyond canal ramifications, including isthmuses, curved canals, oval canals, and irregular canal morphologies, particularly within the apical third which may harbor resistant microorganisms.[2,3]
Sodium hypochlorite is widely regarded as the gold standard root canal irrigant owing to its broad-spectrum antibacterial activity and its ability to dissolve organic tissue. It is highly effective against both Gram-positive and Gram-negative microorganisms, including E. faecalis, the major cause of persistent and refractory endodontic infections. In addition to its potent antimicrobial action, sodium hypochlorite is the only commonly used irrigant capable of dissolving necrotic pulp tissue and organic components of the smear layer, thereby enhancing canal debridement and disinfection.[4]
Despite these advantages, its clinical use is associated with several well recognized drawbacks. Sodium hypochlorite exhibits cytotoxic effects on vital tissues, accidental extrusion beyond the apical foramen can result in severe irritation and inflammation of the periapical tissues, accompanied by pain, swelling, and tissue damage.[5]
In addition, sodium hypochlorite is unable to remove the inorganic component of the smear layer produced during root canal preparation, which necessitates the use of 17% ethylene diamine tetra acetic acid (EDTA) as a final flush. EDTA, a chelating agent, reacts with calcium ions in hydroxyapatite crystals, leading to demineralization and removal of the inorganic smear layer. This process opens the dentinal tubules and facilitates deeper and more effective cleaning of the root canal system.[6] These limitations have driven ongoing research into alternative irrigants and adjunctive strategies, such as dual action irrigants commercially available as; DualRinse (Medcem, Weinfelden, Switzerland), Twin Kleen (MaarcDental, Maharashtra, India), and ChloroQuick (Neelkanth Dental and Surgical Factory, Jodhpur, India), in which an agent is combined with NaOCl to preserve the antimicrobial efficacy of sodium hypochlorite while minimizing its adverse effects. Concurrently, this approach maintains smear layer removal through a concept known as continuous chelation. The solution can then be used as the sole irrigant throughout the entire root canal preparation process.[9,10]
In response to these concerns, a recently introduced all-in-one endodontic irrigant, Triton, combines chelating agents, surfactants, and sodium hypochlorite without the use of EDTA, chlorhexidine, or antibiotics. Delivered via an automix system, its dual-component formulation produces a final 4% sodium hypochlorite solution, enabling concurrent organic and inorganic tissue dissolution.[9]
According to the manufacturer, Brasseler USA, Triton All-in-One Irrigation Solution is a dual component endodontic irrigant. The system includes two separate solutions, Part A and Part B, which are combined using a proprietary automix cap to produce the final irrigant. Part A contains citric acid; the main chelator, Sodium hydroxide; a pH modifier along with a mix of surfactants, stabilizers, and water. While Part B consists of 8% sodium hypochlorite (NaOCl), a pH modifier, and water. When mixed, the resulting solution delivers approximately 4% NaOCl along with the chelators, surfactants, and other additives, enabling concurrent organic tissue dissolution, inorganic debris removal, and smear layer elimination, while maintaining chemical stability.[10]
Up to this date, there are few studies available; Xuyang Sheng et al. 2023 and Mallory Hackbarth et al. 2024 discussing the antibacterial efficacy of Triton irrigation solution. Hence, this in vitro study aims to evaluate the antibacterial efficacy of Triton All-In-One solution compared to Sodium hypochlorite against Enterococcus faecalis in extracted mandibular premolars.
Objectives
To evaluate the antibacterial efficacy of Triton All-In-One irrigation solution compared to Sodium hypochlorite against E. faecalis in extracted mandibular premolars
Research Question
Will the use of Triton All-In-One irrigation solution differ from Sodium hypochlorite in terms of antibacterial efficacy against E. faecalis in extracted mandibular premolars?
Hypothesis
Null Hypothesis (H0): There is no difference in the antibacterial efficacy between Triton All-In-One irrigation solution and sodium hypochlorite against E. faecalis in extracted mandibular premolars
PICO
· P (Population):
Extracted mandibular premolars infected with Enterococcus faecalis
I (Intervention):
I1: Irrigation using Triton All-In-One solution
I2: Irrigation using 5% Sodium Hypochlorite + 17% EDTA
C (Control):
Irrigation using 5% Sodium hypochlorite
O (Outcome):
Antibacterial efficacy against E. faecalis in the root canal system
Trial Design
Type: comparative in vitro study
Allocation ratio: 1:1:1
Framework: superiority
Materials and Methods
Sample Size Calculation
In a previous study by ElSawaf et al. in 2024 the antibacterial effect against Enterococcus faecalis within the 5.25% NaOCl group was normally distributed with a mean and standard deviation of 98.71± 0.25%. If the true difference in the experimental and control means is 0.25%, with a large Cohen's d effect size of 1, we will need to study 17 samples per group to be able to reject the null hypothesis that the population means of the experimental and control groups are equal with probability (power) 0.8. The Type I error probability associated with this test of this null hypothesis is 0.05. Sample size was calculated using PS Power and Sample for windows version 3.1.6 using independent t test.
Sample Description
Fifty-one extracted mandibular premolars due to periodontal or orthodontic problems, collected from the Oral & Maxillofacial department, Faculty of Dentistry, Cairo University
· Eligibility criteria:
Inclusion Criteria:
Mandibular premolars
Teeth with fully developed roots and no visible caries or cracks
Exclusion Criteria:
Teeth with previous endodontic treatment
Teeth with severe curvature
Teeth with calcified canals
Teeth with open apices
Teeth with visible cracks or fractures
Teeth with root resorption
Teeth with perforations
Teeth with extensive restorations
Sample preparation
· A total of 51 extracted mandibular premolars will be collected for this study. The external root surfaces will be debrided using curettes to remove adherent periodontal tissue and calculus, followed by immersion in 5% Sodium hypochlorite (NaOCl) (JK Dental Vision, Mansoura, Egypt) for 30 minutes to eliminate any remaining organic debris.
· The teeth will then be thoroughly rinsed and stored in sterile saline solution until further use.
· The teeth will be decoronated using a diamond disc under copious water irrigation to standardizeroot length to 16 mm.
· A #10 K-file (MANI, INC. Japan) will be used to confirm canal patency, and the working length will be determined using a #15 K-file (MANI, INC. Japan) inserted until it is visible at the apex, then subtracting 1 mm to obtain a standardized working length of 15 mm.
· Root canal instrumentation will be performed using Pepsi Gold (Fanta Dental Materials Co., Ltd, Shanghai, China) rotary multi-file system up to size 40, taper 4%, in accordance with the manufacturer’s instructions.
· The endo motor used will be E-Connect Pro Max (E-Value) by Eighteeth (Eighteeth (Changzhou Sifary Medical Technology Co., Ltd., China)
· Irrigation will be performed using 2 mL of 5% NaOCl (JK Dental Vision, Mansoura, Egypt) after each rotary file, delivered with a 30-gauge side vented needle inserted 2 mm short of the working length.
· Apical patency will be maintained with a #10 K-file between each successive file.
· Upon completion of instrumentation, 5 mL of saline will be used as a final rinse, followed by drying with sterile paper points.
· Teeth will be sterilized in an autoclave at 121°C and 15 psi for 20 minutes.
· The prepared canals will be artificially inoculated with Enterococcus faecalis biofilms under standardized laboratory conditions. A pure culture of E. faecalis (ATCC 29212) will be grown in Brain Heart Infusion (BHI) and incubated at 37°C for 24 hours.
· The sterilized root canals will be inoculated with the bacterial suspension and incubated for 21 days, with fresh BHI broth replenished every 48 hours to maintain bacterial viability.
· This protocol ensures consistent contamination of all specimens with mature biofilms prior to the application of the irrigant regimens.
· After 21 days of E. faecalis incubation inside the root canals, pre-irrigation bacterial samples (S1) will be obtained from each tooth with a sterile size #40 paper point that will be entered to the full working length, moved circumferentially along the walls, and transferred to Brain Heart Infusion (BHI) broth for culturing and Colony Forming Units (CFU) quantification
· Irrigation protocols will be applied to three groups, after which post-irrigation bacterial samples (S2) will be obtained in the same manner to calculate the percentage of the bacterial load reduction.
The specimens will be randomly divided into three experimental groups 17 per group
Intervention for each group
Group A: Triton Solution
- Irrigation will be carried out with 5 mL of Triton solution
- The solution will be delivered using a 30-gauge side vented needle 2 mm short of the working length
- Activation will be done using ultrasonic tips for 60 seconds followed by water rinse
Group B: 5% Sodium Hypochlorite + 17% EDTA
- Irrigation will be carried out with 5 mL of 5% NaOCl
- The solution will be delivered using a 30-gauge side vented needle 2mm short of the working length
- Activation will be done using ultrasonic tips for 60 seconds followed by saline rinse
- 1 mL of 17% EDTA will then be used as a final flush followed by saline rinse
Group C: 5% Sodium Hypochlorite (NaOCl)
- Irrigation will be carried out with 5 mL of 5% NaOCl
- The solution will be delivered using a 30-gauge side vented needle 2mm short of the working length
- Activation will be done using ultrasonic tips for 60 seconds followed by saline rinse
Outcomes
The efficacy of the interventions will be measured using the following primary outcome.
| A | B | C | D | E | |
| Outcome Measured | Description | Method | Unit of Measurement | Timepoint | |
| Antibacterial Efficacy | Bacterial Load Reduction Percentage | Culturing technique (S1 - S2) / S1 X 100Hanafy et al. 2025 | CFUs per mL | S1: Pre-irrigation after bacterial incubation S2: Immediately post-irrigation |
After 21 days of incubation with E. faecalis, pre-irrigation bacterial samples (S1) will be collected from the root canals using sterile size #40 paper points and transferred to Brain Heart Infusion (BHI) broth for culturing and CFU quantification. Following the application of the irrigation protocols to the three groups, post-irrigation samples (S2) will be collected similarly to determine the percentage reduction in bacterial load.
Assignment to intervention
Sequence generation
Random allocation and sequence generation will be performed using computer random sequence generator program. (https//:www.random.org).
Allocation concealment
To maintain allocation concealment, a sealed envelope system will be employed. Each envelope willcontain the allocation group corresponding to a specific tooth number. The envelopes will be opaque to prevent any personnel from anticipating the allocation. The envelopes will be opened only after the teeth have been prepared and ready for intervention to ensure that the allocation sequence remains concealed until the point of intervention.
Implementation
Random allocation, sequence generation and the allocation concealment will be performed by the Co-supervisor.
Blinding
To minimize potential bias, the following blinding strategies will be implemented.
Assessor blinding: Laboratory personnel responsible for conducting the microbial assays evaluation will be blinded to group allocation. They will be informed only that the specimens originate from an experimental study, without disclosure of the specific treatment received.
Statistician blinding: The statistician will be unaware of the identity or allocation of study groups during data analysis, so that the results are analyzed objectively without influence from expectations or prior knowledge about the interventions.
Statistical methods
Data will be analyzed using Medcalc software, version 22 for windows (MedCalc Software Ltd, Ostend, Belgium). Continuous data will be described using mean and standard deviation. Intergroup comparison between continuous variables will be performed using independent t test. Intragroup comparison will be done using paired t test. A p-value less than or equal to 0.05 will be considered statistically significant and all tests will be two tailed. Statistical power of the study will be set at 80 % with 95 % confidence level.
Ethics
The research will be submitted to the Ethics Committee for review and approval. Upon completion of the experiment and analysis of the results, all instruments and tooth samples will be sterilized and disposed of in a designated incinerator under the supervision of the Microbiology Department, Cairo University.
Protocol references
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[2] Mamat R, Nik Abdul Ghani NR. The Complexity of the Root Canal Anatomy and Its Influence on Root Canal Debridement in the Apical Region: A Review. Cureus. 2023;15(11):e49024. Published 2023 Nov 18. doi:10.7759/cureus.49024
[3] Elsawaf, G., Gawdat, S., Dwedar, R., Emara, R. Antibacterial Efficacy of Sodium Hypochlorite at Different Temperatures Against Enterococcus Faecalis in Single Rooted Teeth (A Comparative In-Vitro Study). Advanced Dental Journal, 2024; 6(4): 666-674. doi: 10.21608/adjc.2024.261259.1460
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[9] Vidal-Montolío MI, Sanz JL, Ghilotti J, Folguera S, Llena C. Efficacy of Etidronic Acid for Smear Layer Removal: A Systematic Review of In Vitro Studies. J Funct Biomater. 2026;17(1):48. Published 2026 Jan 16. doi:10.3390/jfb17010048
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[12] Brasseler USA. Triton All‑In‑One Irrigation Solution FAQs. Brasseler USA; 2022. https://brasselerusadental.com/wp-content/uploads/sites/9/2022/01/Triton-FAQs-Final.pdf. Accessed January 8, 2026.
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