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 Table of Contents  
Year : 2017  |  Volume : 4  |  Issue : 3  |  Page : 194-197

Comparative evaluation of four different sterilization methods on contaminated endodontic files

1 Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India
2 Department of Pedodontics and Preventive Dentistry, Narayana Dental College, Nellore, Andhra Pradesh, India
3 Department of Microbiology and Pathology, Nagarjuna University, Guntur, Andhra Pradesh, India

Date of Web Publication13-Jul-2017

Correspondence Address:
Pratap Goud Jai Shankar Margana
Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjhr.cjhr_12_17

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Background: The reuse of instruments in the field of endodontics leads to cross infection due to contamination with microbes as the instruments come into direct contact with saliva, blood, and infected tissues. Since microbes are considered to be the major cause of endodontic failures, sterilization of endodontic instruments is mandatory for maintaining asepsis to prevent cross-contamination from one person to other. Hence, the present study was conducted to compare the effectiveness of four different methods of sterilizing contaminated endodontic files. Materials and Methods: A total of 48 stainless steel K files were divided into four groups based on the sterilization method followed – Group A: Autoclave, Group B: Glass bead sterilization, Group C: Glutaraldehyde, and Group D: Quitanet Plus (aldehyde-free solution). In all the tested groups, half of the files were contaminated with Escherichia coli and remaining with Enterococcus faecalis. Then, presterilization colony counts were recorded, followed by sterilization through respective methods. Later, the sterilized files were rinsed with distilled water and 100 ul of the diluted concentration was transferred and cultured onto the respective agar plates to determine the total microbial reduction. Results: Autoclave showed complete effectiveness in reducing the microbial count followed by Quitanet Plus, glass bead sterilizer, and glutaraldehyde. Conclusion: Autoclave is considered to be the best sterilization technique to prevent cross infection in endodontic therapy.

Keywords: Disinfection, Enterococcus faecalis, Escherichia coli, endodontic files, microorganisms, sterilization

How to cite this article:
Yenni M, Bandi S, Avula SS, Margana PG, Kakarla P, Amrutavalli A. Comparative evaluation of four different sterilization methods on contaminated endodontic files. CHRISMED J Health Res 2017;4:194-7

How to cite this URL:
Yenni M, Bandi S, Avula SS, Margana PG, Kakarla P, Amrutavalli A. Comparative evaluation of four different sterilization methods on contaminated endodontic files. CHRISMED J Health Res [serial online] 2017 [cited 2022 Aug 11];4:194-7. Available from: https://www.cjhr.org/text.asp?2017/4/3/194/210476

  Introduction Top

Virtually, 700 bacterial species reside in the oral cavity of human beings with each individual harboring 100–200 species on an average.[1] Even though most of the oral microorganisms are commensals, under certain circumstances some of these will become pathogenic causing oral infections. Various studies proved that the presence of microbiota was a major determinant in endodontic infections, thus the success of endodontic treatment depends on the eradication of these microbes from the pulp chamber and root canals.[2],[3] This can be achieved by various endodontic instruments such as barbed broaches, reamers, and files.[3]

The biomechanical preparation and intricate anatomy of the root canal may result in accumulation of residual organic and inorganic material during the procedure.[4] These residues may act as antigens, infectious agents, or nonspecific irritants, increasing the likelihood of cross infection from one patient to another; therefore, asepsis of reused instruments is especially important in endodontic therapy.[5],[6]

Autoclave works by utilizing heat in the form of saturated steam under controlled pressure and temperature.[1] Even though it is time-consuming, this method has several advantages such as excellent microbial lethality, cost-effectiveness, lack of toxic residues, and the ability to be physically monitored.

Glass bead sterilizer, which works under the principle of dry heat, is the rapid chairside sterilization technique and is the most commonly used method of sterilization of endodontic files. The beads used should be smaller than 1 mm in diameter because large beads are not effective in transferring heat to the instruments. Moreover, the presence of large air spaces between the beads prevents heat transfer.[7]

The most commonly used agent for cold sterilization is glutaraldehyde. It has a broad spectrum of biocidal activity with pungent odor.[8] It penetrates into blood and exudates due to its low surface tension and permits rinsing.[9] However, contact with glutaraldehyde liquid as well as vapor severely irritates the eyes and burns the skin. Hence, the need for safer chairside cold sterilization method is looked on as an alternative.[10]

Quitanet Plus is recently introduced quaternary ammonium compound which comprises alkyl dimethyl benzyl ammonium chloride and didecyldimethylammonium chloride, commonly called as “quats.”[11] It is an aldehyde-free liquid used for disinfection and cleansing of operatory instruments. It has bactericidal, tuberculocidal actions and also acts against HIV.[12]

Even though there are various techniques for the sterilization of endodontic instruments, studies comparing these were scanty. Hence, the present study was aimed to compare the effectiveness of four different sterilization methods (autoclave, glass bead sterilization, glutaraldehyde, and Quitanet Plus) on contaminated endodontic files.

  Materials and Methods Top

After obtaining the approval for the study design from the Institutional Ethical Committee, this in vitro microbial study was conducted in the Department of Pedodontics and Preventive Dentistry in collaboration with the Department of Botany and Microbiology to evaluate the efficacy of various methods of sterilizing the endodontic hand files. The test microorganisms used in the present study were Enterococcus faecalis (MTCC no. 452) and  Escherichia More Details coli (MTCC no. 439).

Lyophilized forms of these two different bacterial species were activated by growing them on respective selective media (trypticase soy agar for E. faecalis, Davis supplemented minimal medium for E. coli) which favor the growth of individual bacterium as described by the standard procedure of MTCC, Chandigarh, India.

The study sample comprised 48 files (Prime Dental Products Pvt. Ltd., Mumbai, India) which were divided into four groups based on the method of sterilization – Group A: Autoclave, Group B: Glass bead sterilization, Group C: Glutaraldehyde (Merck specialities Pvt. Ltd. Mumbai, India), and Group D: Aldehyde-free solution (Quitanet Plus) (Septodont Healthcare India Pvt. Ltd., Maharashtra, India). All the files included in this study were presterilized in an endodontic instrument box by autoclaving for 30 min at 121°C at a pressure of 15 pounds for standardization.

The presterilized files were placed in the test tubes containing respective bacterial broths and left for 24 h for contamination at 37°C, followed by transfer of these diluted concentrations (100 μl) onto the agar plates using spread plate technique. After incubating these agar plates for 24 h, they were subjected to colony count which served as presterilization values. Once these values were obtained, contaminated files in their respective groups were sterilized using the following methods: In Group A, files were placed in endodontic instrument box and were subjected to autoclave at 121°C, for 15 min at 15 lb pressure; in Group B, files were placed in the periphery of the glass bead sterilizer for 10 s at 240°C with beads of size 1–1.5 mm; in Group C, files were placed in a sterile glass container containing 2% glutaraldehyde solution and left for 30 min; and in Group D, files were placed in a sterile glass container containing aldehyde-free solution (Quitanet Plus) and left for 20 min (as per the manufacturer instructions).

After sterilization, the files were rinsed with distilled water and 100 μl of the diluted concentration was transferred onto the prepared  Petri dish More Detailses and incubated at 37°C. Further, they were checked for growth of microorganisms after 24 h, and the colony forming units (CFU) were counted with the help of colony counter using the following formula: Number of colonies/dilution factor × volume plated.

The whole procedure was conducted by a single investigator. To avoid bias in the result, a second investigator randomly evaluated the samples and calculated the CFU's. As the interexaminer variability was insignificant, the prior results were only considered. The values thus obtained were subjected to statistical analysis using Wilcoxon signed-rank test.

  Results Top

The postoperative samples of Group A showed complete sterilization followed by Group D, B, and C for both the tested organisms. Out of the two tested microorganisms, E. faecalis showed more resistance to all the sterilization methods compared to E. coli [Table 1] and [Figure 1]a, [Figure 1]b.
Table 1: Mean microbial reduction in all the four groups

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Figure 1: (a) Colony-forming units against Escherichia coli with different sterilization methods. (b) Colony-forming units against Enterococcus foecalis with different sterilization methods

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Out of the four sterilization techniques followed in this study, superior results were noticed in Group A samples. However, the remaining three techniques also proved effective as they showed statistically significant difference between pre- and post-sterilization values (P < 0.01).

  Discussion Top

Root canal infection is a dynamic process with diverse microbes such as Gram-positive facultative cocci, lactobacilli, Fusobacterium nucleatum, Actinomyces, Porphyromonas endodontalis, Porphyromonas gingivalis, E. coli, E. faecalis, and Candida, with Actinomyces which are dominating at various stages of disease process.[13] Even though about 500 bacterial species are recognized as normal inhabitants of oral cavity, only 150 microbial species have been isolated from the infected root canals.[14] In the present study, E. faecalis and E. coli were chosen as the test microorganisms because 11.43% of E. coli and 29.77% E. faecalis have been isolated in primary and secondary endodontic infections, respectively.[1],[15],[16] E. faecalis, a Gram-positive facultative anaerobic spherical bacterium, can survive in very harsh environments including extremely alkaline pH (9.6) and salt concentrations. It has been reported to be associated with persistent infections and failed endodontic therapies nine times more than the cases of primary infections.[17] E. coli is a Gram-negative facultative anaerobic rod-shaped bacterium. It was also isolated from persistent periapical infections whose virulence factors are mainly due to “O” antigen of the polysaccharide capsule.[18]

The classic endodontic triad for the success of root canal treatment includes canal instrumentation, i.e., cleaning along with shaping, disinfection, and obturation, out of which canal instrumentation is commonly accomplished by endodontic files. Thus, sterilization of these contaminated instruments is imperative for achieving success in the endodontic treatment.[19]

In this study, autoclave (Group A) showed complete sterilization of all the samples for both the microorganisms. The results were in agreement with the studies conducted by Hurtt and Rossman [20] and Rajkumar and Lakshminarayanan.[21] However, contradictory results were noticed in the study by Schug-Kosters et al. who stated that hot steam fails to reach all the intricate parts of the endodontic instruments resulting in incomplete sterilization.[22]

Quitanet Plus (Group D) is an aldehyde-free solution which acts by disruption of intermolecular interactions within the cell membrane of microorganisms, thereby compromises the cellular permeability and induces leakage of cellular contents.[11] In the present study, Quitanet Plus showed better efficacy next to autoclave. However, a study by Halebathi et al.[11] showed that Quitanet Plus was least efficient in sterilizing the endodontic files and this could be due to varying sterilization protocols followed during the study.

Glass bead sterilizer (Group B) showed incomplete sterilization of the samples for both the tested organisms in spite of the smaller sized (1–1.5 mm) beads used, which results in better conduction of heat.[23] Moreover, intense dry heat damages vegetative and spore forms of bacteria.[24] The results of the present study were alike to the study conducted by Raju et al.[23] The incomplete effectiveness of dry-heat sterilization was due to its low penetrating ability into the microbes compared to moist heat.

The outcome of glutaraldehyde (Group C) sterilization in this study was unsatisfactory because none of the files showed complete sterility. Glutaraldehyde acts by denaturation of proteins and alkylation of nucleic acids of bacteria. The other mode of action involves cross-linking of proteins at outer and inner layers of bacterial cell that leads to inhibition of transport, enzyme activity, and synthesis of RNA, DNA, and proteins.[23] The results were similar to studies conducted by Venkatasubramanian et al.,[24] Kumar et al.[12] who suggested that incomplete sterilization of endodontic files with glutaraldehyde might be due to the insufficient immersion time or may be due to some unknown resistant factor of bacteria to that chemical.

Further studies with larger sample are recommended to evaluate the detrimental effects of endodontic files following sterilization to emphasize the efficient sterilization method without damaging the working efficacy of instruments.

  Conclusion Top

Proper sterilization protocol is mandatory to prevent cross infection among patients. In the present study, autoclave showed superior efficacy for both E. coli and E. faecalis and hence it can be considered as the gold standard method. Quitanet Plus also showed better efficacy on both the tested organisms, so it can be recommended as an alternative technique.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Henderson B, Wilson M. Commensal communism and the oral cavity. J Dent Res 1998;77:1674-83.  Back to cited text no. 1
Gajan EB, Aghazadeh M, Abashov R, Salem Milani A, Moosavi Z. Microbial flora of root canals of pulpally-infected teeth: Enterococcus faecalis a prevalent species. J Dent Res Dent Clin Dent Prospects 2009;3:24-7.  Back to cited text no. 2
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Vinay P, Reddy GY, Hegde N, Darshini P. Sterilization methods in orthodontics. A review. Int J Dent Clin 2011;3:44-7.  Back to cited text no. 10
Halebathi RG, Dodwad PK, Uppin VM, Gandhi B, Sarangi P, Sahni N. Comparative evaluation of efficacy of two methods of sterilization for rotary nickel-titanium files: An in vitro study. World J Dent 2011;2:193-8.  Back to cited text no. 11
Kumar KV, Kiran Kumar KS, Supreetha S, Raghu KN, Veerabhadrappa AC, Deepthi S. Pathological evaluation for sterilization of routinely used prosthodontic and endodontic instruments. J Int Soc Prev Community Dent 2015;5:232-6.  Back to cited text no. 12
Peciuliene V, Maneliene R, Balcikonyte E, Drukteinis S, Rutkunas V. Microorganisms in root canal infections: A review. Stomatologija 2008;10:4-9.  Back to cited text no. 13
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Rani A, Chopra A. Isolation and identification of root canal bacteria from symptomatic non vital teeth with periapical pathosis. Endodontology 2006;18:12-7.  Back to cited text no. 15
Rana V, Baba SM, Pandey A. Bacteriology of infected deciduous root canal – A review. Peoples J Sci Res 2009;2:45-8.  Back to cited text no. 16
Narayanan LL, Vaishnavi C. Endodontic microbiology. J Conserv Dent 2010;13:233-9.  Back to cited text no. 17
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Rajkumar K, Lakshminarayanan L. The effectiveness of two commonly used methods of sterilizing endodontics. J Indian Dent Assoc 2001;72:245-8.  Back to cited text no. 21
Schug-Kosters M, Wiegandt M, Feistl R. Sterilization of endodontic instruments with gasseous formaldehyde. Dent Abstr 1957;2:217-8.  Back to cited text no. 22
Raju TB, Garapati S, Agrawal R, Reddy S, Razdan A, Kumar SK. Sterilizing endodontic files by four different sterilization methods to prevent cross-infection: An in-vitro study. J Int Oral Health 2013;5:108-12.  Back to cited text no. 23
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