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Year : 2015  |  Volume : 5  |  Issue : 2  |  Page : 125-128

Effect of sonic agitation, manual dynamic agitation on removal of Enterococcus faecalis biofilm

1 Department of Conservative and Endodontics, Vananchan dental college. Farathiya, Hoor, Garwah, Jharkhand, India
2 Department of Conservative Dentistry and Endodontics, Sree Siddhartha Dental College and Hospital, Karnataka, India

Date of Web Publication20-Apr-2015

Correspondence Address:
Rajshekhar Chatterjee
C/O: Senior lecturer, Department of Conservative and Endodontics. Vananchan Dental College. Farathiya, Hoor, Garwah. Jharkhand - 822 114
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1658-5984.155451

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Objectives: The aim of the study was to compare manual dynamic agitation with sonic agitation on removal of intra radicular Enterococcus faecalis (E. faecalis) biofilm. Material and Methods: Extracted mandibular premolars for orthodontic purpose were sectioned at cervical level and divided into three groups (n = 30). The root canals were instrumented using Protaper rotary instruments up to apical file F4. Roots were sterilized and E. faecalis bacteria were incubated within their root canal space for four weeks. Confirmation of biofilm was done using scanning electron microscopy and Gram staining. All groups were irrigated with side vented needle by using three percent sodium hypochlorite (NaOCl) for 60 seconds. Two experimental groups were agitated with manual dynamic agitation (with master gutta-percha cone) and sonic agitation (EndoActivator). Remaining bacteria were collected using sterile paper point, which were incubated inside brain-heart infusion (BHI) broth to check turbidity. The turbid broth was streaked on blood agar plate for colony counts. Result: Both experimental groups showed highly significant difference in their mean colony count when compared with control group; with P < 0.001. Conclusion: Passive sonic agitation with EndoActivator has proven to be the best irrigating system followed by manual dynamic agitation and conventional needle irrigation.

Keywords: EndoActivator, Enterococcus faecalis, intraradicular biofilm, manual dynamic agitation

How to cite this article:
Chatterjee R, Venugopal P, Jyothi K N, Jayashankar C M, Kumar S A, Kumar P S. Effect of sonic agitation, manual dynamic agitation on removal of Enterococcus faecalis biofilm. Saudi Endod J 2015;5:125-8

How to cite this URL:
Chatterjee R, Venugopal P, Jyothi K N, Jayashankar C M, Kumar S A, Kumar P S. Effect of sonic agitation, manual dynamic agitation on removal of Enterococcus faecalis biofilm. Saudi Endod J [serial online] 2015 [cited 2023 Feb 1];5:125-8. Available from: https://www.saudiendodj.com/text.asp?2015/5/2/125/155451

  Introduction Top

Root canal infections are polymicrobial in nature. [1]  Enterococcus faecalis Scientific Name Search aecalis) is the most commonly found bacterial species responsible for both primary and secondary root canal infections. [2],[3] It is an established fact that bacteria inside infected root canals exist in the form of a biofilm rather than as free floating organisms. [2],[4],[5] Biofilms are heterogeneous arrangement of microbial cells that are embedded in a self-made extracellular polymeric substance (EPS) matrix. This EPS matrix makes them 1000 times more resistant to phagocytosis and antimicrobial agents. [6] Moreover, as the biofilm matures they get readily anchored on the substrate by the formation of a conditioning layer. [6],[7] Removal of these infected biofilm is always a challenge in endodontics. Antibacterial irrigating solutions alone may not be effective to kill and remove these biofilms. [7],[8] Proper physical agitation with a strong antibacterial solution is necessary to remove these biofilms from root canal walls. [8],[9] In this process, numerous developments have been made to create more effective irrigant delivery systems with proper physical agitation. Sonic and ultrasonic micro energies are predominantly used in contemporary irrigation agitation techniques. [10] There is renewed interest in sonic agitation techniques with the introduction of EndoActivator (Dentsply), where a smooth disposable polymer tip produces a powerful hydrodynamic turbulence within the root canal system. [10],[11] Previous studies have also proven the superiority of this sonic agitation over static irrigation and passive ultrasonic agitation. [12],[13] Although these machine-assisted agitations are effective in debridement, they all require specialized gadgets. Machtou in 1980 proposed a simpler way of agitation technique by moving a well-fitted gutta-percha (GP) point inside prepared root canal. [14] This manual dynamic agitation has also been proved to be effective in removal of debris and smear layer. [15],[16] Although this agitation technique was effective for debridement, it was not evaluated for removal of biofilm.

The aim of this study was to compare manual dynamic agitation with sonic agitation on removal of E. faecalis biofilm.

The null hypothesis was that there would be no significant difference between manual dynamic agitation and sonic agitation in removing intra-radicular E. faecalis biofilm.

  Materials and methods Top

Institutional ethical clearance was obtained before taking up the study. Ninety-eight extracted mandibular premolar teeth were disinfected with 3% sodium hypochlorite (NaOCl) for five minutes and stored in thymol solution. The coronal part of the teeth was sectioned at cervical level by diamond abrasive disc (Dentsply, India) to maintain 13 mm remaining root length. Root canal instrumentation was carried out with Protaper rotary instrument (Dentsply, India) up to F4 master apical file size. Three percent NaOCl was used for irrigation after each instrumentation. A final flush with 17% ethylenediaminetetraacetic acid (EDTA) (Prime Dental, India) was carried out to remove smear layer. Prepared roots were autoclaved at 121°C under 15 lb pressure for 15 minutes to overcome any cross contamination and sterility of the sample was checked by incubating four of them within brain-heart infusion (BHI) broth (HiMedia, India). E. faecalis (ATCC 29212, Microbiologics, USA) was subcultured from frozen bacterial strip. One McFarland standard suspension was prepared in BHI broth and was diluted 30 fold to obtain an initial bacterial suspension of approximately 1 × 10 7 colony-forming units per ml. Then 1.2 ml of this suspension was inoculated inside each sterile root canal space using micropipette (Picission Biotech, India). Eppendorf tubes (Biogenix. India) containing roots with bacterial inoculum were capped and placed vertically into incubator (Labotech, India). Re-inoculation was performed with fresh bacterial broth every four days to allow a constant source of nutrition for the biofilm to grow. Eight samples were randomly selected and formation of biofilm was confirmed in four samples by scanning electron microscopy (Cambridge S 200, Germany) and four samples for Gram staining. Remaining 90 samples were randomly divided into three groups containing 30 teeth in each group (n = 30). Irrigation protocol was carried out in all groups by using 30-gauge side vented needles (Dentazon, Korea) with 3% NaOCl for 60 seconds. A total volume of five ml of three percent NaOCl irrigation was standardized for all groups. Control group didn't receive any agitation, whereas other two experimental groups received agitation.

Control group: Irrigation with 3% NaOCl, no agitation

Experimental group 1: Manual dynamic agitation with well-fitting F3 gutta-percha cone (Dentsply. India) to the working length with gentle push-pull motion; 100 strokes/30 seconds

Experimental group 2: Sonic agitation with EndoActivator (Dentsply, India) at 10,000 cycles per minute.

Final rinse was done with 17% EDTA and carryover effect of NaOCl was nullified by rinsing the root canals with sodium thiosulfate solution. Remaining bacteria were collected from root canal spaces using sterile paper point (Dentsply, India) which were incubated inside BHI broth to check turbidity. Turbid broth was streaked on blood agar plate (HiMedia, India) for colony count using Digital Colony counter (HiMedia, India), which was expressed in CFU/ml. Collected data was tabulated and subjected for statistical analysis by using Kruskal-Wallis analysis of variance (ANOVA) and Mann-Whitney U test.

  Results Top

All the experimental groups show significantly less colony count compared to control group.

[Table 1] shows mean colony count in different groups. Mean colony count is in the following order: Control group > Group 1 > Group 2.
Table 1: Descriptive table showing the mean colony count in different groups

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[Table 2] shows inter group comparison using Kruskal-Wallis ANOVA. Both experimental groups (groups 1 and 2) show highly significant difference in their mean colony count when compared to control group with P value < 0.001.
Table 2: Intergroup comparison using Kruskall-Wallis ANOVA

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[Table 3] shows groupwise comparisons using Mann-Whitney test. Each group shows highly significant difference in its mean colony count when compared to other groups with P < 0.001.
Table 3: Group - wise comparison by Mann-Whitney test

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  Discussion Top

Enterococcus faecalis is the most dominating species among the root canal flora. It has the ability to form biofilm readily on root canal walls even in its starvation phases. [4],[5],[6],[7],[17]

Specific data regarding the minimum time required for the formation of intra-radicular biofilm is not available in scientific literature. [17],[18],[19],[20],[21] Zhejun Wang et al. have confirmed formation of intra-radicular biofilm in three weeks which is also found to be more resistant to root canal disinfecting agents than one-day-old biofilm. [22] Considering this, a mature biofilm of three weeks was selected in the present study to test the efficacy of irrigating systems on an established root canal infection.

Needle irrigation is the most commonly employed irrigation technique. In most of the clinical situations it produces adequate canal debridement but this type of static irrigation techniques do not create any hydrodynamic turbulence energy within the irrigants. [10],[23],[24]

To overcome the problems of passive needle irrigation, various agitation methods are advocated. Manual dynamic agitation is one among them where a simple gutta-percha master cone is used to manually agitate the irrigants within the canal. [10],[14] In the present study, manual dynamic agitation was found to remove significantly more biofilm than passive needle irrigation, which is in accordance with the study results of Huang et al.[15],[16] The energy created by the moving gutta-percha point in this technique could have been the main reason to physically disrupt the biofilm from the root canal walls. [10],[15],[16]

In the present study passive sonic agitation with EndoActivator has proved to achieve maximum disinfection by removing intraradicular biofilm significantly compared to other methods. This is similar to the research result of Cameron et al.[12],[13]

EndoActivator produces powerful hydrodynamic intracanal waves. The random explosion of these waves creates bubbles which oscillate within the given solution. These bubbles expand and collapse as an implosion leading to radiating shockwaves that dissipate at a speed of 25,000-30,000 times per second. These shock waves serve to detach the biofilm from root canal surfaces. [10],[11] The bio-acoustic effect of sonic irrigation was also reported by Ya Shen et al. where the shock waves created by EndoActivator have been found to transport disinfecting agents deep into biofilm by breaking its protective mechanism and leading to bacterial death. [25] This action failed to totally remove the calcium hydroxide particles from the middle and apical third of the root canals. [26]

In the present study manual dynamic agitation has not performed as effectively as sonic agitation. The reason behind this could be, the energy created by push-pull motion of the GP point (3.3 Hz) is much lesser than sonic energy of 1-6 kHz, but manual dynamic agitation is a simple, cost-effective way of root canal agitation technique, which removes significantly more bacterial biofilm than syringe irrigation in the absence of any gadgets. [10],[15],[16] Clinical randomized controlled trials are awaited to observe the impact of all these irrigation systems in achieving clinical success.

  Conclusion Top

Within the parameters of the study, it can be concluded that:

  • None of the groups showed complete removal of E. faecalis biofilm from the root canal
  • Manual dynamic agitation and sonic agitation performed better in removing intra radicular E. faecalis biofilm compared to syringe irrigation
  • Maximum removal of E. faecalis biofilm was achieved by using sonic agitation with EndoActivator.

  Acknowledgment Top

I hereby acknowledge Dr. Kishor Bhat, Professor and Head of department Oral Microbiology, Maratha Mandala Dental college, Belgum, India for his help and support.

  References Top

Hargreaves KM, Cohen S. Cohen's pathway of the pulp. 10 th ed.. India: Elsevier; 2011. p. 529-55.  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
Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: Its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:51-6.  Back to cited text no. 3
Ricucci D, Siqueira JF Jr. Biofilm and apical periodontitis: Study of prevalence and association with clinical and histopathologic findings. J Endod 2010;36:1277-88.  Back to cited text no. 4
George S, Kishen A, Song KP. The role of environmental changes on monospecies biofilm formation on root canal wall by Enterococcus faecalis. J Endod 2005;31:867-72.  Back to cited text no. 5
Usha HL, Kaiwar A, Mehta D. Biofilm in endodontics: New understanding to an old problem. Int J Contemp Dent 2010;1:44-51.  Back to cited text no. 6
Ingle JI, Baumgartner LK. Ingle's Endodontics. 6 th ed. India: CBS Publishers; 2013. p. 268-86.  Back to cited text no. 7
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Haapasalo M, Shen Y, Qian W, Gao Y. Irrigation in endodontics. Dent Clin North Am 2010;54:291-312.  Back to cited text no. 9
Gu LS, Kim JR, Ling J, Choi KK, Pashley DH, Tay FR. Review of contemporary irrigant agitation techniques and devices. J Endod 2009;35:791-804.  Back to cited text no. 10
11. Ruddle CJ. Hydrodynamic disinfection tsunami endodontics. Dent Today 2007;26:110, 112, 114-711. .  Back to cited text no. 11
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. 12
Kanter V, Weldon E, Nair U, Varella C, Kanter K, Anusavice K, et al. A quantitative and qualitative analysis of ultrasonic versus sonic endodontic systems on canal cleanliness and obturation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:809-13.  Back to cited text no. 13
Machtou P. Irrigation investigation in endodontics. Paris VII University, Paris, France: Master's Thesis; 1980.  Back to cited text no. 14
Huang TY, Gulabivala K, Ng YL. A bio-molecular film ex-vivo model to evaluate the influence of canal dimensions and irrigation variables on the efficacy of irrigation. Int Endod J 2008;41:60-71.  Back to cited text no. 15
Paragliola R, Franco V, Fabiani C, Mazzoni A, Nato F, Tay FR, et al. Final rinse optimization: Influence of different agitation protocols. J Endod 2010;36:282-5.  Back to cited text no. 16
Arias-Moliz M, Ferrer-Luque C, Espigares-Garcia M, Baca P. Enterococcus faecalis biofilms eradication by root canal irrigants. J Endod 2009;35:711-4.  Back to cited text no. 17
Liu H, Weix X, Ling J, Wang W, Huang X. Biofilm formation capability of Enterococcus faecalis cells in starvation phase and its susceptibility to sodium hypochlorite. J Endod 2010;36:630-5.  Back to cited text no. 18
Bhuva B, Patel S, Wilson R, Niazi S, Beighton D, Mannocci F. The effectiveness of passive ultrasonic irrigation on intra radicular Enterococcus faecalis biofilms in extracted single rooted human teeth. Int Endod J 2010;43:894-8.  Back to cited text no. 19
Portenier I, Waltimo T, Haapasalo M. Enterococcus faecalis-the root canal survivor and star in post treatment disease. End Top 2003;6:135-59.  Back to cited text no. 20
Soares JA, Roque de Carvalho MA, Cunha Santos SM, Mendonça RM, Ribeiro-Sobrinho AP, Brito-Júnior M, et al. Effectiveness of chemomechanical preparation with alternating use of sodium hypochlorite and EDTA in elimination intra canal Enterococcus faecalis biofilm. J Endod 2010;36:894-8.  Back to cited text no. 21
Wang Z, Shen Y, Haapasalo M. Effectiveness of endodontic disinfecting solutions against young and old Enterococcus faecalis biofilm in dentin canals. J Endod 2012;38:1376-9.  Back to cited text no. 22
Ram Z. Effectiveness of root canal irrigation. Oral Surg Oral Med Oral Pathol 1977;44:306-12.  Back to cited text no. 23
Huffaker SK, Safavi K, Spãngberg LS, Kaufman B. Influence of passive sonic irrigation systems on the elimination of bacteria from root canal systems: A clinical study. J Endod 2010;36:1315-8.  Back to cited text no. 24
Shen Y, Stojicic S, Qian W, Olsen I, Haapasalo M. The synergistic antimicrobial effect by mechanical agitation and two chlorhexidine preparations on biofilm bacteria. J Endod 2010;36:100-4.  Back to cited text no. 25
Al-Garni S, Al-Shahrani S, Al-Nazhan S, Al-Maflehi N. Evaluation of calcium hydroxide removal using EndoActivator system: An in vitro study. Saudi Endod J 2014;4:13-7.  Back to cited text no. 26
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  [Table 1], [Table 2], [Table 3]

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