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Year : 2018  |  Volume : 21  |  Issue : 4  |  Page : 438-442
An in vitro comparison of the antimicrobial efficacy of positive pressure and negative pressure irrigation techniques in root canals infected with Enterococcus faecalis

1 Department of Conservative Dentistry and Endodontics, JCD Dental College, Sirsa, India
2 Private Practitioner, Dental Solutions Clinic and Implant Centre, Ludhiana, India
3 Private Practitioner, Hasija Dental Clinic, Hissar, Haryana, India

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Date of Submission21-Nov-2017
Date of Decision20-Dec-2017
Date of Acceptance19-Mar-2017
Date of Web Publication27-Jul-2018


Aim: The aim of this in vitro study was to compare the efficacy of irrigation with the negative pressure and the traditional positive pressure in eradicating Enterococcus faecalis from the root canal.
Materials and Methods: A total of 40 extracted mandibular premolars were sterilized and divided into four groups with 10 samples each. Then, they were prepared to apical size #35 (Group 1, 3) and 45 (Group 2, 4) and inoculated with E. faecalis for 7 days and then irrigated with 3% sodium hypochlorite and 17% ethylenediaminetetraacetic acid. The positive pressure irrigation was performed in Group 1 and Group 2 while the negative pressure irrigation (NPI) using the Endovac in Group 3 and Group 4. Samples taken after the root canal irrigation procedures were cultured and the colony-forming units were counted. Data were statistically analyzed using the Fisher's exact test.
Results: Results were statistically significant when comparing apical negative-pressure irrigation size 45 (Group 4) to positive-pressure irrigation size 35 (Group 1).
Conclusion: The results of this in vitro study showed that the apical NPI method (Endovac) was more effective in removing E. faecalis from the root canal at the larger apical preparation size.

Keywords: Endodontics; endovac; Enterococcus faecalis; irrigation; negative pressure irrigation

How to cite this article:
Kumar T, Dhillon JS, Gill GS, Singla R, Rani S, Dhillon M. An in vitro comparison of the antimicrobial efficacy of positive pressure and negative pressure irrigation techniques in root canals infected with Enterococcus faecalis. J Conserv Dent 2018;21:438-42

How to cite this URL:
Kumar T, Dhillon JS, Gill GS, Singla R, Rani S, Dhillon M. An in vitro comparison of the antimicrobial efficacy of positive pressure and negative pressure irrigation techniques in root canals infected with Enterococcus faecalis. J Conserv Dent [serial online] 2018 [cited 2020 Feb 18];21:438-42. Available from:

   Introduction Top

The primary aim of endodontic therapy in necrotic teeth is the reduction in the number of bacteria and the other microbes in the root canal system that prevent healing of periradicular periodontitis.[1]

The complex root canal morphology makes complete mechanical debridement a clinical challenge.[2] It has been shown that 35% or more of the canal system remains untouched by endodontic instruments.[3] This finding highlights the importance of the root canal irrigation and the chemical debridement and disinfection of the root canal system.

Sodium hypochlorite (NaOCl) is the most commonly used root canal irrigant due to its antimicrobial properties. It is introduced passively into the canal through a notched end or side-vented needle which is placed 2–3 mm short of the working length. However, if the needle is placed too close to the apical foramen or the irrigant is forcibly expressed, the chances of extrusion of the solution increases.[4]

These concerns advocate search of newer irrigation delivery systems which place the irrigant up to working length without any risk of periapical extrusion. To address these objectives; Endovac (Discus Dental, Culver City, CA, USA) an apical negative-pressure irrigation was developed. Various studies using the Endovac have shown the increased efficiency of irrigation at working length, increased sealer penetration and to avoid extrusion, and complications.[5],[6],[7]

Among the different microorganisms, Enterococcus faecalis is the most difficult bacteria to eradicate from the root canals. It is found in 4%–40% of primary endodontic infections.[8] In root-filled teeth with periradicular lesions it has a prevalence ranging from 30% to 78%.[9],[10]

Various studies [11],[12],[13] have been conducted to check the antimicrobial efficacy of the negative pressure irrigation (NPI) system but with conflicting results. While some studies have shown better results, others have reported no or little effect. This study was conducted with the aim of comparing the antimicrobial efficacy of the positive pressure and the NPI techniques in root canals infected with E. faecalis.

   Materials and Methods Top

Forty intact human single-rooted teeth with one root canal and one apical foramen (mandibular premolars) were collected and stored in distilled water until further use. The presence of a single canal was verified radiographically by buccolingual and mesiodistal radiographs. All teeth crowns were flattened so that only 5 mm of coronal structure remained and the root canal length remains at 16–17 mm. All teeth were divided into four groups of 10 samples each.

  • Group 1: Positive pressure irrigation (PPI) with apical preparation up to no. 35
  • Group 2: PPI with apical preparation up to no. 45
  • Group 3: NPI with apical preparation up to no. 35
  • Group 4: NPI with apical preparation up to no. 45.


The simplified outline of complete procedure is presented in flowchart of methodology [Flowchart 1]. Cultivation of E. faecalis: pure isolated colonies of E. faecalis (ATCC 29212) were grown on sheep blood agar plates. These colonies were suspended in brain–heart infusion (BHI) broth and incubated for 48 h at 37°C to form the inoculum [Figure 1].
Figure 1: Inoculation and Enterococcus faecalis colonies. (a) Inoculation of infected brain heart infusion broth on sheep blood agar plate. (b) Colonies of Enterococcus faecalis on sheep blood agar plates

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Group I

Conventional access cavities were prepared and working length was determined using no. 10 K file. All teeth were prepared to an apical size 35 using the step-back technique. The samples were than sterilized by autoclaving. To check the sterility of the sample each sample was dipped in a test tube containing BHI broth. These test tubes were incubated at 37°C and checked for turbidity after 24 h. If no turbidity was present it was further incubated for another 24 h. If still there was no turbidity, the sample was considered sterile. In case turbidity was present the sample was reprocessed. The sample was held with the help of sterile tweezer. BHI broth containing E. faecalis (see the cultivation of E. faecalis) was taken in a sterile insulin syringe and injected into the prepared canal of the sample until it started trickling from the periapical opening. This infected sample was dipped in a test tube containing 5 ml of sterile BHI broth and incubated at 37°C for 1-week. Then, it was rinsed with freshly prepared distilled water and its external surface was wiped with gauge dipped in alcohol. This sample was then dipped into 5 ml of sterile BHI broth and incubated at 37°C. The test tubes were checked for turbidity every 24 h for a maximum period of 72 h. The appearance of turbidity indicated that the sample is infected with E. faecalis. In this case, no turbidity appeared it was reinfected.

Irrigation of the infected samples

For Group 1 irrigation was performed using the positive pressure technique using 3% NaOCl and 17% ethylenediaminetetraacetic acid (EDTA). Irrigation was performed with a 30-G needle and a 5 ml syringe [Figure 2]a. Irrigation was performed with 3% NaOCl for 2 min, followed by 17% EDTA for 1 min, and 3% NaOCl for 2 min. The total final irrigation time was 5 min.
Figure 2: Positive and negative pressure irrigation. (a) Positive pressure irrigation with 30 G needle. (b) Endovac irrigation

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Checking the antimicrobial efficacy of irrigation

With the help of sterile H-file dentinal shavings were collected from the irrigated sample. The portion of the H-file with the shavings was cut into a small pieces of approximately 2 mm and dipped into sterile BHI broth and incubated for 48 h at 37°C. In case no turbidity appeared within this period, it was an indicator that the sample has been completely sterilized by the irrigation. In case of turbidity 20 μL of this suspension was inoculated on to the sheep blood agar plates which were incubated at 37°C for 48 h. Colony-forming units were counted after the purity of cultures was confirmed by Gram staining and colony morphology. Observations were recorded and put to the statistical analysis.

Group 2 – All the steps were same as Group 1 except that all samples in this group were prepared to an apical size of no. 45.

Group 3 – All the steps were similar to those of Group 1 except irrigation of infected samples.

Irrigation was done using Endovac (or the negative pressure technique) using 3% NaOCl and 17% EDTA following the manufacturer's recommendations [Figure 2]b. Macroirrigation of each canal was accomplished with 3% NaOCl over 30 s. The open-ended macrocannula was moved up and down in the canal from a point where it started to bind to a point only below the orifice. The macrocannula was then removed quickly so that no air was drawn into the canal space and the canal was left full of irrigant. Microirrigation began immediately after the macro-irrigation with three cycles of irrigation. During a cycle of microirrigation, the pulp chamber was kept full with irrigant, whereas the microcannula was taken to the full working length for 6 s, then lifted 2 mm coronally for 6 s, and then moved back to the working length for 6 s. The second cycle of irrigation used 17% EDTA and the third used 6% NaOCl again. The total final irrigation time was 3 min 30 s for the Endovac groups. The total volume of irrigant used was 10 ml in both groups.

Group 4 – All the steps were same as Group 3, except that the biomechanical preparation for the samples was done up to no. 45.

Data were statistically analyzed using the Fisher's exact test with the two-sided P value and level of significance established at P < 0.05.

   Results Top

Results are tabulated in [Table 1]. No statistically significant difference was found between Groups 1 and 2, between Groups 3 and 4, and Group 1 and Group 3. Nonsignificant results were obtained when Group 2 and 3 were compared. The statistically significant difference was observed between Groups 1 and 4 with a value of P < 0.01.
Table 1: Results showing positive and negative samples obtained after irrigation

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   Discussion Top

Microorganisms and their end products are the main cause of pulpal and periapical pathosis. Owing to the complex root canal system mechanical debridement only reduces the half of bacterial load from the root canals. Along with irrigation, this bacterial load decreases by 50%–80%.[14] This underscores the importance of irrigation in endodontics.

Syringe irrigation (PPI) is the most common and widely used method of irrigant delivery.[15] This method is economical, requires a minimal apparatus and has universal availability. Limitations of syringe irrigation include its inability to clean beyond the main root canal,[16] vapor lock,[17] apical extrusion of irrigant,[18] or hypochlorite accidents.[19] Endovac or the NPI was developed to minimize the risks associated with syringe irrigation. Although costly in comparison it is found to be effective in removing debris from the apical third, smear layer removal, and minimize the risk of apical extrusion of irrigant.[20],[21] However, conflicting results have been obtained by the studies reporting its clinical antimicrobial efficacy.[11],[12],[13]

The present study was conducted to ascertain the antimicrobial efficacy of the positive and negative irrigation techniques. E. faecalis was selected for the purpose of the present study, because it is believed to be one of the intracanal bacteria which is most resistant to elimination by disinfecting agents.[22]

The current study found the nonsignificant difference between syringe irrigation groups (Group 1 and 2) although a number of negative cultures were more in Group 2. This is expected as preparing the apical portion of the root canal to the larger instrument size (#45 in Group 2) has been shown to enhance root canal disinfection by reaching inaccessible areas.[23] This provides the potential to remove the intratubular bacteria and open the dentinal tubules to allow antimicrobials to penetrate more effectively. Same holds true for the nonsignificant difference between Group 3 and 4.

Similarly nonsignificant difference was seen between Group 1 and Group 3 which could be due to the reason that although the Endovac microcannula effectively aspirates irrigants in the most apical area of the canal, the clogging of its small perforations by debris may reduce its disinfection effect by reducing the fluid flow in the apical canal.[12],[24]

Significant results are observed in Group 1 (PPI #30) and Group 4 (NPI #45). The study results are in concurrence with the study of Hockett et al.[11] who found the significant reduction in E. faecalis numbers when compared to the positive control needle group. This can be due to the improved efficiency of irrigation with an increase in apical size preparation,[23] and superior removal of debris leading to better disinfection.[20] These results are in conflict with Brito et al.[12] and Miller and Baumgartner [13] who found no significant differences among the conventional irrigation and Endovac irrigation. In Brito et al. the root canals were instrumented to apical size 25 only. Furthermore, paper points were used to assess the microbial load in vitro which has known limitations.[25] In Miller et al. different methodology was used. They bypassed sampling and pulverized the apical 5 mm of each specimen.

Comparison of Group 2 and 4 also yielded nonsignificant results which indicates that preparing a canal to the larger size may leads to better disinfection [22] irrespective of the method of the delivery of irrigant.

The study design may be favorable to the negative-pressure irrigation groups since the microcannula was introduced to working length while the positive-pressure irrigation needle was introduced 2 mm short of working length. However, this simulates the clinical situation that most practitioners feel comfortable and safe with when using syringe irrigation. Thus, the Endovac can act as a better option when cleaning teeth with the large apical preparations but its high cost may act as a deterrent.

   Conclusion Top

Within the limitations of this in vitro study, an apical negative pressure method (Endovac) may be more effective in removing E. faecalis from the root canal at the larger apical preparation size (no. 45).


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Conflicts of interest

There are no conflicts of interest.

   References Top

Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321-8.  Back to cited text no. 1
Giardino L, Ambu E, Becce C, Rimondini L, Morra M. Surface tension comparison of four common root canal irrigants and two new irrigants containing antibiotic. J Endod 2006;32:1091-3.  Back to cited text no. 2
Mitchell RP, Yang SE, Baumgartner JC. Comparison of apical extrusion of NaOCl using the EndoVac or needle irrigation of root canals. J Endod 2010;36:338-41.  Back to cited text no. 3
Sjögren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 1997;30:297-306.  Back to cited text no. 4
Venumbaka NR, Baskaran P, Mungara J, Chenchugopal M, Elangovan A, Vijayakumar P, et al. Comparative evaluation of the efficacy of EndoVac and conventional irrigating systems in primary molars – An in vitro study. J Clin Pediatr Dent 2017. [Epub ahead of print]. doi:10.17796/1053-4628-42.2.10.  Back to cited text no. 5
Kara Tuncer A, Unal B. Comparison of sealer penetration using the EndoVac irrigation system and conventional needle root canal irrigation. J Endod 2014;40:613-7.  Back to cited text no. 6
Siu C, Baumgartner JC. Comparison of the debridement efficacy of the EndoVac irrigation system and conventional needle root canal irrigation in vivo. J Endod 2010;36:1782-5.  Back to cited text no. 7
Rôças IN, Siqueira JF Jr., Santos KR. Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod 2004;30:315-20.  Back to cited text no. 8
Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1-7.  Back to cited text no. 9
Murad CF, Sassone LM, Faveri M, Hirata R Jr., Figueiredo L, Feres M, et al. Microbial diversity in persistent root canal infections investigated by checkerboard DNA-DNA hybridization. J Endod 2014;40:899-906.  Back to cited text no. 10
Hockett JL, Dommisch JK, Johnson JD, Cohenca N. Antimicrobial efficacy of two irrigation techniques in tapered and nontapered canal preparations: An in vitro study. J Endod 2008;34:1374-7.  Back to cited text no. 11
Brito PR, Souza LC, Machado de Oliveira JC, Alves FR, De-Deus G, Lopes HP, et al. Comparison of the effectiveness of three irrigation techniques in reducing intracanal Enterococcus faecalis populations: An in vitro study. J Endod 2009;35:1422-7.  Back to cited text no. 12
Miller TA, Baumgartner JC. Comparison of the antimicrobial efficacy of irrigation using the EndoVac to endodontic needle delivery. J Endod 2010;36:509-11.  Back to cited text no. 13
Gulabivala K, Patel B, Evans G, Ling NG. Effects of mechanical and chemical procedures on root canal surfaces. Endod Topics 2005;10:103-22.  Back to cited text no. 14
Willershausen I, Wolf TG, Schmidtmann I, Berger C, Ehlers V, Willershausen B, et al. Survey of root canal irrigating solutions used in dental practices within Germany. Int Endod J 2015;48:654-60.  Back to cited text no. 15
Jiang LM, Lak B, Eijsvogels LM, Wesselink P, van der Sluis LW. Comparison of the cleaning efficacy of different final irrigation techniques. J Endod 2012;38:838-41.  Back to cited text no. 16
Tay FR, Gu LS, Schoeffel GJ, Wimmer C, Susin L, Zhang K, et al. Effect of vapor lock on root canal debridement by using a side-vented needle for positive-pressure irrigant delivery. J Endod 2010;36:745-50.  Back to cited text no. 17
Druttman AC, Stock CJ. An in vitro comparison of ultrasonic and conventional methods of irrigant replacement. Int Endod J 1989;22:174-8.  Back to cited text no. 18
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. 19
Kungwani ML, Prasad KP, Khiyani TS. Comparison of the cleaning efficacy of EndoVac with conventional irrigation needles in debris removal from root canal. An in-vivo study. J Conserv Dent 2014;17:374-8.  Back to cited text no. 20
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Kumar VR, Bahuguna N, Manan R. Comparison of efficacy of various root canal irrigation systems in removal of smear layer generated at apical third: An SEM study. J Conserv Dent 2015;18:252-6.  Back to cited text no. 21
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Ahangari Z, Samiee M, Yolmeh MA, Eslami G. Antimicrobial activity of three root canal irrigants on Enterococcus faecalis: An in vitro study. Iran Endod J 2008;3:33-7.  Back to cited text no. 22
Rodrigues RC, Zandi H, Kristoffersen AK, Enersen M, Mdala I, Orstavik D, et al. Influence of the apical preparation size and the irrigant type on bacterial reduction in root canal-treated teeth with apical periodontitis. J Endod 2017;43:1058-63.  Back to cited text no. 23
Kurtzmann GM. Improving endodontic success through the use of the EndoVac irrigation system. Endod Pract 2009;10:17-20.  Back to cited text no. 24
Sathorn C, Parashos P, Messer HH. How useful is root canal culturing in predicting treatment outcome? J Endod 2007;33:220-5.  Back to cited text no. 25

Correspondence Address:
Dr. Tarun Kumar
Department of Conservative Dentistry and Endodontics, JCD Dental College, Sirsa, Haryana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JCD.JCD_328_17

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