Indian Journal of Dental Research

: 2013  |  Volume : 24  |  Issue : 4  |  Page : 451--455

Assessment of precipitate formation on interaction of irrigants used in different combinations: An in vitro study

Amarnath Shenoy1, Nagesh Bolla2, Sayish2, Raj K Sarath2, CH Sunil Ram2, Sumlatha2,  
1 Department of Conservative Dentistry and Endodontics, Yenepoya Dental College, Mangalore, India
2 Department of Conservative Dentistry and Endodontics, SIBAR Dental College, Guntur, Andhra Pradesh, India

Correspondence Address:
Amarnath Shenoy
Department of Conservative Dentistry and Endodontics, Yenepoya Dental College, Mangalore


Introduction: Irrigants play an essential role in the successful debridement and disinfection of pulp space. Various combination of irrigants used during root canal treatment enhance their efficacy, but some form precipitates which affects the diffusion of intracanal medicaments and the seal of the obturated root canal. Aim: To evaluate the combination of various irrigants whether it forms the precipitate and also to quantify the amount of precipitate formed. Materials and Methods: Five different irrigants were taken in ten test tubes each having the combination of two irrigants and observed for 2 min for precipitate formation. Results: One-way-ANOVA test revealed more amounts of precipitate with 2% Chlorhexidine (CHX) 5% Tulsi followed by 2% CHX, 5% Neem and 3% Sodium hypochlorite 2% CHX. Conclusion: Precipitate formation is seen more with the 2% CHX-5% Neem followed by 2% CHX-5% Tulsi when compared with the control group. When these combinations of irrigants were used, intermediate flushes of saline or distilled water reduced the amount of precipitate.

How to cite this article:
Shenoy A, Bolla N, Sayish, Sarath RK, Sunil Ram C H, Sumlatha. Assessment of precipitate formation on interaction of irrigants used in different combinations: An in vitro study.Indian J Dent Res 2013;24:451-455

How to cite this URL:
Shenoy A, Bolla N, Sayish, Sarath RK, Sunil Ram C H, Sumlatha. Assessment of precipitate formation on interaction of irrigants used in different combinations: An in vitro study. Indian J Dent Res [serial online] 2013 [cited 2021 Nov 28 ];24:451-455
Available from:

Full Text

Root canal treatment has a major objective, which is to prevent or cure apical periodontitis caused by bacteria in the root canals that can initiate and cause periapical inflammatory lesions. [1] Furthermore, by cleaning, shaping, and obturating the complex anatomy of the root canal system a three dimensionally seal can be obtained in order to facilitate healing at the peri-radicular area.

Success in endodontic treatment depends on how well the pulp space is shaped and cleaned. Cleaning includes complete debridement of pulp space from bacteria, debris, smear layer, and other organic and inorganic tissue in the pulp space.

Cleaning can be done either mechanically using hand and rotary instruments or chemically using various combinations of irrigants. However, the anatomy of the root canal system is so complex, complete debridement using mechanical instruments may not be possible. [2] In such teeth with complex anatomy such as fins or other irregularities that might be missed by instrumentation, chemical debridement facilitates proper cleaning of the root canal space and is important for successful root canal treatment. [3]

Several studies have been conducted for an irrigant that meets four major requirements:

Antimicrobial activityNon-toxicity to Periapical tissuesWater solubilityCapacity to dissolve organic matterAs no irrigant has all the ideal properties, it is mandated to use combinations of irrigants in a sequential manner to enhance their antimicrobial effect and dissolution of organic and inorganic tissues. Endodontic irrigants have been well characterized individually; but their combined use is not well-understood. Individually, no irrigant forms precipitate but when irrigants are combined; due to acid-base reaction form a neutral and insoluble substance referred to as precipitate. [4] It is difficult to remove the precipitate from the root canals as it occludes dentinal tubules, preventing the intracanal medicament penetration into dentinal tubules and thus compromises the seal of the obturated root canal. [5]

The most commonly used irrigants are Chlorhexidine (CHX), sodium hypochlorite (NaOCl) and citric acid (CA). The constant increase in antibiotic resistant strains and side effects caused by synthetic drugs has prompted researchers to look for herbal or natural alternatives. Natural irrigants like Neem and Tulsi are used for chemical debridement recently because of their biocompatibility and anti-bacterial effect. There are no studies related to interaction between these irrigant combinations; therefore, the aim of this study was to evaluate whether precipitate was formed by combining various irrigants and also to determine the amount of precipitate formed.

 Materials and Methods

Five different types of irrigants namely 2% CHX gluconate, 3% NaOCl, 17% CA, 5% Neem, and 5% Tulsi were used in this study.

The Neem and Tulsi aqueous irrigants were obtained by placing solutions in a soxhet apparatus for 24 hr to get a 100% aqueous extract. These were diluted with distilled water to get the concentration of 5% rather than 4% concentrations for our convenience. These five irrigants were grouped into 10 of various combinations. In each group, different combinations of irrigants were prepared for making 10 test groups of 10 each [Figure 1] and [Table 1]. Two irrigants, 1 ml of each, in all the groups were mixed in a test tube and were observed for 2 min for precipitate formation.{Table 1}{Figure 1}

The formed precipitate was filtered; air dried and weighed using weighing electronic balance (Keryo, Lab, India). The data obtained were statistically analyzed using One-way ANOVA test.


Group-2 containing 1 ml 2% CHX + 1 ml 5% Neem [Figure 2] and group-3 containing 1 ml 2% CHX + 1 ml 5% Tulsi [Figure 3] and the control group 1 ml 2% CHX + 1 ml 3% NaOCL [Figure 4] showed precipitate formation immediately.{Figure 2}{Figure 3}{Figure 4}

One-Way ANOVA test was conducted for the weights of the precipitate obtained having a significant difference (P < 0.5) which revealed that more amount of precipitate of a mean weight 30 mg is observed with group-3 (1 ml 2% CHX – 1 ml 5% Tulsi) followed by group-2 (1 ml 2% CHX – 1 ml 5% Neem) with a mean weight of 12 mg which was compared with a positive control group-1 (1 ml 2% CHX – 1 ml 3% NaOCL) showing a mean weight of 4 mg. [Table 2] significance was at P < 0.05.{Table 2}

One-way ANOVA: 1 ml 2% CHX + 1 ml 3% NaOCl, 1 ml 2% CHX + 1 ml 5% Neem, 1 ml 2% CHX + 1 ml 5% Tulsi [Graph 1] [SUPPORTING:1] shows the weight of precipitate of the 3 groups.


Effective disinfecting in endodontics is only achieved by mechanical preparation in presence of antimicrobial chemical agents and this is due to the vast complexity of the entire root canal system. The chemical agents that are used for irrigation can improve mechanical debridement by flushing out debris, dissolving tissue, disinfecting root canal system as well as remove the smear layer formed during the shaping the root canal. [6]

While selecting any irrigating solution or medication, the knowledge of its mechanism of action on the predominant bacterial micro flora is of great value. Proper information about morphology, structure, and physiology of micro-organisms which are responsible for pain and destruction of Periapical tissues led to several therapeutic trends. The knowledge of the interrelation between micro-organisms and host, together with the chemical and biological dynamics of antimicrobial medications constitutes the first step in endodontic treatment.

The use of aqueous NaOCL as a main irrigant in endodontics became popular since 1920's and this was based on H. Dakin's meticulous studies on the efficacy of different solutions on infected necrotic tissue during World War I.

NaOCL has tissue dissolving and antimicrobial properties. [7] The formation of hypochlorous acid when in contact with organic debris is the reason for its germicidal activity. 3% and 6% NaOCL eliminates and disrupts biofilms where as 1% NaOCl only disrupts biofilms. At high concentrations, it is toxic and causes Periapical inflammation, [8] whereas at low concentrations, it is ineffective against specific microorganisms. The effect of NaOCL is short, and it does not impart antimicrobial substantivity.

Irrigation with a tissue dissolving anti-bacterial solution is a pre-requisite for a successful root canal treatment but still does not guarantee the absence of viable bacteria in the root canal dentin continuum. [9] So CHX with anti-bacterial properties is used in combination with NaOCL.

CHX as an irrigant has been used as an alternative to or in combination with NaOCL. This is because of its lower toxicity and substantivity. It has an affinity to dental hard tissues. Once bound to a surface it has shown prolonged antimicrobial activity, a phenomenon called substantivity. [10]

CHX lacks tissue dissolving and smear layer removal properties. [11]

Complete cleaning of root canal system requires irrigating solutions that dissolve both organic and inorganic tissues. As hypochlorite is active only against the organic, other substances must be used to complete the removal of the smear layer and dentin debris. [12],[13],[14]

Ethylene di-amine tetra acetic acid (EDTA)and CA effectively dissolves inorganic material, including hydroxyapatite. [15],[16],[17] They have little or no effect on organic tissue and when used alone they do not have any antimicrobial activity. EDTA is most commonly used as a 17% neutralized solution and CA as a 10% solution. Removal of smear layer by EDTA or CA improves the antimicrobial effect locally and improves the action of disinfecting agents in deeper layers of dentin. [18]

EDTA and CA will reduce the amount of chlorine in the NaOCL solution instantaneously making hypochlorite irrigant ineffective on bacteria and necrotic tissues. Hence, EDTA and CA should never be mixed with NaOCl. [19]

Mixing CHX and EDTA immediately produces a white precipitate. [20] Hence in this study 10% CA was used in combination with test groups.

Because of the cytotoxic reactions of the most of the commercial irrigants and their complete inability to eliminate bacteria from dentinal tubules recent trend is to use biologic medication extracted from natural plants as irrigants. [21]

Tulsi, scientifically known as Ocimum sanctum, is a time tested premier medicinal herb. In India it is worshipped by the Hindus and used in ayurvedic medicine since ancient times. It contains enormous antimicrobial substances and is used in variety of illness ranging from diabetes mellitus, arthritis, bronchitis, skin diseases etc. [22]

Neem (Azadirachta indica) also known as Indian Neem/ margosa tree is also one of the most versatile medicinal plants known to have a wide spectrum anti-bacterial activity. In Sanskrit, it is called "aristha" meaning reliever of sickness. In dentistry, it has been looked up as highly efficacious alternative to CHX in cases of periodontal disorders. [23]

Its antioxidant and antimicrobial properties makes it a potential agent for root canal irrigation as an alternative to NaOCl. [23]

As no single irrigant has all the ideal requirements, combinations of irrigants are used sequentially to enhance their antimicrobial effect. However, by doing so, there are chances of precipitate formation that will hinder their antimicrobial effect, and if precipitate is left behind, raises potential concerns with respect to leaching of the precipitate into the surrounding tissues and the seal of the root canal. [4]

In the present study, the irrigants, namely 2% CHX, 3% NaOCl, 10% CA, 5% Neem and 5% Tulsi were used as combinations of two and were evaluated for formation and amount of precipitate. In this study, the 2% CHX with 5% Neem, 2% CHX with 5% Tulsi, and the control group i.e., 2% CHX with 3% NaOCl formed precipitate.

More amount of precipitate with a mean weight of 30 mg were observed with 2% CHX + 5% Tulsi (green precipitate) followed by 2% CHX + 5% Neem (green precipitate) with a mean weight of 12 mg and 2% CHX + 3% NaOCl (reddish brown precipitate containing the suspected carcinogen p-chloroaniline) [24] with a mean weight of 4 mg.


As we expect that combinations of irrigants synergistically increase their properties rather when using only one of the irrigants, it was not always true. Due to the acid-base reaction sometimes the combination of irrigants leads to precipitate formation which shows unfavorable properties.

In this study, precipitate formation was seen with 1 ml of 2% CHX in combination with 1 ml of 5% Neem,1 ml of 2% CHX + 1 ml of 5% Tulsi and 1 ml of 2% CHX + 1 ml of 3% NaOCl.

Though, these natural irrigants show effectiveness in cleaning similar to the chemical irrigants, further studies have to be conducted on the cytotoxicity of these natural irrigants and their combination products. Within the limitations of this study we would like to conclude that before using a combination of irrigants we should check for its compatibility.


1Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965;20:340-9.
2Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321-8.
3Safavi KE, Spangberg LS, Langeland K. Root canal dentinal tubule disinfection. J Endod 1990;16:207-10.
4Bui TB, Baumgartner JC, Mitchell JC. Evaluation of the interaction between sodium hypochlorite and chlorhexidine gluconate and its effect on root dentin. J Endod 2008;34:181-5.
5Krishnamurthy S, Sudhakaran S. Evaluation and prevention of the precipitate formed on interaction between sodium hypochlorite and Chlorhexidine. J Endod 2010;36:1154-7.
6Haapasalo M, Shen Y, Qian W, Gao Y. Irrigation in endodontics. Dent Clin North Am 2010;54:291-312.
7Abou-Rass M, Piccinino MV. The effectiveness of four clinical irrigation methods on the removal of root canal debris. Oral Surg Oral Med Oral Pathol 1982;54:323-8.
8Spangberg L, Engström B, Langeland K. Biologic effects of dental materials. 3. Toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg Oral Med Oral Pathol 1973;36:856-71.
9Byström A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 percent sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol 1983;55:307-12.
10White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229-31.
11Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium hypochlorite as antimicrobial endodontic irrigants. J Endod 1994;20:276-8.
12Yamada RS, Armas A, Goldman M, Lin PS. A scanning electron microscopic comparison of a high volume final flush with several irrigating solutions: Part 3. J Endod 1983;9:137-42.
13Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147-57.
14Torabinejad M, Khademi AA, Babagoli J, Cho Y, Johnson WB, Bozhilov K, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170-5.
15Czonstkowsky M, Wilson EG, Holstein FA. The smear layer in endodontics. Dent Clin North Am 1990;34:13-25.
16Baumgartner JC, Brown CM, Mader CL, Peters DD, Shulman JD. A scanning electron microscopic evaluation of root canal debridement using saline, sodium hypochlorite, and citric acid. J Endod 1984;10:525-31.
17Loel DA. Use of acid cleanser in endodontic therapy. J Am Dent Assoc 1975;90:148-51.
18Zaccaro Scelza MF, da Silva Pierro VS, Chagas MA, da Silva LE, Scelza P. Evaluation of inflammatory response of EDTA, EDTA-T, and citric acid in animal model. J Endod 2010;36:515-9.
19Zehnder M, Schmidlin P, Sener B, Waltimo T. Chelation in root canal therapy reconsidered. J Endod 2005;31:817-20.
20Rasimick BJ, Nekich M, Hladek MM, Musikant BL, Deutsch AS. Interaction between chlorhexidine digluconate and EDTA. J Endod 2008;34:1521-3.
21Palombo EA. Traditional Medicinal Plant Extracts and Natural Products with Activity against Oral Bacteria: Potential Application in the Prevention and Treatment of Oral Diseases. Evid Based Complement Alternat Med 2011;1-15.
22Agarwal P, Nagesh L, Murlikrishnan. Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro study. Indian J Dent Res 2010;21:357-9.
23Aarti Bohra, Vibha Hegde. Comparison of antibacterial efficiency of Neem leaf extract and 2% Sodium hypochlorite against E. faecalis, C. albicans and mixed culture-An in vitro study Endodotology 2010;22:8.
24Basrani BR, Manek S, Sodhi RN, Fillery E, Manzur A. Interaction between sodium hypochlorite and Chlorhexidine gluconate J Endod 2007;33:966-9.