He In Vitro Antibacterial Activity Biology Essay

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The aim of the present study was to compare the in vitro antibacterial activity of common root canal irrigants with a combination technique against intratubular Enterococcus faecalis.


In this experimental in vitro study, 75 single-rooted teeth were selected and their crowns and root-ends were dissected to obtain specimens with 5mm in length. Enterococcus faecalis strains were inoculated in the brain-heart infusion (BHI) medium and incubated for 21 days to contaminate the teeth. The contaminated teeth were divided into 5 groups (n=15) which were irrigated with 2% chlorhexidine (2% CHX), 3% hydrogen peroxide (3% H2O2), 5.25% sodium hypochlorite (5.25% NaOCl), 2% CHX + 3% H2O2 and sterile saline (control). Surface and deep dentinal chips were collected in trypticase soy broth (TSB) for each sample. After incubation, the number of colony-forming units (CFUs) were counted for dentinal surface and deep areas. Kruskal-wallis and Mann-whitney U tests were used for statistical analysis (α=0.05).


In the surface and deep dentine areas, the least CFUs were counted in 2% CHX+ 3% H2O2, 5.25% NaOCl and 2% CHX groups respectively. Statistically, 5.25% NaOCl and 2% CHX + 3% H2O2 had similar antibacterial effect in both surface and deep dentine areas.The least antibacterial ability was reported for the 3% H2O2 in both areas.


2% CHX + 3% H2O2 showed the most antibacterial action while 3% H2O2 alone demonstrated the least antibacterial efficacy against Enterococcus faecalis.

Key words

Antibacterial effect, Chlorhexidine plus hydrogen peroxide, Enterococcus faecalis, endodontic irrigants, in vitro


The removal of the remaining pulp tissues and eliminating microorganisms are the main fundamentals of a successful endodontic therapy. Microorganisms infecting the root canal may survive endodontic procedures (1) due to anatomical complexities and limited access of instruments(2). Therefore, the use of an effective irrigant is necessary to eradicate microorganisms from the area which is not accessible to instruments. A variety of irrigants have been introduced in an attempt to reduce or eliminate the number of bacteria in the root canal system. An irrigant at the very least should have antibacterial activity and capacity of dissolving tissues (3). Also, these solutions must be compatible with the periradicular tissues. An ideal irrigant should possess substantive effect to preserve its therapeutic effect for a longer period of time.

Sodium hypochlorite (NaOCl) is currently the most commonly used irrigant during the mechanical instrumentation phase of endodontic therapy. Its tissue-dissolving and antibacterial property has been well proven. However, it is not substantive and it is highly irritating in higher concentrations to the periradicular tissues (4). In addition, factors like concentration (5), temperature (6) and pH (7) greatly affect its efficacy.

Substantivity is an important factor in the selection of a suitable irrigant. Chlorhexidine (CHX) is a broad-spectrum antimicrobial agent active against aerobic and anaerobic bacteria. It has been shown that CHX has antibacterial efficacy comparable to that of sodium hypochlorite (8). CHX has strong binding affinity to the hydroxyapatite in dentine, enamel and cementum which can be slowly released. This property results in the longer bacteriostatic activity of CHX (9). Antibacterial efficacy of 2% CHX has been shown to continue 72 hours after instrumentation (10). On the other hand, CHX lacks the ability to dissolve organic matters which negatively affects its cleaning capacity (11).

Hydrogen peroxide (H2O2) is another agent used for sterilization and disinfection purposes. H2O2 is an active agent with potential inhibitory effect on bacteria, viruses, fungi, yeast and spores (12). It produces hydroxyl free radicals which attacks protein and DNA of microbes (12). It has been shown that H2O2 lacks antibacterial activity and there is no reason to use it alone as an irrigant in endodontic treatments (3). In order to increase the antibacterial effectiveness of irrigating solutions, combination techniques have been suggested. It has been proposed that combining H2O2 with CHX significantly increases the antibacterial activity of the mixture compared to the compounds alone (3, 13, 14).

Many studies have investigated the effectiveness of different irrigants against Enterococcus faecalis (E.faecalis) (14-16). However, there has not been conclusive results regarding the efficacy of combination techniques of irrigants in endodontic treatments. In addition, limited data are available on the penetrating ability of endodontic irrigants into depts of dentinal tubules. As a result the present study was designed to compare the effectiveness of common endodontic irrigants with a combination technique against E.faecalis in surface and deep dentine areas.

Materials and Methods

Specimen Preparation

In this in vitro experimental study, 75 extracted single-rooted human teeth were cleaned and placed in 2.5% NaOCl for 24 hours. After surface disinfection, the teeth were stored in normal saline. Specimen preparation in this study is a modification of methods previously described by Haapasalo and Orstavik (17). The teeth crowns and root-ends were removed to obtain uniform specimens with 5mm root length. The canals were enlarged using Gates Glidden(GG) #3 bur (Maillefer, Ballaigues, Switzerland) with a 0.9 mm diameter to standardize the internal diameters. As a result, cylindrical roots with 5mm in length and 0.9 mm inner diameter were obtained. The outer surfaces of the specimens were covered with an apoxy resin (3M Dental Products, Bracknell, UK). The canals were irrigated with 17% EDTA for 5 minutes followed by 5.25% sodium hypochlorite for another 5 minutes to remove the smear layer. Then the roots were sterilized by autoclave for 30 min at 121 °C .

Root Canal Contamination

Pure E.faecalis cultures (ATCC 29212) were cultivated in brain heart infusion (BHI, HiMedia, Mumbai, India) broth medium and then suspended in 4.0 mL of BHI. The cell suspension was adjusted spectrophotometrically to match the turbidity of 6.3x108 CFU/mL of E.faecalis (equivalent to ï‚»2.0 McFarland standards). One end of root cylinders was sealed with temporary cement (Cimpat, Septodont, Saint-Maur-des-Fossés, France) and 3µL of the suspension was placed into the root canals by an automatic pipette. The cylinders' other ends were sealed with temporary cement.The specimens were placed into Petri dishes covered with humid sterile gauze and incubated aerobically for 21 days at 37 °C. On 7th and 14th days, the temporary cement was removed and fresh BHI broth was added to the canals to ensure viability of bacteria.

Experimental Groups

The contaminated roots were randomly assigned to 5 groups (n=15) according to the irrigant used for canal disinfection as follows:

Group 1: 2% CHX (Marca, FGM product, Brasil)

Group 2: 3% H2O2 (Merck, Darmstadt, Germany)

Group 3: 5.25% NaOCl (Merck, Darmstadt, Germany)

Group 4: 2% CHX + 3% H2O2 (1:1 mixing ratio)

Group 5: Control (sterile saline)

The canals were irrigated using 2mL of each solution for 10 minutes. A 27-gauge syringe was used to irrigate the canals and a #30 file (Mani Inc., Utsunomiya, Japan) was used to agitate the irrigants inside the canals for one minute. After irrigation procedure, the root canals were washed with 5mL sterile saline. 0.5% Tween 80 (Merck, Darmstadt, Germany) + Lessitin (Merck, Darmstadt, Germany), Acetanilid (Merck, Darmstadt, Germany), Sodium thiosulfate (Merck, Darmstadt, Germany) and 0.5% Tween 80 + Lessitin + Acetanilid were used respectively to neutralize remaining irrigants inside the root canals. Finally, the root canals were irrigated with sterile saline and dried with sterile paper points.

Dentine Samples

Canals of root cylinders were enlarged using #4 (1.1mm in diameter) and #5 (1.3mm in diameter) GG burs to collect surface and deep dentine samples respectively (fig.1). Each bur was used three times throughout the entire extention of the root canals. The dentinal chips were transferred into a tube containing 1mL trypticase soy broth (TSB, HiMedia, Mumbai, India) medium. The samples were mixed for 1 minute and then 25µL of the samples was poured on blood agar culture medium (HiMedia, Mumbai, India) and incubated at 37°C for 24 hours. The growing CFUs were counted by a blinded microbiologist. Bacterial identity was confirmed by colony morphology and gram staining.

Data Analysis

Kruskal-Wallis test was used to analyze CFU differences among experimental groups. Paired comparisons were done using Mann-whitney U test (=0.05).


In the surface and deep dentine samples, the minimum and maximum E. faecalis CFUs were counted in 2% CHX + 3% H2O2 and control groups respectively (Tables 1 and 2).

The use of various irrigants in this study significantly reduced the number of bacterial CFUs recovered from surface and deep dentine samples when compared with control treatment (p<0.05).

Surface Dentine Samples

2% CHX, 5.25% NaOCl and 2% CHX + 3% H2O2 groups were significantly more effective in reducing the number of bacterial CFUs when compared with 3% H2O2 group (p<0.05). There was no significant difference between 2% CHX, 5.25% NaOCl and 2% CHX + 3% H2O2 groups (p>0.05).

Deep Dentine Samples

5.25% NaOCl and 2% CHX + 3% H2O2 groups were significantly more effective in reducing bacterial CFUs when compared with 2% CHX and 3% H2O2 groups (p<0.05). There was no significant difference between 5.25% NaOCl and 2% CHX + 3% H2O2 groups (p>0.05).

2% CHX was significantly more effective in reducing bacterial CFUs when compared with 3% H2O2 (p<0.05).


Since bacteria are the main cause of pulpal infections, an appropriate treatment protocol which is capable to completely eradicate the microorganisms from the root canal system can ensure the success of root canal therapy. The use of irrigants as chemical adjuncts has been recommended because the complete cleaning of the root canal system is not guaranteed by the mechanical means alone (18). It has been shown that the use of irrigants during root canal treatment can dramatically enhance the effectiveness of the mechanical debridement (5). An ideal irrigant must be capable of adhering to the dentinal walls and directly applying its antibacterial effects (19). CHX, NaOCl and H2O2 are canal irrigants with well-known advantages and drawbacks. Since there are conflicting results regarding the antimicrobial efficacy of these solutions, in the present study, the antibacterial synergistic effect of a combination technique was compared with these established irrigants in surface and deep dentine areas.

E. faecalis is a treatment-resistant bacteria frequently found in infected root canals, especially in the secondary and persistent endodontic infections (20). This bacteria is highly resistant to various harsh environmental conditions like alkaline environment, bile salts, starvation and many antibacterial agents (20, 21). Therefore, it is very difficult to eradicate this strain from the infected root canals. Since this bacteria is highly resistant against different antibacterial agents, the antimicrobial efficacy of endodontic irrigants has been assessed against this bacteria in various studies (5-7, 14, 16, 22). Because of its high resistance and ease of handling in microbiological samplings, this bacteria was selected as a measure for antibacterial activity of selected irrigants in the present study.

In the present study, 2% CHX + 3% H2O2 showed the highest antibacterial effectiveness against E.faecalis followed by 5.25% NaOCl, 2% CHX and 3% H2O2. Furthermore, all of these irrigants were significantly more effective in reducing bacterial CFUs than sterile saline (control group). The results of current study indicates that: 1) irrigation, as a concept during root canal treatment, can be effective in reducing bacterial loads; 2) irrigants, even with short exposure time, can have antibacterial effects beyond the root canal lumen in depts of dentinal tubules; 3) the penetrating ability and thus the antibacterial effectiveness of endodontic irrigants is reduced in deep dentine compared with surface dentine; and 4) the penetrating ability and thus the antibacterial effectiveness of 2% CHX + 3% H2O2 and 5.25% NaOCl into depts of dentinal tubules were more than other irrigants used in this experiment.

In deep dentine area, NaOCl showed significantly higher antibacterial effect compared with CHX. This significant difference may highlight the higher penetrability of NaOCl compared with CHX into dentinal tubules. However, when CHX was combined with H2O2, its antibacterial effect was enhanced. This improvement may be due to better penetration into dentinal tubules or the synergistic additive effect of the two irrigants. NaOCl solution is the most commonly used root canal irrigant in endodontic treatment (3). In addition to antibacterial effectiveness and its low toxicity in the lower concentrations, NaOCl provides good necrotic and organic tissue solvent action (23). The major shortcoming of NaOCl is its inadequate effect against some microorganisms in the lower concentrations. Furthermore, there are limited conclusive studies recommending optimal concentrations in root canal irrigation (3).Various studies have investigated the antibacterial efficacy of 5.25% NaOCl and 2% CHX against E.faecalis and have demonstrated no significant difference between these two agents (8, 16, 24, 25). The results of the present study are also in agreement with those studies in surface dentine area as no significant difference was observed between 5.25% NaOCl and 2% CHX.

In the present study, 2% CHX demonstrated higher antibacterial activity against E.faecalis compared with H2O2 in surface and deep dentine areas. CHX has a broad antibacterial spectrum (26) and has a very low toxicity even at high concentrations (27). It has been proposed that the antibacterial effect of CHX stems from its ability to denature the bacterial cell wall while forming irreversible defects in the membrane (13). Since H2O2 exerts its antimicrobial action against intracellular organelles like DNA, the use of other agents in combination with H2O2 has been recommended (3). It has been postulated that the efficacy of H2O2 can be increased by enhancing its permeability through cell wall (13). The results of this investigation are in accordance with the findings of Shahriari et al. study (14) as the use of H2O2 alone showed significantly lower antibacterial activity against E.faecalis compared with CHX.

The use of synergism between two active agents seems to be a logical method to maximize their effectiveness while reducing their side effects. The antibacterial effect of CHX and H2O2 has been proved in different studies; however, these two agents show some shortcomings when used alone. CHX does have a bitter taste and can stain teeth (28). H2O2 can cause mucosal irritations (29), induce pathologic alterations (30) and pulp cytotoxicity (31). Considering the oxidative properties of H2O2, Steinberg et al. postulate that this combination may reduce the teeth-staining side effect of CHX (13). In the present study, the combination of CHX and H2O2 was used to test the hypothesis that the combination of these two agents can enhance their overall antibacterial effect. It was shown that the combination of 2% CHX + 3% H2O2 had higher antibacterial action against E.faecalis compared with CHX and H2O2 used alone. This increase in the antibacterial activity can be explained by the fact the CHX can increase the permeability of the bacterial cell membrane by denaturing cell wall and this change facilitates H2O2 penetration through cell membrane(13). Heling and Chandler reported the synergic activity of CHX and H2O2 in certain concentrations using similar methodology to the present study (32). Steinberg et al. indicated that the antibacterial effectiveness of CHX and H2O2 combination is increased compared with each of the irrigants alone (13). Shahriari et al. (14) showed the least number of E.faecalis CFUs following irrigation by CHX and H2O2 combination which concur with our findings.

There are limitations to this study. First, the present study used an in vitro model, whereas, in vivo, the nature of endodontic infections is significantly different. Second, a single microorganism was used to infect the root canals in the current investigation, but endodontic infections are usually polymicrobial with different dynamics. In vivo, other microorganisms may interact with E.faecalis and thus alter the behavior of this bacteria. Third, the combinations of various concentrations of CHX and H2O2 may have different antibacterial effectiveness. Fourth, in vivo conditions, the remaining tissues and fluids in the root canal system may reduce the efficiency of irrigants.

In conclusion, under the limitations of this study, 2% CHX and 3% H2O2 combination can be considered as a potential useful irrigant in root canal treatments, but further research should focus on its biocompatibility and in vivo clinical efficiency.

Table and figure legends:

Table 1: E. faecalis CFUs using selected irrigants in surface dentine

Table 2: E. faecalis CFUs using selected irrigants in deep dentine

Figure 1: Sequential removal of the dentinal chips from surface (GG #4) and deep (GG #5) dentine