one possible reason for persistent endodontic infection may be retention of microorganisms in the dentine tissue of the root canal walls . an infection of the pulp can result in microbial colonization of the entire root canal system , together with the dentinal tubules adjacent to the canal . selective pressures related to oxidation  reduction potential , nutrient supply , and microbial interactions are related to the maintenance of endodontic infections . it is the dominant microorganism in root - filled teeth presenting post - treatment apical periodontitis and can be isolated from the root canal in pure culture . e. faecalis has been the focus of attention as a recognized pathogen , isolated both in mixed microbiota and in monocultures . several virulence factors ( aggregation substance , enterococcal surface proteins ( esp ) , gelatinase , cytolysin toxin , extracellular superoxide production , capsular polysaccharides , antibiotic resistance determinant ) can facilitate tissue invasion , immunomodulation , and cause toxin - mediated damage . sodium hypochlorite presents antimicrobial activity with action on bacterial essential enzymatic sites promoting irreversible inactivation originated by hydroxyl ions and chloramination action . dissolution of organic tissue can be verified in the saponification reaction when sodium hypochlorite destroys fatty acids and lipids resulting in soap and glycerol . chlorhexidine gluconate solutions of varying concentrations have recently been recommended as endodontic irrigants and dressings.[810 ] chlorhexidine is a cationic agent ( biguanide group ; 4-chlorophenyl radical ) , which exhibits antibacterial activity . the cationic nature of the compound promotes connection with anionic compound at the bacterial surface ( phosphate groups from teicoic acid at gram - positive and lipopolysaccharide at gram - negative bacteria ) capable of altering its integrity . the potassium ion , being a small entity , is the first substance to appear when the cytoplasmic membrane is damaged . various research results have been inconclusive when comparing the antimicrobial effect of these solutions.[1113 ] disadvantages of conventional root canal treatments include their skill - dependent nature , long treatment time , possible weakening of teeth due to widening of the root canal , and use of medicaments such as sodium hypochlorite . the current problem of bacterial drug resistance perhaps best illustrates the continuing requirement both for new agents and new approaches to eliminate infection from root canal system . the commercial phenothiazine dye , toluidine blue ( tb ) , is an effective photosensitizing agent for the inactivation of pathogenic organisms , including viruses , bacteria , and yeast . in recent years , tb has been reported as an antifungal and antibacterial drug for inactivation of yeast and some gram - positive and gram - negative bacteria . photodynamic therapy ( pdt ) matured as feasible medical technology in 1980s at several institutions throughout the world to eradicate premalignant and early - stage cancer and reduce the tumor size in end - stage cancers involving 3 components : 1 . tissue oxygen . the word photodynamics means the study of activating effects of light on living organisms . employing the same principle , pdt can be described as a medical treatment that utilizes light to activate a photosensitizing agent in presence of oxygen . pdt or photoactivated disinfection uses light of a specific wavelength to activate a nontoxic photoactive dye ( photosensitizer ) in the presence of oxygen . the photoactivated sensitizer can interact with the biological substrate , leading to the production of highly reactive oxygen species , such as singlet oxygen and free radicals , which can kill microorganisms by damaging essential cellular molecules , including proteins , membrane lipids , and nucleic acid . the aim of this study was to compare the effectiveness of 2% chlorhexidine , 2.5% naocl , pdt , and combination of two factors ( pdt plus 2.5% naocl ) as intracanal disinfectants in extracted teeth against e. faecalis . ninety freshly extracted , intact , adult , human , single - rooted , mature teeth with a single canal were collected and stored in sterile saline . calculus and stains were removed from the root surface using an ultrasonic scaler ( cavitron , dentsply ltd , weybridge , uk ) . after preparation of coronal two - thirds of all canals using gates glidden files number 4 , 3 and 2 , they were sequentially prepared within 1 mm apical end of the canal , using hedstrm files ( antaeos , munich , germany ) up to size 40 . the canal was irrigated with physiologic saline after the use of each size file . to remove the smear layer that had developed on the canal wall as a result of the instrumentation , each canal the assembled tooth , lid , and bottle were covered with aluminium foil and autoclaved at 121c , for 15 min . the bottle was then aseptically filled with sterile brain heart infusion broth ( bhi ) ( merk , poole , uk ) so that the root was covered . a clinical strain of e. faecalis strain atcc 29212 , isolated from a treated root canal at the department of endodontics of shahid beheshti university of medical sciences was used in this experiment . bacterial suspension was prepared by a pure culture of this e. faecalis strain , grown in 1 ml of sterile bhi broth to obtain an optical turbidity of 0.5 mcfarland standard , and incubated for 1 hour at 37c in the incubator . the inoculums were injected into the prepared root canal using a sterile syringe and incubated for 48 h at 37c . teeth were randomly divided into five experimental groups ( each composed of 15 teeth ) according to the post - instrumentation procedures . groups were as follow : the canals were filled with naocl ( 2.5% v / v ) for 5 min , removed with sterile paper points , and irrigated with normal saline solution ( .85% v / v ) . the canals were filled with naocl ( 2.5% v / v ) for 5 min , removed with sterile paper points and then was irradiated with diode laser . the irradiation source was a diode laser ( fotoson cms dental , denmark ) with a total power of 200 mw / cm and 625 nm of wavelength . total irradiation time was 60 s. root canal infected with e. faecalis was subjected to lethal photosensitization with toluidine blue with concentration 15 g / ml and diode laser for 60 s. the irradiation source was a diode laser ( fotoson cms dental , denmark ) with a total power of 200 mw / cm and 625 nm of wavelength . the canals were filled with naocl ( 2.5% v / v ) for 5 min , removed with sterile paper points , and then subjected to pdt as described above . root canals were injected with chlorhexidine ( 2% v / v ) ( altrincham , cheshire , wa14 5dh , uk ) for 5 min , removed with sterile paper points , and irrigated with normal saline solution ( .85% v / v ) . controls consisted of no treatment ( positive control ) and without inoculation of bacterium ( negative control ) for five experimental groups . following all treatments , the liquid content of the root canal canals were filled with sterile 0.85% normal saline solution , and sample was taken by the sequential use of three paper points placed to the working length . paper points were transferred to tubes containing 1 ml of 0.85% normal saline solution and agitated for 1 minute . after 10-fold serial dilutions in saline , aliquots of 0.1 ml were plated onto blood agar plates and incubated at 37c for 48 hours . the colony forming units ( cfus ) grown were counted and then transformed into actual counts based on the known dilution factors . ninety freshly extracted , intact , adult , human , single - rooted , mature teeth with a single canal were collected and stored in sterile saline . calculus and stains were removed from the root surface using an ultrasonic scaler ( cavitron , dentsply ltd , weybridge , uk ) . after preparation of coronal two - thirds of all canals using gates glidden files number 4 , 3 and 2 , they were sequentially prepared within 1 mm apical end of the canal , using hedstrm files ( antaeos , munich , germany ) up to size 40 . the canal was irrigated with physiologic saline after the use of each size file . to remove the smear layer that had developed on the canal wall as a result of the instrumentation , each canal the assembled tooth , lid , and bottle were covered with aluminium foil and autoclaved at 121c , for 15 min . the bottle was then aseptically filled with sterile brain heart infusion broth ( bhi ) ( merk , poole , uk ) so that the root was covered . a clinical strain of e. faecalis strain atcc 29212 , isolated from a treated root canal at the department of endodontics of shahid beheshti university of medical sciences was used in this experiment . bacterial suspension was prepared by a pure culture of this e. faecalis strain , grown in 1 ml of sterile bhi broth to obtain an optical turbidity of 0.5 mcfarland standard , and incubated for 1 hour at 37c in the incubator . the inoculums were injected into the prepared root canal using a sterile syringe and incubated for 48 h at 37c . teeth were randomly divided into five experimental groups ( each composed of 15 teeth ) according to the post - instrumentation procedures . groups were as follow : the canals were filled with naocl ( 2.5% v / v ) for 5 min , removed with sterile paper points , and irrigated with normal saline solution ( .85% v / v ) . the canals were filled with naocl ( 2.5% v / v ) for 5 min , removed with sterile paper points and then was irradiated with diode laser . the irradiation source was a diode laser ( fotoson cms dental , denmark ) with a total power of 200 mw / cm and 625 nm of wavelength . total irradiation time was 60 s. root canal infected with e. faecalis was subjected to lethal photosensitization with toluidine blue with concentration 15 g / ml and diode laser for 60 s. the irradiation source was a diode laser ( fotoson cms dental , denmark ) with a total power of 200 mw / cm and 625 nm of wavelength . the canals were filled with naocl ( 2.5% v / v ) for 5 min , removed with sterile paper points , and then subjected to pdt as described above . root canals were injected with chlorhexidine ( 2% v / v ) ( altrincham , cheshire , wa14 5dh , uk ) for 5 min , removed with sterile paper points , and irrigated with normal saline solution ( .85% v / v ) . controls consisted of no treatment ( positive control ) and without inoculation of bacterium ( negative control ) for five experimental groups . following all treatments , the liquid content of the root canal was carefully absorbed with paper points . canals were filled with sterile 0.85% normal saline solution , and sample was taken by the sequential use of three paper points placed to the working length . paper points were transferred to tubes containing 1 ml of 0.85% normal saline solution and agitated for 1 minute . after 10-fold serial dilutions in saline , aliquots of 0.1 ml were plated onto blood agar plates and incubated at 37c for 48 hours . the colony forming units ( cfus ) grown were counted and then transformed into actual counts based on the known dilution factors . the canals were filled with naocl ( 2.5% v / v ) for 5 min , removed with sterile paper points , and irrigated with normal saline solution ( .85% v / v ) . the canals were filled with naocl ( 2.5% v / v ) for 5 min , removed with sterile paper points and then was irradiated with diode laser . the irradiation source was a diode laser ( fotoson cms dental , denmark ) with a total power of 200 mw / cm and 625 nm of wavelength . root canal infected with e. faecalis was subjected to lethal photosensitization with toluidine blue with concentration 15 g / ml and diode laser for 60 s. the irradiation source was a diode laser ( fotoson cms dental , denmark ) with a total power of 200 mw / cm and 625 nm of wavelength . the canals were filled with naocl ( 2.5% v / v ) for 5 min , removed with sterile paper points , and then subjected to pdt as described above . root canals were injected with chlorhexidine ( 2% v / v ) ( altrincham , cheshire , wa14 5dh , uk ) for 5 min , removed with sterile paper points , and irrigated with normal saline solution ( .85% v / v ) . controls consisted of no treatment ( positive control ) and without inoculation of bacterium ( negative control ) for five experimental groups . following all treatments , the liquid content of the root canal was carefully absorbed with paper points . canals were filled with sterile 0.85% normal saline solution , and sample was taken by the sequential use of three paper points placed to the working length . paper points were transferred to tubes containing 1 ml of 0.85% normal saline solution and agitated for 1 minute . after 10-fold serial dilutions in saline , aliquots of 0.1 ml were plated onto blood agar plates and incubated at 37c for 48 hours . the colony forming units ( cfus ) grown were counted and then transformed into actual counts based on the known dilution factors . table 1 depicts the mean , median , range , and percent reduction of cfus observed for all groups . counting of all cfu of e. faecalis remaining in the control ( no treatment ) and groups was done to determine effectiveness of treatments . there was a significant reduction in the cfu counts ( p < 0.05 ) compared with the initial numbers recorded in positive control , photodynamic therapy alone , and chlorohexidine therapy ; both had mild antibacterial effects . a maximum of viable bacterial reduction was observed with a combination of pdt plus 2.5% naocl that resulted in 100% bacterial kill [ figure 1 ] . the group analyses were performed to investigate the ability of each procedure for reducing the bacterial counts when compared with the previous conditions . a reduction in the number of cfus was statistically significant for all groups ( p < 0.05 for all groups ) . the reduction factors were significantly higher in group 2 ( naocl plus diode laser ) compared to group 3 ( pdt ) and group 5 ( chlrohexidine ) ( p < .05 ) . the mann - whitney u test did not show significant differences when comparing samples from group 2 and group 1 ( p = 0.09 ) . however , group 4 ( pdt plus naocl ) was significantly more effective in eliminating bacteria from the root canals when compared to all [ figure 1 ] ( p < 0.05 ) . highest cfu of viable bacteria was from positive control group , while the negative control group was free of microorganisms under the experimental conditions [ table 1 ] . effect of antibacterial agents against e. faecalis counts of enterococcus faecalis colony - forming units after effect of antibacterial agents although this species has been recently questioned as to its importance in the etiology of treatment failures , such a role can not be surely disregarded in the light of current evidence . moreover e. faecalis has been widely used as a valuable microbiological marker for in vitro studies because it has been shown to successfully colonize the root canal in a biofilm - like style , invade dentinal tubules , and resist to some endodontic treatment procedures . in present study , e. faecalis was chosen as the test microorganism because it is one of the most resistant microorganism found in infected root canals , and it has been reported that cases with refractory endodontic treatment were associated with this bacterial strain.[2426 ] from the results obtained with the control teeth , it is clear that e. faecalis was capable of migrating up to 400 m and deeper into the dentinal tubules . in the present study , 2% chlorhexidine solution was used because previous results have shown that 2% solution was more efficient in the shortest period of time than all other concentrations . previous reports have shown that chlorhexidine has a marked effect against e. faecalis[2730 ] and could be effective even at low concentrations against the microorganisms most frequently present in infected root canals , anaerobic bacteria , and candida albicans . the 2.5% naocl ( 5 min ) alone exhibited reduction of e. faecalis in the root canal system of extracted human teeth . however when combined with diode laser , 2.5% naocl was more effective on e. faecalis killing . the effect of naocl plus pdt was the most effective therapy and achieved 100% kill of e. faecalis after 2-min contact time . in this study 2.5% naocl was used because it was shown to denature bacterial toxins and dissolve organic tissues . studies have shown that the magnitude of the antimicrobial efficacy of a 2.5% naocl and chlorhexidine can be influenced by the methodology , microbial characteristics in the biofilm , exposure time , and concentration of the substance tested . the antimicrobial effect of naocl by direct contact on e. faecalis occurred after 2 min . our results demonstrate that 2% chlorhexidine and 2.5% naocl had a significant effect on the viability of e. faecalis . thus , our result confirms those of previous studies on oral microorganisms . in this study , in previous studies of bactericidal activity against e. faecalis biofilm extracted from human teeth , it was reported that the total energy output of a diode laser was 36 j and total energy level was 76 j and combined with a photosensitizer was adequate for a high level of sterilization . nagayoshi et al . , found that irradiation at 5 w for 60 seconds in the presence of a photosensitizer decreased lesion defect area . in the present study , irradiation with laser , tbo and irrigation with naocl 2.5% ( 5 min ) with an absorption maxima ( 625 nm ) had an excellent efficacy against e. faecalis , because in the total of 15 canals incubated with this bacterial strain and then treated with pdt / laser / naocl , no bacteria could be detected in 100% cases . seal and coworkers ( 2002 ) reported effect of 100% killing teeth infected with s. intermedius biofilm treated with 3% naocl for 10 min . previous results have shown that singlet oxygen production was not the most relevant parameter to consider when evaluating the antimicrobial activity of pdt against s. mutans . a major advantage of pdt in treatment of root canal infections is the absence of thermal side - effects in the tissues surrounding the roots , as associated with the use of high power lasers . the anticipated benefits of access of laser light and photosensitizer were more limited than hypothesized . refinement of the laser delivery system by introduction of the laser beam into the root canal and/or increased energy delivery may be needed to achieve a better antimicrobial effect . overall pdt of single species grown in a tooth model was interestingly effective considering the light dose at the access cavity . but ultimately , combination of it with 2.5% naocl and pdt is the best option to maximize root canal disinfection and can predictably disinfect root canals in clinical settings . there are many variables to be taken into account when developing a pdt protocol , including light parameters , photosensitizers , and light delivery techniques . the peak of absorption of a photosensitizer should match the wavelength of the light used for irradiation in order to promote formation of singlet oxygen , a highly reactive oxygen molecule responsible for pdt - mediated bacterial killing . both methylene blue and toluidine blue , are phenothiazine dyes that have a maximum wavelength absorption of 656 nm and 625 nm , respectively . for endodontic disinfection , photosensitizers have been tested at concentrations ranging from 6.25 mg / ml to 25 mg/ ml for methylene blue and from 10 mg / ml to 100 mg/ ml for toluidine blue . souza and workers used tbo with concentration of 15 g / ml against e. faecalis . in this study , we used concentration of 15 g / ml . souza reported that pdt with either methylene blue or toluidine blue did not have a significant additional effect to the chemo - mechanical preparation using 2.5% naocl as an irrigant in the reduction of e. faecalis populations . the additional antibacterial effect of pdt with either photosensitizer ( methylene blue and toluidine blue ) was reported , but it was statistically significant only for toluidine blue . the results of our study showed that reduction in the number of cfus was statistically significant for all groups : chlorhexidine , naocl , laser plus tbo ( pdt ) , laser / naocl , and pdt / naocl against e. faecalis . one of the aims of root canal treatment is to eliminate the bacteria , their products , and the substrate from the root canal system . the result of this research suggests that effect of pdt plus 2.5% naocl simultaneously is effective in the elimination of e. faecalis from dentinal tubules under the conditions of this study .