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 .
 background : enterococcus faecalis has been widely used as a valuable microbiological pathogen for in vitro studies due to its ability to successfully colonize the root canal in a biofilm - like style , invade dentinal tubules , and resist endodontic treatment procedures.the aim of this study was to compare the bactericidal efficacy of photodynamic therapy ( pdt ) , 2% chlorhexidine , 2.5% naocl , and combination of pdt and 2.5% naocl against e. faecalis.materials and methods : sixty single - rooted teeth had their canals contaminated with e. faecalis in brain heart infusion broth and were incubated for 48 hours.the canals were then subjected to 2% chlorhexidine , 2.5% naocl , pdt ( red light emitting diode 625 nm+ toludine blue ) and pdt + 2.5% naocl . 
 controls consisted of no treatment ( positive control ) and without inoculation of bacterium ( negative control ) . following treatment , 
 the canal contents were sampled with sterile paper points.the samples were dispersed in transport medium , serially diluted , and cultured on blood agar to determine the number of colony forming units ( cfu ) . 
 data were analyzed by mann - whitney u test at 5% significance level . 
 the significance level for all analyses was set at p < .05.results : combination of pdt and 2.5% naocl achieved maximum reduction in recovered viable bacteria , no viable bacteria was observed after treatment of pdt + 2.5% naocl.conclusion:combination of pdt and 2.5% naocl simultaneously is effective in the elimination of e. faecalis from dentinal tubules under the conditions of this study . 
INTRODUCTION
MATERIALS AND METHODS
Preparation of specimens
Testing procedures
Sodium hypochlorite (NaOCl) irrigation
Diode laser plus 2.5% NaOCl
Photodynamic therapy
2.5% NaOCl plus PDT
Chlorhexidine irrigation
Control groups
Sampling procedures
RESULTS
DISCUSSION
CONCLUSION