in endodontic literature , the importance of chemomechanic preparation has long been emphasized . in particular , the irrigation procedure has been a crucial step in endodontic treatments . sodium hypochlorite is the most commonly used irrigant for non - surgical endodontic procedures , despite its limitations . its allergenic potential , corrosive potential , and toxicity to periapical tissues are the main problems associated with sodium hypochlorite ( 1 , 2 ) . enterococcus faecalis is often isolated from infected teeth presenting with persistent disease ( 3 ) . enterococci are the leading resistant bacteria in different endodontic treatments ( 4 , 5 ) . this type of bacteria can penetrate into dentinal tubules ( 6 ) , resist against high ph values ( 7 ) , exhibit virulence factors ( 8) , and form biofilms . moreover , biofilms can show strong antimicrobial tolerance through four mechanisms : the physical barrier properties of the extracellular polysaccharide matrix ( 10 ) , slow growth of bacterial cells residing within a biofilm ( 11 ) , deep locations of cells that stay alive under decreased oxygen tension ( 12 ) , and highly resistant phenotypic states ( 13 ) . , super - oxidized solutions have been shown to be potent antimicrobial agents and disinfectants through oxidative damage ( 14 ) . electrolyzed water contains a mixture of inorganic oxidants , such as hypochlorous acid ( hclo ) , hypochlorous acidic ion ( clo ) , chlorine ( cl2 ) , hydroxide ( oh ) , and ozone ( o3 ) , which are effective for inactivating a variety of microorganisms responsible for endodontic infections ( 15 ) . anolyte solution has been termed super - oxidized water ( spo ) ( 17 ) . the authors also noted that eca resulted in more numerous open dentin tubules throughout the whole length of canals according to naocl . one of these disinfectants is spo , which includes the highly reactive superoxide ion o2 , which is a common form of oxygen that is created when molecular oxygen gains a single electron . superoxide radicals can attack susceptible biological targets , including lipids , proteins , and nucleic acids ( 18 ) . one of the other advantages of spo is its lower level of toxicity when compared to naocl ( 19 ) and hydrogen peroxide ( 20 ) . moreover , spo has been investigated as an irrigation solution in root canals ( 16 , 21 ) and recommended as a disinfectant for dental unit water lines and endoscopes ( 17 ) . the aim of the present study was to evaluate and compare the antibacterial efficacy of spo on e. faecalis biofilms in human root canals at different irrigation times . informed consent was obtained from the patients before the study , and the study was approved by the local ethics committee on human research of cumhuriyet university ( ethics committee code : 09/02/2013 ) . one hundred twenty - six human mandibular premolar teeth with a single canal were used for the study . after the residues were removed , following the extraction , teeth were kept in a 0.9% saline solution at 4c . sterile diamond discs ( horico , hopf , ringleb & co. , gmbh & cie , berlin , germany ) were used to remove the coronal portions of the teeth , below the level of the cemento - enamel junction , under water cooling . the root canals were prepared using protaper next ( dentsply , tulsa endodontics , oklahoma , usa ) ni - ti instruments with crown - down technique with an electric motor ( sendoline , perfect endo , australia ) . pn instruments were used with a gentle in - and - out brushing motion at a speed of 300rpm , with light apical pressure . for optimum usage , the following instrumentation sequence was used : x1 ( size 17 , 0.4 taper ) , x2 ( size 26 , 0.6 taper ) , x3 ( size 30 , 0.7 taper ) , and x4 ( size 40 , 0.6 taper ) instruments at the wl . one ml of 5.25% naocl solution was used after each instrumentation in the root canals . finally , next , three layers of nail polish ( loreal jet - set diamond , paris , france ) were applied to the whole external root surface of each tooth . finally , the rubber caps were sterilized by eto ( melag , euroklav 23v - s , germany ) . enterococcus faecalis ( atcc 29212 ) strains were cultured in blood agar ( brain - heart infusion agar , acumedia manufactures , inc . , lansing , michigan , usa ) . the incubation procedure was performed at 37c for 24 hours . before each experiment , 0.5 mcfarland turbidity was set with a kristalspectm device . then , the strains were subcultured in a trypticase soy broth ( detroit , michigan , usa ) and incubated aerobically at 37c for 24 hours . the value of 10l of the bacterial suspension ( final concentration of about 1.510 ) was transferred to the mechanically expanded lumen of the root canals , using a sterile micropipette ( blaubrand , wertheim , germany ) , except for ten canals , which were used as negative controls . the orifices of the root canals were sealed with temporary filling material ( 3 m espe , dental products , usa ) . all samples were stored at 37c for ten days in a humid atmosphere , and the re - inoculation procedure was repeated every 72 hours with fresh cultures on the first , fourth , seventh , and tenth days . scanning electron microscopy analysis was performed to identify the biofilm formation at the end of the tenth day ( figure 1 ) . the sem images of the remaining bacteria obtained from the root canals after super - oxidized water irrigation of groups 3 and 5 are shown in figures 2 and 3 . for sem preparation , the root canals were immersed in a fixative solution containing 4% buffered paraformaldehyde for 24 hours . the root canals were dehydrated in ascending degrees of ethanol series , following their separation with a diamond disc ( horico , hopf , ringleb & co. , gmbh & cie , berlin , germany ) longitudinally . the x20.000 magnification with a scanning electron microscope ( jeol jsm 6400 , noran instruments , tokyo , japan ) was preferred for specimen examination . digital images were taken at the center of the coronal ( 9 mm from apex ) , middle ( 6 mm from apex ) , and apical ( 3 mm from apex ) thirds of each canal in both root segments . twenty teeth with biofilm processed canals were chosen for each group and randomly divided into six groups , as follows . group 1 : root canal irrigation was performed with 0.9% saline solution ( negative control ) with a 10ml / min flow rate for 2 minutes . group 2 : root canal irrigation was performed with spo ( medilox ; o - m medical dental textile , ankara , turkey ) that consists of a mixture of oxidizing substances , including hypochlorous acid at a concentration of 50 - 80mg / l , with a ph of 5.5 and a redox potential > 850 - 1000mv . group 3 : root canal irrigation was performed with spo with a 10ml / min flow rate for 2 minutes . group 4 : root canal irrigation was performed with spo with a 10ml / min flow rate for 3 minutes . group 5 : root canal irrigation was performed with spo with a 10ml / min flow rate for 5 minutes . group 6 : root canal irrigation was performed with 5.25% naocl ( positive control ) with a 10ml / min flow rate for 2 minutes . bacterial cultures were taken with paper points ( dentsply , maillefer , usa ) that were inserted into the root canals , before and after root canal irrigation , to examine the bacteriological status . for standardization , finally , a total of 120 teeth were evaluated after root canal irrigation , and cfu counts of the breeding colonies of microorganisms were calculated on blood agar plates . variation data were analyzed using the spss statistical software program ( version 22.0 , spss inc . the data were subjected to statistical analysis using one - way analysis of variance ( anova ) and tukey s post hoc test to examine pairwise differences at a significance level of 0.05 . informed consent was obtained from the patients before the study , and the study was approved by the local ethics committee on human research of cumhuriyet university ( ethics committee code : 09/02/2013 ) . one hundred twenty - six human mandibular premolar teeth with a single canal were used for the study . after the residues were removed , following the extraction , teeth were kept in a 0.9% saline solution at 4c . sterile diamond discs ( horico , hopf , ringleb & co. , gmbh & cie , berlin , germany ) were used to remove the coronal portions of the teeth , below the level of the cemento - enamel junction , under water cooling . the root canals were prepared using protaper next ( dentsply , tulsa endodontics , oklahoma , usa ) ni - ti instruments with crown - down technique with an electric motor ( sendoline , perfect endo , australia ) . pn instruments were used with a gentle in - and - out brushing motion at a speed of 300rpm , with light apical pressure . for optimum usage , the following instrumentation sequence was used : x1 ( size 17 , 0.4 taper ) , x2 ( size 26 , 0.6 taper ) , x3 ( size 30 , 0.7 taper ) , and x4 ( size 40 , 0.6 taper ) instruments at the wl . one ml of 5.25% naocl solution was used after each instrumentation in the root canals . finally , next , three layers of nail polish ( loreal jet - set diamond , paris , france ) were applied to the whole external root surface of each tooth . finally , the rubber caps were sterilized by eto ( melag , euroklav 23v - s , germany ) . enterococcus faecalis ( atcc 29212 ) strains were cultured in blood agar ( brain - heart infusion agar , acumedia manufactures , inc . , the incubation procedure was performed at 37c for 24 hours . before each experiment , 0.5 mcfarland turbidity was set with a kristalspectm device . then , the strains were subcultured in a trypticase soy broth ( detroit , michigan , usa ) and incubated aerobically at 37c for 24 hours . the value of 10l of the bacterial suspension ( final concentration of about 1.510 ) was transferred to the mechanically expanded lumen of the root canals , using a sterile micropipette ( blaubrand , wertheim , germany ) , except for ten canals , which were used as negative controls . the orifices of the root canals were sealed with temporary filling material ( 3 m espe , dental products , usa ) . all samples were stored at 37c for ten days in a humid atmosphere , and the re - inoculation procedure was repeated every 72 hours with fresh cultures on the first , fourth , seventh , and tenth days . scanning electron microscopy analysis was performed to identify the biofilm formation at the end of the tenth day ( figure 1 ) . the sem images of the remaining bacteria obtained from the root canals after super - oxidized water irrigation of groups 3 and 5 are shown in figures 2 and 3 . for sem preparation , the root canals were immersed in a fixative solution containing 4% buffered paraformaldehyde for 24 hours . the root canals were dehydrated in ascending degrees of ethanol series , following their separation with a diamond disc ( horico , hopf , ringleb & co. , gmbh & cie , berlin , germany ) longitudinally . the x20.000 magnification with a scanning electron microscope ( jeol jsm 6400 , noran instruments , tokyo , japan ) was preferred for specimen examination . digital images were taken at the center of the coronal ( 9 mm from apex ) , middle ( 6 mm from apex ) , and apical ( 3 mm from apex ) thirds of each canal in both root segments . twenty teeth with biofilm processed canals were chosen for each group and randomly divided into six groups , as follows . group 1 : root canal irrigation was performed with 0.9% saline solution ( negative control ) with a 10ml / min flow rate for 2 minutes . group 2 : root canal irrigation was performed with spo ( medilox ; o - m medical dental textile , ankara , turkey ) that consists of a mixture of oxidizing substances , including hypochlorous acid at a concentration of 50 - 80mg / l , with a ph of 5.5 and a redox potential > 850 - 1000mv . group 3 : root canal irrigation was performed with spo with a 10ml / min flow rate for 2 minutes . group 4 : root canal irrigation was performed with spo with a 10ml / min flow rate for 3 minutes . group 5 : root canal irrigation was performed with spo with a 10ml / min flow rate for 5 minutes . group 6 : root canal irrigation was performed with 5.25% naocl ( positive control ) with a 10ml / min flow rate for 2 minutes . bacterial cultures were taken with paper points ( dentsply , maillefer , usa ) that were inserted into the root canals , before and after root canal irrigation , to examine the bacteriological status . for standardization , specimens under 1.510 cfu / ml were excluded . finally , a total of 120 teeth were evaluated after root canal irrigation , and cfu counts of the breeding colonies of microorganisms were calculated on blood agar plates . variation data were analyzed using the spss statistical software program ( version 22.0 , spss inc . , chicago , illinois , usa ) . the data were subjected to statistical analysis using one - way analysis of variance ( anova ) and tukey s post hoc test to examine pairwise differences at a significance level of 0.05 . the comparisons and mean values of bacterial counting obtained before and after spo irrigation in root canals are shown in table 1 . when the antibacterial effects of all disinfection agents were compared with each other , there were statistically significant differences between group 1 ( negative control ) and all groups ( p < 0.05 ) . table 1 . the comparisons and mean values of bacterial counting obtained before and after spo irrigation in root canalsgroups , n = 20before irrigation ( sx ) , cfu mlafter irrigation ( sx  sd ) , cfu mlgroup 1 , saline ( negative control)1.5  101500000  0group 2 , super - oxidized water 1 min1.5  104400  1960group 3 , super - oxidized water 2 mins1.5  10805  123group 4 , super - oxidized water 3 mins1.5  1039.5  19.6group 5 , super - oxidized water 5 mins1.5  107.50  5.96group 6 , naocl ( positive control)1.5  100  0cfu = colony - forming units ; by the one - way anova , f = 115.425 ; p = 0.000 ( p < 0.05).values with the same capital superscript letter are statistically no different at p > 0.05 by tukey s test.although group 6 ( positive control ) showed no statistically significant difference when compared with groups 4 and 5 ( p > 0.05 ) , group 6 showed a statistically significant difference compared to all other groups ( p < 0.05 ) . cfu = colony - forming units ; by the one - way anova , f = 115.425 ; p = 0.000 ( p values with the same capital superscript letter are statistically no different at p > 0.05 by tukey s test . although there was no statistically significant difference between groups 2 and 3 ( p < 0.05 ) , both groups showed statistically significant differences when compared with other experimental groups ( p > 0.05 ) . stereomicroscopic images obtained from the root canals in groups 3 and 5 are shown in figures 2 and 3 . the sem images of group 3 indicated that some bacteria became intertwined with dentin tubules ( figure 2 ) . the sem images of group 5 demonstrated a low number of bacteria ( figure 3 ) . moreover , the orifices of the dentinal tubules were observed more clearly , and the surfaces of the intertubular dentin showed roughness . many researchers in endodontics have investigated the effects of various disinfectants on e. faecalis ( 22 - 24 ) . e. faecalis still poses a challenge from a clinical point of view , as it is a resistant species that is one of the major causes of endodontic treatment failure . appropriate endodontic irrigation during root canal preparation is therefore necessary to adequately debride the canal system . the actual aim of the present study was to examine the importance of bacterial biofilm during root canal irrigation procedures . the predominating species in post - treatment infection is e. faecalis , which is the most resistant to intracanal medications and invades dentinal tubules as biofilm forms ( 25 ) . the biofilm of e. faecalis may have a significant effect on the rate and extent of attachment by microorganisms and may be created under conditions that simulate conditions in vivo ( 9 ) . moreover , a biofilm can show strong antimicrobial tolerance , with physical barrier properties of the extracellular polysaccharide matrix ( 4 ) . estrela et al . ( 26 ) put forth three aspects that are necessary for biofilm formation : the bacterial colonization structure , the biological indicator , and time . biofilm formation was obtained in the present study . finally , development and maturation of the bacterial biofilm was observed by sem examination . thus , the biofilm form of bacteria can be helpful in facilitating examination of the antimicrobial potential of endodontic materials . the aim of irrigation by chemical solutions should be to eliminate both the smear layer formed during instrumentation and the biofilm , which is difficult to remove when compared with bacteria in a planktonic state . furthermore , the irrigating solutions must indicate strong antimicrobial properties to remove the exopolymeric substance and to guarantee disruption of the biofilm ( 27 ) . 22 ) examined the antibacterial efficacy of 5.25% naocl against e. faecalis at different times . ( 23 ) investigated the antibiofilm and antibacterial effects of 2.5% naocl on e. faecalis biofilms , and most of the bacteria was eliminated in root canals . in another study using 5% naocl , subbiya et al . ( 24 ) found consistent results with former studies , namely 100% biofilm reduction . in the present study , naocl irrigation showed insufficient bacterial elimination at 1 minute ; therefore , 2 minutes irrigation time was preferred . as a result , complete elimination of e. faecalis biofilm was achieved with 5.25% naocl for 2 minutes . the present study purposed to evaluate the antimicrobial activity of spo against e. faecalis biofilm in human root canals ex vivo . the antimicrobial activity of spo was previously examined against bacteria , mycobacteria , viruses , fungi , and spores ( 17 , 19 , 28 , 29 ) . the microbicidal activity of spo , when using membrane filters , was found to be highly active against several microorganisms on endoscope after 2 minutes ( 17 - 19 ) . ( 21 ) investigated the antimicrobial activity of naocl and spo as irrigating solutions on e. faecalis in bovine root canals for 3 minutes . in the present study , spo was used at 3 and 5 minutes in human root canals infected by e. faecalis biofilms . the present study indicated a high antimicrobial effect of spo after 3 and 5 minutes contact time in root canals . spo also showed a similar antibacterial effect with 5.25% naocl in the present study . despite some variables , such as irrigation period , experimental samples , and types of microorganisms , the results of the present study are consistent with the above - mentioned studies . these results show a directly proportional relationship between the antibacterial effect of super - oxidized water and exposure time . in light of this in vitro study , spo showed antimicrobial properties against e. faecalis biofilm , and its prolonged use in root canal was seen to enhance its activity . detailed understanding of the mode of action of various solutions is important for optimal irrigation . under the circumstances of the present study , spo exhibited antimicrobial activity on e. faecalis biofilm in root canals with a 10 ml / min flow rate for 3 minutes . spo is effective in terms of reduction of e. faecalis , and it has potential to be an efficient root canal irrigant . moreover , spo is an effective agent for eliminating the debris and smear layer ( 3 ) and is nontoxic in the apical third of the canal . more investigation is needed to examine irrigants in terms of toxicity , tissue dissolving ability , and strong antibacterial efficacy . none of the available irrigants has all of the characteristics of an ideal irrigant . further research is warranted to determine an ideal material and/or technique to completely clean infected root canals . within the limitations of this in vitro study , it can be concluded that , as a new product , super - oxidized water had a highly antibacterial effect against e. faecalis biofilms in root canals . moreover , super - oxidized water indicated a remarkable and similar bactericidal effect to that of traditional naocl against e. faecalis biofilms . within the limitations of this in vitro study , it can be concluded that , as a new product , super - oxidized water had a highly antibacterial effect against e. faecalis biofilms in root canals . moreover , super - oxidized water indicated a remarkable and similar bactericidal effect to that of traditional naocl against e. faecalis biofilms .
backgroundthe success of endodontic treatment depends on a few crucial factors . 
 one of these factors is the complete chemomechanic preparation of root canal against various bacteria . in particular 
 , the effect of resistant bacteria may cause intense pain with flare - up and formation of periapical lesions . 
 therefore , the strong effect of irrigants plays an important role in terms of the complete elimination of these bacteria to achieve long - term successful treatment.objectivesthe aim of this study was to investigate the antibacterial effects of super - oxidized water ( spo ) in root canals infected with enterococcus faecalis biofilms.methodsone hundred twenty single - root , premolar teeth were selected . 
 initially , the teeth were prepared and then disinfected . 
 e. faecalis were inoculated and kept at 37c for 24 hours in the root canals . 
 the re - inoculation procedure was repeated on the first , fourth , seventh , and tenth days . 
 the infected root canals were divided into one negative ( saline ) and one positive ( sodium hypochlorite ) control group and four experimental groups ( super - oxidized water : 1 , 2 , 3 , or 5 minutes ) ( n = 20 ) . 
 paper points were placed in the root canals to control and evaluate the biofilm formation . 
 biofilms were counted on blood agar plates , and data was evaluated and statistically analyzed using one - way anova and tukey s test.resultsalthough sodium hypochlorite ( naocl ) showed no statistically significant difference when compared with three and five minutes of spo irrigation ( p > 0.05 ) , naocl showed statistically significant differences among all other groups ( p < 0.05).conclusionssuper - oxidized water indicated a remarkable and similar bactericidal effect to that of traditional naocl against e. faecalis biofilms . in terms of successful endodontic treatment approaches 
 , super - oxidized water may be used as an effective irrigation solution in clinics .