endodontic irrigants should have , amongst other properties , a broad antimicrobial 
 spectrum of activity against anaerobic and facultative micro - organisms growing in 
 biofilms and a relative absence of toxicity against periapical tissues and oral 
 mucosa .
sodium hypochlorite ( naocl ) is recommended as the main root canal irrigant because of 
 its broad antimicrobial activity , the capacity to prevent formation of and dissolve the 
 smear layer , in association with chelating agents , and its ability to dissolve tissue 
 remnants .
however , naocl has 
 been shown to have a cytotoxic effect on vital tissue and can therefore elicit 
 inflammatory reactions if it reaches the periapex .
furthermore , naocl has been shown to cause a change in the force 
 required to fracture dentin , and a 
 reduction of the elastic modulus and flexural strength of dentin .
furthermore naocl corrodes protaper niti rotary ( dentsply / maillefer , baillagues , 
 switzerland ) and carbon steel instruments , cause early fractures of protaper ( dentsply / maillefer ) 
 instruments and , when heated , 
 reduce resistance to cyclic fatigue of nickel - titanium files .
sterilox 's aquatine alpha electrolyte ( optident dental , ilkley , west 
 yorkshire , uk ) is a super - oxidized water that consists of a mixture of oxidizing 
 substances including hypochlorous acid ( hocl ) at a concentration of 144 mg / l , with a ph 
 of 5.0 - 6.5 and a redox potential of > 950 mv20 .
the manufacturer suggests that the 
 production of hocl in the sterilox dental system ( optident dental ) does not produce free 
 radical cl and that the available free chlorine in the solution is 200 ppm , that is 
 larger than the concentration reported in literature .
super - oxidized water has been suggested as an alternative to naocl , as it provides 
 efficient cleaning of root canal walls , and has been recommended for the disinfection of 
 endoscopes , dental unit water 
 lines and dental impression 
 materials .
the aim of this study was to compare the antimicrobial action against enterococcus 
 faecalis of naocl , optident sterilox electrolyte solution ( optident 
 dental ) and sterilox 's aquatine alpha electrolyte when used as 
 irrigating solutions in an e. faecalis infected bovine root canal model .
bovine incisors were used throughout this study . the study exerted no influence on the 
 animal 's fate at any stage as they were previously slaughtered in an italian 
 slaughterhouse for commercial purposes .
the apical 5 mm and the crown of each incisor 
 were dissected and the remaining root was cut into 1 cm slices with a diamond disc 
 ( abrasive technology inc , westerville , oh , usa ) . subsequently the canal lumen was 
 widened to a minimal diameter of 1.4 mm using the parapost xp 
 endodontic post system drills ( coltene / whaledent , konstanz , germany ) .
finally the smear layer was removed via copious irrigation in an edta solution ( smear 
 clear , sybronendo , scafati , italy ) ( 4 min ) and naocl ( teepol bleach , teepol , orpington , 
 uk ) ( 4 min ) in an ultrasonic bath .
fifteen roots were placed individually in 10 ml of brain heart infusion ( bhi ) broth 
 ( oxoid , basingstoke , uk ) and autoclaved .
these were left to cool to room temperature and 
 then incubated overnight at 37c to verify the sterility of the samples .
the bhi 
 broths containing the roots were inoculated with 100 l of an overnight culture of 
 e. faecalis jh2 - 2 
 and incubated for 10 days at 37c to allow for bacterial growth , infiltration of 
 the dentin tubules and e. faecalis jh2 - 2 biofilm formation .
the roots were divided into 3 groups , according to the irrigant used : group 1 was 
 irrigated with the optident sterilox electrolyte solution ( this is 
 essentially saline and was used as our negative control ) , group 2 was irrigated with 4% 
 naocl ( teepol bleach ) and group 3 was irrigated with freshly prepared sterilox 's 
 aquatine alpha electrolyte solution .
after sealing the apical portion with autoclaved physiowax ( ra lamb ltd , eastbourne , 
 uk ) , 5 cc of the selected irrigant was dispensed using a 27 gauge monoject syringe 
 ( kendall , tyco , mansfield , ma , usa ) in an up - and - down motion , and left in 
 situ for 3 min . following the removal of the apical seal to allow for the irrigation solution to drain , 
 the coronal 5 mm portion of the specimen was sampled by grinding dentin and canal 
 contents using parapost xp endodontic post system drill 
 ( coltene / whaledent ) with a diameter of 1.5 mm .
debris collected in the flutes of each drill was placed in a 1.5 ml microcentrifuge tube 
 containing 1 ml bhi broth .
after vortexing for 10 sec , a serial dilution of the debris 
 containing bhi broth was made and 100 l of neat , 10 , 10 and 
 10 dilutions were plated in duplicate onto fresh bhi agar plates and 
 incubated overnight at 37c . to confirm the morphology and gram group of the 
 bacterial cells ,
if the carry - over of naocl could prevent the growth of cells in 
 the broths an additional experiment was carried out on sterile bovine teeth .
these had 
 been treated the same as the teeth used above except that they had not been inoculated 
 with e. faecalis .
after irrigation with naocl , the debris from the 
 drill flutes were put into 900 l of bhi and 100 l of stationary phase e. faecalis 
 culture was added .
in addition , one group of teeth was irrigated with sterile water to provide a 
 negative control .
colony - forming units ( cfu ) with too many 
 to count ( tmtc ) ( defined here as > 800 cfu per agar plate ) were attributed the highest 
 rank in a non - parametric approach with anova on ranks with a duncan 
 post - hoc .
the kruskal - wallis test was used to compare the 
 three groups involved followed by mann - whitney 's test as a post - hoc 
 procedure adjusted with bonferroni correction for multiple comparisons .
data were analyzed using spss software v. 15.0 for 
 windows ( spss inc . ,
the overnight incubation of the sectioned bovine root canals resulted in no growth in 
 any of the samples .
this indicates that all the root sections were sterile at the start 
 of the experiment .
gram staining of a number of the resulting colonies showed the presence of gram - positive 
 cocci , consistent with the e. faecalis inoculum .
results of the dilution series are presented in table 
 1 , while statistical analysis of the raw data for neat and 10 
 dilution is presented in figure 1 .
each group contained 5 roots 
 and serial dilutions and plating were carried out in duplicate . whilst naocl 
 ( group 2 ) was the only irrigant to eradicate the e. faecalis a significant 
 difference
was seen between the optident sterilox electrolyte 
 solution ( group 1 ) and the sterilox 's aquatine alpha 
 electrolyte irrigation ( group 3 ) tmtc = too many to count ( > 800 cfu per plate ) .
mean number of colony - forming units recovered from debris after dentine grinding 
 and root canal irrigation with naocl ( a ) - sterilox 's aquatine alpha 
 electrolyte ( b ) - or optident sterilox electrolyte solution ( c ) - 
 1a ; neat broth ( no dilution ) .
the vertical dashed line shows 
 significant difference between the groups on either side ( p<0.05 ) .
naocl was the only irrigant to eliminate all bacteria and was significantly better at 
 killing e. faecalis than both the optident sterilox electrolyte 
 solution ( saline ) and sterilox 's aquatine alpha 
 electrolyte .
additionally sterilox 's aquatine alpha 
 electrolyte solution was superior to optident sterilox electrolyte 
 solution .
the experiment to determine the effect of carry - over of naocl resulted in similar colony 
 counts for the samples from teeth irrigated with naocl and water ( results not shown ) 
 indicating that carry over of naocl had no noticeable effect in our experiment .
this study evaluated the antimicrobial action of sterilox 's aquatine alpha 
 electrolyte , a commercially available super - oxidized water in the 
 united kingdom , in bovine root canals .
e. faecalis was selected as the test species , because it is commonly 
 detected in asymptomatic , persistent root canal infections .
the bovine 
 root model was chosen as it is clinically relevant , although the large root canal 
 preparation size allows for more favorable dynamics of irrigation for the solution 
 tested than is likely to occur in vivo .
additionally the number of 
 bacteria present is likely to be artificially high compared to the in 
 vivo situation . despite these limitations , the ex vivo 
 model has been successfully used previously to test the ability of e. 
 faecalis to survive diverse root canal irrigations .
our study suggested that the protocol followed was either able to prevent carry - over of 
 the antimicrobial effect of naocl onto the bhi plates , possibly due to drainage of the 
 solutions after irrigation or any carry over had no effect on the viability of the 
 organisms , possibly due to the immediate dilution of the samples in the bhi broth . 

furthermore , based on pilot studies , it was decided to collect samples at a single depth 
 as no difference was found between different depths of sampling when naocl was used as 
 irrigant ; this is consistent with the results form other investigators on a similar 
 bovine tooth model .
sterilox 's aquatine alpha electrolyte is obtained by passing a sodium 
 chloride solution ( optident sterilox electrolyte solution ) over coated titanium 
 electrodes at 9 amps in a specifically made device ( optident sterilox dental 
 generator ; optident dental ) .
optident sterilox electrolyte solution 
 ( non - activated ) was used as the negative control as we did not expect any antimicrobial 
 action from this irrigant .
naocl was tested , because it is largely recommended as the 
 main root canal irrigant .
endodontic literature suggests that infection of the root canal at the time of 
 obturation has a negative influence on the prognosis of endodontic treatment ; naocl was the only irrigant tested 
 which was consistently associated with negative cultures in our study . however , 
 sterilox 's aquatine alpha electrolyte might be able to reduce the 
 bacterial load to levels that could influence treatment outcome .
these results are not consistent with those of a previous study where different 
 irrigants , including naocl , where tested against e. faecalis in a 
 bovine tooth model , in fact krause , et al .
( 2007 ) suggest that 5.25% naocl was not able to render the dentinal 
 shavings obtained sterile , it was however , significantly more effective than the other 
 solutions tested .
the major 
 difference between the models is the difference in volumes of irrigation used ; 60 l 
 twice against 5 cc in our study , therefore suggesting a role for the amount of irrigant 
 used on the ability to eliminate root canal infection in the bovine root model ; in the 
 same way a previous investigation indicates that the volume of irrigation has a 
 significant influence in removing a bio - molecular film from root canal walls .
the disinfecting actions of super - oxidized water are heavily reduced in the presence of 
 organic contamination .
the model used in our study allows for 
 a greater bacterial growth than one might expect in an in vivo 
 situation .
it further excludes the mechanical aspect of root canal preparation , so that 
 the bacterial biomass present in the root canal is likely to be greater than in normal 
 clinical conditions .
consequently , its elimination will depend exclusively on the 
 flushing and chemical effects of the irrigation solution tested .
the importance of 
 instrumentation in obtaining a significant reduction in bacterial content has been 
 shown .
therefore we 
 hypothesize that , in the presence of a reduced bacterial load , as a result of a 
 chemo - mechanical preparation super - oxidized water irrigation might have the ability to 
 eradicate a more clinically relevant root canal infection .
the result of a previous study showed 
 that super - oxidized water had no ability to prevent the growth of e. faecalis 
 using paper disks as the delivery method on petri dishes , a protocol more 
 favorable to the irrigant when compared to the bovine root model due to long time of 
 contact with the micro - organisms , absence of interaction with dentin and cells in a 
 metabolically active phase , therefore more susceptible to antimicrobials .
nonetheless , a different irrigation 
 source was tested ( dermacyn , oculus innovative sciences , petaluma , ca , usa ) .
different 
 super - oxidized waters are produced by a similar electrolysis process but , due to a 
 difference in the active concentration and the ph of the final solution , the product can 
 have a different anti - microbial activity .
one of the suggested advantages of super - oxidized water , when compared to naocl , is its 
 level of toxicity .
it is worth 
 noting , that the mechanism of action of super - oxidized water involves oxidative damage 
 which might cause ageing and irreversible dysfunctions that eventually produce cellular 
 death .
a ph - neutral 
 super - oxidized solution ( microcyn ; dermacyn , oculus innovative sciences , petaluma , ca , 
 usa ) has been tested .
it was found to be significantly less cytotoxic than antiseptic 
 hydrogen peroxide concentrations ( used as a positive control for oxidative damage ) 
 because it does not induce genotoxicity or accelerated ageing in 
 vitro .
however , 
 microcyn has a different ph than sterilox 's aquatine alpha 
 electrolyte and this needs to be taken into account when comparing 
 the two irrigants .
under the conditions of this study sterilox 's aquatine alpha 
 electrolyte appeared to have significantly more antimicrobial 
 action when used as an irrigant in the root canal system compared to the non - activated 
 optident sterilox electrolyte solution , but naocl was the only 
 irrigant able to eliminate all bacteria in our experiments .
sterilox 's aquatine alpha 
 electrolyte caused a bacterial load decrease although being less 
 effective than naocl .
 ideally root canal irrigants should have , amongst other properties , antimicrobial 
 action associated with a lack of toxicity against periapical tissues . sodium 
 hypochlorite ( naocl ) 
 is a widely used root canal irrigant , however it has been shown 
 to have a cytotoxic effect on vital tissue and therefore it is prudent to investigate 
 alternative irrigants . 
 sterilox 's aquatine alpha electrolyte 
 belongs to the group of the super - oxidized waters ; it consists of a mixture of 
 oxidizing substances , and has been suggested to be used as root canal irrigant . 
 
 super - oxidized waters have been shown to provide efficient cleaning of root canal 
 walls , and have been proposed to be used for the disinfection of medical 
 equipment.objectiveto compare the antimicrobial action against enterococcus faecalis of naocl , 
 optident sterilox electrolyte solution and sterilox 's aquatine 
 alpha electrolyte when used as irrigating solutions in a bovine 
 root canal model.methodologyroot sections were prepared and inoculated with e. faecalis jh2 - 2 . 
 after 10 days 
 of incubation the root canals were irrigated using one of three solutions ( naocl , 
 optident sterilox electrolyte solution and sterilox 's aquatine 
 alpha electrolyte ) and subsequently sampled by grinding dentin 
 using drills . 
 the debris was placed in bhi broth and dilutions were plated onto 
 fresh agar plates to quantify growth.resultssodium hypochlorite was the only irrigant to eliminate all bacteria . when the 
 dilutions were made , although naocl was still statistically superior , sterilox 's 
 aquatine alpha electrolyte solution was superior to optident 
 sterilox electrolyte solution.conclusionunder the conditions of this study sterilox 's aquatine alpha 
 electrolyte appeared to have significantly more 
 antimicrobial action compared to the optident sterilox electrolyte 
 solution alone , however naocl was the only solution able to 
 consistently eradicate e. faecalis in the model . 
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS