sample selection and preparation of tooth samplespreparation of inoculumassignment of tooth samples into experimental groupsculture apparatus . 
  sample selection and preparation of tooth samples preparation of inoculum assignment of tooth samples into experimental groups fifty extracted single - rooted human teeth which were intact , noncarious , nonfractured , standardized canal preparation was done with protaper rotary niti files ( dentsply - maillefer , ballaigues , switzerland ) in a crown down technique 1 mm short of the apical foramen to a 50 size that is up to f5 . the apical foramina are enlarged and kept patent using # 15 k - file ( mani co , tokyo , japan ) approximately 1 mm beyond the apical foramen . final flush was completed with aqueous 17% edta ( b n laboratories , mangalore ) to open all dentinal tubules to facilitate bacterial ingress . canal was then dried with a paper point , cementum was coated with two layers of nail polish and apical patency was confirmed . ligature wire was wrapped around a cervical portion of the teeth to suspend the teeth into the culture media [ figure 1 ] . patency files coated with streptococcus sanguis passed into the teeth with sodium hypochlorite , and teeth was suspended into culture medium with ligature wire initially , a stock of streptococcus sanguis ( himedia laboratories , mumbai ) was streaked onto a plate of tryptic soy agar ( himedia laboratories mumbai ) with 5% defibrinated sheep blood . single colony was obtained from this plate and inoculated into tryptic soy broth in a test tube and cultured overnight under the same condition . fifty teeth were randomly divided into five groups of 10 each : 
 group 1 [ figure 2 ] : experimental group  contaminated files with s. sanguis were placed 23 mn beyond apical foramen in teeth filled with naocl and into the culture mediumgroup 2 [ figure 3 ] : experimental group  contaminated files with s. sanguis were placed in a fresh solution of naocl for 10 s and then placed into the teeth filled with naoclgroup 3 [ figure 4 ] : first positive control group  contaminated file passed through salinegroup 4 [ figure 5 ] : second positive control group  contaminated file placed adjacent to teeth filled with naoclgroup 5 [ figure 6 ] : negative control group - sterile files passed through naocl in the root canal . group 1 [ figure 2 ] : experimental group  contaminated files with s. sanguis were placed 23 mn beyond apical foramen in teeth filled with naocl and into the culture medium group 2 [ figure 3 ] : experimental group  contaminated files with s. sanguis were placed in a fresh solution of naocl for 10 s and then placed into the teeth filled with naocl group 3 [ figure 4 ] : first positive control group  contaminated file passed through saline group 4 [ figure 5 ] : second positive control group  contaminated file placed adjacent to teeth filled with naocl group 5 [ figure 6 ] : negative control group - sterile files passed through naocl in the root canal . group 1 specimens showing no growth group 2 specimens showing no growth group 3 ( first positive control ) specimens showing 100% growth group 4 ( second positivecontrol ) specimens showing 100% growth group 5 specimen ( sterile files  negative control ) showing no growth each test tube contains 2 ml of fresh tryptic soy broth . 1 ml of sodium thiosulfate ( b n laboratories , mangalore ) was added to the tubes to neutralize any residual chlorine that may have leaked into the broth . teeth were suspended into test tubes with ligature wires wrapped around its cervical portion so that the roots are 34 mm into the broth . 15 k - type file coated with s. sanguis was placed into the root canal and extended 23 mm beyond the apical foramen . test tubes were capped , and cultures were allowed to grow for 24 h at 37c . 100 l of culture from test tube were spread onto plates of tryptic soy agar with 5% sheep blood and allowed to culture for 48 h at 37c under anaerobic condition . fifty extracted single - rooted human teeth which were intact , noncarious , nonfractured , un - restored were used in this study . standardized canal preparation was done with protaper rotary niti files ( dentsply - maillefer , ballaigues , switzerland ) in a crown down technique 1 mm short of the apical foramen to a 50 size that is up to f5 . the apical foramina are enlarged and kept patent using # 15 k - file ( mani co , tokyo , japan ) approximately 1 mm beyond the apical foramen . final flush was completed with aqueous 17% edta ( b n laboratories , mangalore ) to open all dentinal tubules to facilitate bacterial ingress . canal was then dried with a paper point , cementum was coated with two layers of nail polish and apical patency was confirmed . ligature wire was wrapped around a cervical portion of the teeth to suspend the teeth into the culture media [ figure 1 ] . patency files coated with streptococcus sanguis passed into the teeth with sodium hypochlorite , and teeth was suspended into culture medium with ligature wire initially , a stock of streptococcus sanguis ( himedia laboratories , mumbai ) was streaked onto a plate of tryptic soy agar ( himedia laboratories mumbai ) with 5% defibrinated sheep blood . single colony was obtained from this plate and inoculated into tryptic soy broth in a test tube and cultured overnight under the same condition . fifty teeth were randomly divided into five groups of 10 each : 
 group 1 [ figure 2 ] : experimental group  contaminated files with s. sanguis were placed 23 mn beyond apical foramen in teeth filled with naocl and into the culture mediumgroup 2 [ figure 3 ] : experimental group  contaminated files with s. sanguis were placed in a fresh solution of naocl for 10 s and then placed into the teeth filled with naoclgroup 3 [ figure 4 ] : first positive control group  contaminated file passed through salinegroup 4 [ figure 5 ] : second positive control group  contaminated file placed adjacent to teeth filled with naoclgroup 5 [ figure 6 ] : negative control group - sterile files passed through naocl in the root canal . group 1 [ figure 2 ] : experimental group  contaminated files with s. sanguis were placed 23 mn beyond apical foramen in teeth filled with naocl and into the culture medium group 2 [ figure 3 ] : experimental group  contaminated files with s. sanguis were placed in a fresh solution of naocl for 10 s and then placed into the teeth filled with naocl group 3 [ figure 4 ] : first positive control group  contaminated file passed through saline group 4 [ figure 5 ] : second positive control group  contaminated file placed adjacent to teeth filled with naocl group 5 [ figure 6 ] : negative control group - sterile files passed through naocl in the root canal . group 1 specimens showing no growth group 2 specimens showing no growth group 3 ( first positive control ) specimens showing 100% growth group 4 ( second positivecontrol ) specimens showing 100% growth group 5 specimen ( sterile files  negative control ) showing no growth . 1 ml of sodium thiosulfate ( b n laboratories , mangalore ) was added to the tubes to neutralize any residual chlorine that may have leaked into the broth . teeth were suspended into test tubes with ligature wires wrapped around its cervical portion so that the roots are 34 mm into the broth . 15 k - type file coated with s. sanguis was placed into the root canal and extended 23 mm beyond the apical foramen . test tubes were capped , and cultures were allowed to grow for 24 h at 37c . 100 l of culture from test tube were spread onto plates of tryptic soy agar with 5% sheep blood and allowed to culture for 48 h at 37c under anaerobic condition . none of the teeth in the negative control ( group 5 ) group exhibited growth . all of the teeth in the first positive control group ( contaminated files placed in teeth filled with saline  group 3 ) exhibited growth . all of the teeth in the second positive control group ( contaminated files placed adjacent to teeth filled with naocl  group 4 ) exhibited growth . for experimental groups 1 and 2 , 100% of the ten teeth in each group showed no positive bacterial growth . fishers exact test showed that group i , group ii and the negative control group were statistically significant from the positive control groups ( p = 0.0005 ) . currently , the role of microorganisms in pulpal and periapical diseases is well known , and the anaerobic bacteria are recognized as important pathogens . the predominance of anaerobic microorganisms in the apical third , including the cemental canal , is a common trait in most studies . further , the presence of bacteria in the cemental canal has been strongly demonstrated in cases of necrotic pulps with the periapical lesion . therefore , from a biological standpoint , it does not seem acceptable to preclude the instrumentation of this portion of the canal . patency is leaving the apical foramen accessible , free from dentin chips , pulp fragments , and other debris . therefore , from a biological standpoint , it is recommendable that , in cases of pulp necrosis ( with or without periapical lesion ) , both patency and cleaning of the apical foramen are performed . several instrumentation methods that are currently used to debride and shape root canals advocate the use of patency and recapitulation files . because patency files are used before the canal space has been thoroughly debrided , the files are subject to contamination by bacteria initially present in the canal . if these contaminated files are reused , bacteria can be introduced into the tooth after the canals have been thoroughly debrided . because patency files are taken through the apical constricture this may affect the outcome of endodontic treatment and may decrease the chances of success . this study demonstrated that patency files contaminated with s. sanguis can be disinfected by 2.5% naocl present in the root canal after irrigation . it is a gram - positive asporogenous , facultatively anaerobic , catalase - negative cocci . culture studies have shown that in teeth with clinical and radiographic signs of apical periodontitis , streptococci appeared to survive following root canal treatment . it has been shown to invade cervical and mid root dentin and 60  at apical dentin . finally , s. sanguis is easily grown on tryptic soy agar with 5% sheep blood . because of alpha hemolysis , they reduce iron in hemoglobin , resulting in greenish color on blood agar , which is clearly evident in this study . naocl is used in concentration varying from 0.5% to 7% . the antibacterial effect and tissue dissolving capacity of aqueous hypochlorite is a function of its concentration , but so is its toxicity . at high concentration , severe irritation have been reported , when the solution were inadvertently forced into periapical tissues . on the other hand , one natural explanation to poorer in vivo performance of naocl is complex root anatomy in the apical third of the canal . in addition , the chemical milieu in the canal is different from a test tube . haapasalo using dentine powder showed that the presence of dentine caused marked delays in the killing of the test organism enterococcus faecalis ( group d streptococci ) , by 1% naocl . considering all these factors , it would be rational to use naocl in the concentration of 2.5% , which was the concentration used in this study . sodium thiosulfate was added in order to neutralize any leakage of residual chlorine into the culture medium . the first positive control group consisted of contaminated files placed through teeth that did not contain naocl . however , it was possible that leakage of naocl into the culture medium could prevent bacterial growth despite the addition of sodium thiosulfate . therefore , sterilized teeth in the second positive control group were filled with naocl to account for possible leakage into the culture medium , and the files were placed on the outside of the teeth . the present study focused on the ability of a single solution to prevent inoculation of periapical tissues with patency files . other solutions may be just as effective as naocl , using the experimental model outlined above . also , when the experimental design was chosen , it was assumed that the naocl would not be able to prevent inoculation of periapical tissues simply by passing the contaminated file through the naocl in the tooth . therefore , group 2 was added to the design to show the results of prolonged exposure the test organism to naocl . obviously , naocl exceeded the expectations of the experiment , and its effectiveness could have been shown without immersing the contaminated files for 10 s. the present study demonstrated that patency files contaminated with s. sanguis can be effectively disinfected by 2.5% naocl present in the root canal after irrigation which means instrumentation process is completed in this case . however , further studies are needed to show the ability of test solutions to prevent inoculation of periapical tissues throughout the instrumentation process .
the purpose of this in vitro study was to analyze the effectiveness of 2.5% sodium hypochlorite ( naocl ) in preventing inoculation of periapical tissue with contaminated patency files . 
 fifty single - rooted teeth with single canals were used in the study . 
 they were randomly divided into five groups of which two were experimental groups , two positive controls , and one negative control group . after root canal preparation , teeth in group 
 i ( experimental ) were filled with 2.5 naocl and # 15 stainless steel k - files contaminated with streptococcus sanguis ( atcc # 10556 ) were allowed to pass through the root canal into the culture medium and cultured . 
 teeth in group ii ( experimental ) were also filled with naocl , but contaminated files used in this group were immersed in 2.5% naocl for 10 s prior to being placed in the canal . 
 the negative control used sterile files pass through 2.5% naocl into the culture medium . the first positive control 
 used contaminated patency files in teeth with saline . 
 the second positive control group placed contaminated files into broth next to teeth filled with naocl ( to evaluate potential chlorine leakage ) . 
 the results were as follows . 
 both the experimental groups and the negative control group showed no growth . both the positive control groups 100% growth for s. sanguis . 
 this indicates that the naocl present in the canal after irrigation was sufficient to kill the test organism .