the goals of endodontic instrumentation include thorough debridement and disinfection of root canal . in an effort to achieve these goals debris such as dentinal filings , necrotic pulp tissue , bacteria and their products
may be extruded from apical foramen into the periradicular region which has been referred to as  worm of necrotic debris .
bacteria extruded from the root canal are mainly gram positive , gram negative , and obligate anaerobes .
enterococcus fecalis , propionibacterium alactolyticus , propionibacterium propionicum were found in root canal failure cases .
recently e. fecalis has been identified as a species most commonly recovered from post treatment disease .
seltzer s and naidrof , tanlap j et al . studied the immunological aspects of postoperative flare - ups and concluded that antigens originating from the root canal result in the formation of antigen  antibody complex when forced beyond the apical foramen , which may lead to severe inflammatory response .
the intensity of inflammatory response will depend on the number ( quantitative factor ) and virulence ( qualitative factor ) of bacteria .
when virulent clonal types of bacterial species are propelled into periapical tissue , even small amount of infected debris can exacerbate the periapical inflammation .
er et al . and kustarci a et al . reported that the intracanal bacteria can be extruded apically along with debris , during instrumentation utilizing a new experimental model .
various studies have been used in the past in evaluating the apical extrusion of debris and irrigant and but very few studies are reported in the literature about bacterial extrusion using an experimental model .
a total of 80 freshly extracted human mandibular premolars were used for the study . teeth with complete root formation , having single canal and mature apical foramen , length of roots approx 21 mm , canal curvature between 0 to10 , apical diameter conforming to 15 k file were selected .
teeth were observed under stereomicroscope and digital radiographs in buccal and proximal directions to determine the presence of single canal .
the tooth was forced through the rubber stopper of a vial after endodontic access cavity preparation .
two coats of nail varnish applied to the external surface of the root and then the tooth with the rubber stopper fitted into mouth of the vial .
23-gauge needle was inserted into the vial through the rubber stopper to equalize the air pressure .
the hole was created in nail varnish that covered the apical foramen using 10 k - file . in this way ,
the tooth with rubber stopper needle unit was placed into the mouth of the vial .
a pure culture of e. fecalis ( atcc 29212 ) was used to contaminate the root canal .
suspension was prepared by adding 1 ml of pure culture of e. fecalis grown in brain - heart infusion broth for 24 h to fresh brain  heart infusion broth .
the mcfarland standard number 0.5 was used to evaluate the broth to ensure that number of bacteria was 1.5  10 colony forming units ( cfu ) ml / l .
canals were hand instrumented with # 10 k - file to carry the bacteria down the length of the canal .
the contaminated root canal was dried at 37c for 24 h. single operator , using aseptic techniques , carried out the preparation and sampling procedures on each specimen under a class i laminar airflow cabinet to prevent airborne bacterial contamination .
group i  teeth in this group were instrumented with the step - back technique .
the procedure was divided into two phases : phase i apical preparation starting at the apical constriction till # 30 k - file .
phase ii preparation of remainder of the canal , gradually stepping back while increasing the instrument # 30 to # 50 .
frequent recapitulation using # 10 , # 15 , # 20 , and # 25 k - files as larger size files are used for apical preparation .
instrumentation was done with hand protaper , a set of six instruments : three shaping files ( sx , s1 , and s2 ) for crown down procedure and three finishing files ( f1 , f2 , and f3 ) for apical shaping . followed by frequent recapitulation with # 10 , # 15 , # 20 , and # 25 k - files as the larger - sized files were used for apical preparation .
ti technique , canals were prepared with 0.12 taper k-3 instrument to the resistance followed by 0.10 taper and 0.08 taper instruments .
initial glide path was achieved using # 10 , # 15 , and # 20 k - files .
canals were further prepared with 0.06 taper with # 45 k-3 instruments to the resistance from largest instrument to smallest reaching the working length . after middle
third scouting with # 10 k - files # 40 , # 35 , # 30 k-3 instruments were used in crown - down fashion till the working length and with frequent recapitulation # 15 , # 20 , and # 25 k - files .
instrumentation with rotary protaper . after achieving a straight - line access ; a smooth glide path was achieved with , # 10 or # 15 k - file used till two - third the working length . s1 shaping file was used and moved apically 3 mm short of working length .
sx files were than used sequentially until resistance was encountered ( 45 mm from working length ) followed by s1 and s2 to working length for shaping of coronal two thirds of the canal .
using f1 , f2 , and f3 files sequentially to the working length finishing of apical third was done . during root canal instrumentation
the rubber stopper was placed on the needle and the needle was advanced into the root canal 3 mm short of working length .
the apical preparation was done till # 30 k - file in all instrumentation techniques . subsequently after root canal preparation 0.1 ml of saline was taken from experimental vial in order to count the bacteria and incubated in brain 
heart infusion agar at 37c for 24 h. colonies of bacteria were counted using a colony counter ( yarco colony counter ) following a classical bacterial counting technique as described by collins et al .
the results indicated that all instrumentation techniques tested caused a measurable apical extrusion of bacteria .
mean number of apically extruded intracanal bacteria the hand instrumentation techniques ( step - back and hand protaper techniques ) extruded more number of bacteria compared to engine driven k-3 and rotary protaper systems .
the step - back technique showed greatest extrusion of bacteria and there was a statistically significant difference ( p<0.05 ) found between the step - back technique and other hand and rotary ni  ti instrumentation techniques .
endodontic flare - up is true complication characterized by development of pain , swelling , and discomfort which commences within few hours or a days after root canal procedures and require an emergency treatment .
the major reason cited for such distressing occurrence is extrusion of debris present within and created during instrumentation of root canal system into periapical tissues resulting in persistent periapical inflammation .
shovelton ds , , seltzer and naidorf , and siqueria have reported that along with debris , the bacteria are also extruded through the apical foramen .
the number of bacteria extruded apically has a direct correlation with weight of debris ( quantitative factor ) , type , and virulence of bacteria ( qualitative factor ) .
naidorf studied the immunological aspects of flare - ups and stated that various mediators released during flare - up will cause damage to the cell membrane resulting in prostaglandin release , bone resorption , amplification of the kinin system and ultimately pain for the patient .
furthermore , perrini and fonzi have found numerous mast cells in human periapical lesions discharging vasoactive amines into the periapical tissues and initiate an inflammatory response .
the aim of present study was to assess the extrusion of intracanal bacteria as a result of canal shaping by different instrumentation techniques .
e. fecalis was chosen as the bacteriological marker because it can survive alone without symbiotic support from other bacteria .
large amount of bacteria extruded apically in step - back technique was due to the watch winding and in - and - out filing motion that acted as piston extruding more amount of bacteria compared with other instrumentation techniques .
difference in the root canal preparation using hand and rotary protaper files is the time of contact between file and the root canal wall .
the engine driven protaper file contacts the apical area for lesser period of time and also the rotational speed and torque is fixed , whereas hand protaper file prepares the apical area for extended period of time and rotational movement of the file is an  operator - controlled variable factor ,  and may extrude more amount of bacteria . the extrusion of bacteria using k-3 ( group - iii ) engine - driven instrumentation technique was the least , which could be related to its use in crown - down fashion .
the presence of variable helix angle and pitch , improves the removal of debris formed by instrumentation coronally .
protaper extruded a significantly higher amount of bacteria , as compared to the k-3 system .
the protaper system is faster system with less number of instruments , which removes substantial amount of dentin in short period of time .
elmsallati et al . , reported that the file with short pitch design extruded less debris than the medium and long ones .
protaper is a long - pitch design instrument while k-3 is short - pitch instrument .
every effort should be made to limit the periapical extrusion of intracanal material during treatment that has the potential to bring about serious systemic disease such as endocarditis , brain abscess , and septicemia , particularly in compromised patients .
further in vivo research in this direction could provide more insight into the biologic factors associated with correlations and consequences of apically extruded debris and may focus on bacterial species that essentially play a major role in post instrumentation flare - ups .
hand instrumentation techniques ( step - back and hand protaper ) extruded large number of bacteria than engine driven techniques ( rotary protaper and k-3 system ) .
the step - back technique extruded highest number of bacteria and among the rotary systems the k-3 system extruded least number of bacteria .
 aim : to evaluate the bacteria extruded apically during root canal preparation using two hand and rotary instrumentation techniques.materials and methods : eighty freshly extracted mandibular premolars were mounted in bacteria collection apparatus . 
 root canals were contaminated with the pure culture of enterococcus fecalis ( atcc 29212 ) and dried at 37c for 24 h. bacteria extruded were collected , incubated in brain heart infusion agar for 24 h at 36c and the colony forming units ( cfu ) were counted.statistical analysis : the mean number of colony forming units were calculated by one - way anova and comparison between the groups made by multiple comparison ( dunnet d ) test.results:the step - back technique extruded highest number of bacteria in comparison to other hand and rotary ni  
 ti systems.conclusion:under the limitation of this study all hand and rotary instrumentation techniques extruded bacteria . 
 among all the instrumentation techniques step - back technique extruded more number of bacteria and k-3 system the least . 
 further in vivo research in this direction could provide more insight into the biologic factors associated and focus on bacterial species that essentially play a major role in post instrumentation flare - ups . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION