are polymicrobial in nature with predominant anaerobic microorganisms . bacteria and their metabolic products , enzymes , and toxins play a vital role in the initiation , propagation , and persistence of pulpal and periradicular pathosis . although several factors contribute to endodontic failures , the literature reported that persistent intraradicular or secondary infection is the major etiology for failed endodontic therapy . studies have shown that microflora in failed root canal therapy are different from those found in untreated teeth . enterococcus faecalis ( e. faecalis ) and candida albicans ( c. albicans ) are considered to be the most resistant species in infected root canals and they are often associated with endodontic treatment failures . e. faecalis is a gram positive , facultative anaerobic cocci commonly found in persistent root canal infection and commonly recovered in over one - third of the canal of endodontically treated teeth with persisting periapical lesions . c. albicans , the most predominant and commonly isolated yeast in failed root canal treatment with periradicular pathosis . it may gain access to the root canal from the oral cavity as a result of poor asepsis during endodontic treatment procedures or because of coronal leakage . it is a dentinophilic aerobic microorganism able to survive in harsh ecologic environment of root canal . due to complex anatomy of root canal system , irrigants help in removal of debris and microorganisms form the canal that can not be reached by endodontic instruments . an ideal root canal irrigant should have good antimicrobial efficacy , nontoxic to periapical tissues , capable of dissolving necrotic tissue , able to lubricate the canal , and help in smear layer removal . sodium hypochlorite ( naocl ) has been widely used as root canal irrigant due to its potent antimicrobial activity and tissue dissolving ability . but it has several undesirable characteristics such as tissue toxicity , risk of subcutaneous emphysema , allergic potential , and disagreeable smell and taste . chlorhexidine ( chx ) digluconate has been suggested as root canal irrigant because of its good antimicrobial efficacy and substantivity . it seems to act by adsorbing onto the cell wall of microorganisms resulting in leakage of intracellular components . at low concentration , it has bacteriostatic effect . at higher concentration , qmix 2 in 1 , an experimental irrigating solution , contains a mixture of a bisbiguanide antimicrobial agent , a polyamino carboxylic acid calcium chelating agent ; saline ; and a surfactant . it was evaluated as an effective irrigant similar to 17% ethylenediaminetetraacetic acid ( edta ) in removing canal wall smear layer after the use of 5.25% naocl as the initial rinse . qmix had stronger antibacterial effects against young and old e. faecalisbiofilm in dentin and had stronger antibacterial effect against e. faecalis present in deep dentin . studies are needed to evaluate the antimicrobial efficacy of qmix 2 in 1 in comparison with naocl and chx against e. faecalis and c. albicans . thus , the aim of the present study is to compare the antimicrobial efficacy of qmix 2 in 1 with 5.25% naocl and 2% chx digluconate against e. faecalis and c. albicans . teeth were cleansed using ultrasonic scalers to render them free from calculus and tissue tags , following which they were stored in physiological saline until use . the teeth were decoronated at the cementoenamel junction using diamond disc ( isomet 2000 ; buehler ltd , lake bluff , il ) and the root lengths was standardized to approximately 14 mm . pulp was extirpated and working length was established 1 mm short of apex where the file exited the apical foramen . gates glidden drills # 3 ( mani inc , tachigi - ken , japan ) were used to prepare the root canal orifices ( 3 mm ) . apical preparation was done using k - file ( dentsply maillefer , ballaigues , switzerland ) up to the iso size # 40 . canals were irrigated copiously with physiologic saline ( 0.9 w / v nacl ) after each instrument . each canal was then rinsed with 1ml of 17% edta ( prime dental product ) for 1 min followed by 3 ml of 5.25% naocl ( prime dental product , mumbai , india ) to facilitate the removal of smear layer . the roots were coated with nail varnish and apical foramen sealed with type ii glass ionomer cement ( gic ; gc fuji , tokyo , japan ) . the roots were sterilized in an autoclave for 15 min at 121c and 15 lb of pressure to ensure complete sterilization within the canal space . the paper points were placed into a test tube containing 1 ml reduced transport fluid , vortexed for 10 s , placed onto brain heart infusion ( bhi ) agar ( himedia laboratories , mumbai , india ) and sabouraud dextrose agar ( himedia laboratories , mumbai , india ) , incubated at 37c for 48 h , and examined for growth . a suspension of e. faecalis ( atcc 29212 ) and c. albicans ( courtesy ibms institute , chennai , india ) were adjusted to 0.5 turbidity on the mcfarland scale ( 1.5  10 bacteria / ml ) . e. faecalis suspension of 10 l was injected into group i samples containing 40 teeth and 10 l suspension of c. albicans into group ii samples containing 40 teeth using a micropipette in a class ii vertical laminar airflow cabinet to prevent any airborne contamination . then inoculated specimens with e. faecalis were placed in vials filled with bhi agar and inoculated specimens with c. albicans were placed in vials filled with sabouraud dextrose agar , then inoculum was added every day and incubated aerobically at 37c for 3 days . after 3 days , aliquots were taken from each tooth using a syringe and plated on bhi agar to verify the growth of e. faecalis and sabouraud dextrose agar to verify the growth of c. albicans . at the end of 3 days , teeth were removed from the vials and excess fluid in canal was removed with sterile paper points . the specimens from each group were divided into four subgroups ( a , b , c , and d ) of 10 teeth each depending on the experimental irrigating solution used . test solutions used were : subgroup ia and iia : 3 ml of 5.25% naocl ( prime dental product , mumbai , india ) , subgroup ib and iib : 3 ml of 2% chx ( dentochlor , ammdent , pb , india ) , subgroup ic and iic : 3 ml of qmix 2 in 1 ( dentsply tulsa dental specialities , tulsa , ok ) , subgroup i d and iid : 3 ml of 0.9% saline ( positive control ) . irrigation was accomplished using 30 gauge prorinse needle ( dentsply ) placed 1 - 2 mm short of the working length . the time of contact of each irrigant was 1 min and a final flush was done with 5ml of distilled water to terminate the action of irrigant . a small amount of saline solution was introduced into the canal , and an endodontic hand file was used in a filing motion to a level 1 mm short of the root apex . sterile 30 gauge needle was used to collect 0.01ml ( 10 l ) of the sample from the canal . using a bacterial inoculation loop ; the plates were incubated at 37c for 24 h and then number of colony forming units ( cfus ) was counted . mean log cfus were compared among the groups using analysis of variance ( anova ) followed by post - hoc games - howell test . results were analyzed with the statistical package for social sciences ( spss)/predictive analytics software ( pasw ) version 18 software . qmix 2 in 1 was the most effective and statistically significant when compared with 5.25% naocl and 2% chx ( p < 0.001 ) . there is no statistical significant difference between 5.25% naocl and 2% chx ( p > 0.001 ) . the main goal of chemomechanical preparation of infected root canals is to completely eliminate intracanal bacteria and prevention of recontamination after treatment . this laboratory study was aimed to evaluate the antimicrobial efficacy of qmix 2 in 1 , naocl , and chx against e. faecalis and c. albicans . the in vitro model developed by orstavik and haapasalo has been used to assess antimicrobial efficacy of irrigants . e. faecalis and c. albicans are frequently isolated in root canals with pulpal infection and in secondary / persistent endodontic infections . in the present study qmix 2 in 1 has shown good antimicrobial efficacy and statistically superior to all other irrigants ( p < 0.01 ) . surface active agent lowers the surface tension of solution and increases their wett ability and enables better penetration of an irrigant in the root canal . the potential benefit of bisbiguanide in this mixture is that it prevents the microbial colonization on dentin surface . calcium chelating agent can cause cell wall damage in gram negative bacteria by chelating and removing divalent cations ( mg and ca ) from bacterial cell membrane and increasing its permeability . wang et al . , concluded that qmix and 6% naocl had stronger antibacterial effects against young and old e. faecalis biofilm in dentin than 2% naocl and 2% chx . qmix and 6% naocl had stronger antibacterial effect against e. faecalis which resides deep in dentin than 1% and 2% naocl and 2% chx . qmix 2 in 1 showed statistically superior antimicrobial efficacy against c. albicans . c. albicans was more resistant to irrigating solutions when the smear layer was present . edta present in qmix 2 in 1 effectively removes the smear layer and chx prevents the colonization of fungi on dentinal walls . there have been very few published studies examining the effect of antimicrobial agents on fungi . those articles that have been published are difficult to compare because of variations in methodology and the agents tested . but there was statistically significant difference present between qmix 2 in 1 and naocl ( p < 0.001 ) . naocl exerts its antibacterial effect by inducing the irreversible oxidation of sulfhydryl groups of essential bacterial enzymes , resulting in disulfide linkages , with consequent disruption of the metabolic function of bacterial cells . many in vitro and in vivo studies have proved naocl has good antimicrobial efficacy against e. faecalis . fidalgo et al . , evaluated the antimicrobial activity of naocl , citric acid , and edta against e. faecalis , c. albicans , and staphylococcus aureus and concluded that 2.5% and 5.25% naocl are microbiocides against s. aureus , while 0.5% and 1% naocl are only microbiostatic against the tested e. faecalis and c. albicans . the positively charged ions released by chx can adsorb into dentin and prevent microbial colonization . chx has shown good antimicrobial efficacy against e. faecalis and c. albicans , but it is inferior to qmix 2 in 1 in this study ( p < 0.001 ) . pavlovic and zivkovic concluded that 2% chx solution was effective against s. aureus , streptococcus mutans , e. faecalis , escherichia coli , and c. albicans . there was no statistical significant difference between chx and naocl ( p > 0.01 ) . arslan et al . , concluded that propolis , biopure mtad , 5% naocl , and 2% chx showed good antimicrobial activity against e. faecalis and c. albicans . ruff et al . , investigated the efficacy of 6% naocl , 2% chx , 17% edta , and biopure mtad against c. albicans and concluded that 2% chx and 6% naocl were equally effective and statistically superior to 17% edta and biopure mtad . saline has also shown considerable reduction in the cfu counts of e. faecalis and c. albicans . the mechanical shaping and cleaning of the canals and irrigation with saline served to flush out the debris from the canals . kuruvilla and kamath reported a significant reduction in microbial counts with usage of saline in canals without mechanical preparation . within the limitations of this study , qmix 2 in 1 demonstrated significant antimicrobial efficacy against e. faecalis and c. albicans .