## 4 Discussion  
The goal of endodontic treatment is to prevent or eliminate apical pathosis by means of shaping, cleaning, disinfecting, and sealing of the root canal system. Studies have proven that mechanical cleaning alone is limited in its ability to render the canal walls free from biofilm, since at least 10% of the walls remain untouched following instrumentation with the most modern rotary nickel-titanium file systems [16; 17]. Thus, reduction of the microbial load and disruption of biofilms are commonly achieved by a combination of mechanical instrumentation and irrigation with tissue dissolving and antimicrobial irrigants [18]. NaOCl is currently the most widely used irrigation solution due to its antibacterial and tissue-dissolving properties [6; 18]. It has been previously shown that these properties are affected by the pH of the NaOCl solution [7; 8; 9; 15]. Various concentrations of NaOCl are being used clinically, ranging from 0.5-5.25% and above, with pH levels of 12 and above [6; 18]. Solutions with lower pH levels are not commercially available since buffering NaOCl renders the solution unstable with a decrease in its shelf life [6; 9; 15]. In the present study, acidified NaOCl was prepared by adding acetic acid, according to the protocol of [PERSON] et al. [9]. Acetic acid is a suitable buffer solution since it has no effect on the available chlorine [8]. Nevertheless, acidification of NaOCl should be done carefully, as excessive pH reduction may negatively affect the dentin. NaOCl at pH 12 has the capability to reduce biofilm-related matter [11], break glycosidic bonds, dissolve glycoconjugates in the biofilm matrix and cause lysis to bacterial cells [11]. [PERSON] et al. [1], used a similar model to the one used in the present study and reported that 2.5% NaOCl substantially reduced the total biovolume of the biofilm compared to biofilms exposed to distilled water. This result was attributed to the disruption of the structure of the biofilm. In the present study, no such reduction of biovolume was found, and the total biovolume of the biofilms exposed to either of the NaOCl irrigants was not different from the biofilm exposed to saline. A potential explanation could be a difference in the bacteria and conditions used to grow the biofilm. In the former study, microbial samples were collected from infected root canals, and the polymicrobial biofilms (of undefined nature) were grown for three weeks with a weekly refreshment of the BHI medium. It could be that the _E. faecalis_ biofilm grown, in the present study, for four weeks while replenishing the BHI medium every two days created a biofilm more resistant to disruption. Figure 2: Representative confocal laser scanning microscopy images. (**a**) After exposure to saline (control); (**b**) after exposure to 4% NaOCl pH 12; (**c**) after exposure to acidified 4% NaOCl pH 6.5; and (**d**) after exposure to 4% NaOCl pH 12, followed by acidified 4% NaOCl at pH 6.5. Additionally, based on previous studies, one could expect to find a more effective reduction in the total biovolume after treatment with NaOCl at pH 12 compared to the acidified irrigant at pH 6.5 [9; 10; 11; 12; 14]. Such a difference was not found in the present study. The above potential explanation may be in line with previous findings that _E. faecalis_ is a relatively resistant bacterium [19; 20]. Nevertheless, this issue warrants further investigation. _E. faecalis_ was chosen for the present study because it has the ability to survive in vivo as a single organism without the support of other bacteria and because it has been extensively used in antimicrobial endodontic studies [9; 19; 21; 22]. The benefit of the simplified in vitro system used in the present study is its ability to isolate and study a single factor (pH of the NaOCl solutions) and avoiding many confounding factors that could result from variations in the biofilm constituents if a mixed biofilm of unidentified nature was used.