## 4 Discussion  
_E. faecalis_ is a Gram-positive, facultatively anaerobic bacterium with considerable clinical significance. It is commonly isolated in endodontic infections and can cause periradicular inflammation. The bacterium has the capacity to penetrate deep into denitnal tubules to establish communities that are organized in biofilms. It can survive as a monoinfection of the root canal under unusual environmental stress and is resistant to chemomechanical preparation [17,21]. Therefore, it serves as a model microorganism for studies utilizing a bacteriological assessment method and was selected as a bacteriological marker in this study. In previous studies, _E. faecalis_ has been incubated in root canals for variable time periods ranging from 24 h to 30 days [21, 22, 23]. The incubation period selected for in vitro research may have an effect on the elimination of microorganisms from root canals. A longer incubation period allows the bacteria to penetrate deeper into denitnal tubules. In light of the data available in the literature, in this study, specimens were infected for 21 days in order to ensure the effective penetration of _E. faecalis_ into the denitnal tubules [5,6,24]. The most common technique for bacteriological assessment is CFU quantification employing culturing techniques, which is followed by molecular methods utilizing DNA detection. Molecular methods have higher sensitivity. However, they may also detect non-viable microorganisms in the root canal, which may be problematic when examining samples obtained right after treatment procedures. In this study, the CFU counting method was preferred due to its effectiveness and simplicity [1,25,26]. The primary cause of a failure of a root canal treatment is the presence of bacteria. Therefore, microbiological elimination is of utmost importance during treatment procedures. Several methods have been used to evaluate this concept. There are numerous studies in the literature evaluating the effectiveness of mechanical instrumentation with various types of file systems in microbial reduction [15,26]. However, only a few studies have compared both endodontic access cavities and file systems [1,2,18]. Our research aimed to assess the impact of different endodontic access cavities and file systems on bacterial reduction in infected root canals [1,2,27]. Bacteriological sampling was performed with a sterile paper point. Sampling using paper points has the advantage of being usable during both in vitro and in vivo experimentation. On the other hand, it has certain limitations, such as providing the opportunity for sampling microorganisms only within the root canal, not those inside dentin tubules [8]. In the current research, prior to sampling, the hand file was inserted into the root canals with an in-and-out motion to allow for enhanced collection of microorganisms from all regions of the possible sampling areas. It has previously been demonstrated that sterile distilled water used as a root canal irrigation during mechanical preparation does not eradicate microorganisms within infected root canals [8]. Since the present study focused on the comparison between the mechanical effects of different file systems on _E. faecalis_ reduction, an antimicrobial irrigation solution was not used [14,24]. Bacterial counts indicated a significant reduction in microbial load following all instrumentation methods in each sample. The null hypothesis was rejected. The use of the WaveOne Gold and Reciproc Blue file systems resulted in a smaller percentage reduction in _E. faecalis_ compared to the ProTaper Gold in the TEC and TAC design groups. In accordance with this study, the ProTaper file system has been previously demonstrated to be significantly more effective in bacterial reduction than other systems [8,28,29]. [PERSON] et al. [30] also found that ProTaper Gold showed superior bacterial reduction compared to WaveOne Gold. Moreover, [PERSON] et al.