## 4 Discussion  
This study was designed to review the depth to which bacteria penetrate into human dental tubules after needle irrigation with NaOCl or CHX. _E. faecalis_, the bacteria assessed, is strongly associated with persistent infections and endodontic treatment failure [31; 32]. The root canal wall represents a surface to which bacteria can adhere and where a biofilm can thrive [6]. Bacteria tend to organize in biofilms and studies have shown the presence of varied microbiota in endodontic infections [33; 34; 35; 36]. Today, alongside chemical irrigation, mechanical shaping is still one of the main ways of root canal disinfection, as it allows the use of an easy and quick three-dimensional obturation technique [37]. Only studies that evaluated human teeth were included in the present review [38]. A study with an in vitro model of dentinal tubule infection of bovine incisors with _E. faecalis_ introduced in 1987 [38] reported that bacteria could invade the tubules from the pulpal and outer side of the tooth. The basic morphology of bovine teeth is similar to that of human teeth [39], although [PERSON] and [PERSON] reported in 1984 [40] that bovine teeth have giant tubules, which are likely to affect the depth of bacterial penetration and the time it takes for bacteria to reach full dentin thickness. In this context, bacteria were found to penetrate 300-400 \(\upmu\)m inside the canal after only one day of incubation. After 3 weeks of incubation, there was a dense infection at a depth of 300-400 \(\upmu\)m and a moderate infection at 400-500 \(\upmu\)m, while the front of the infection could reach 800-1000 \(\upmu\)m.  
CLSM and analysis of dentinal shavings analysis are two methods that can be used to obtain a quantitative assessment of the penetration depth of bacteria inside dentinal tubules. When the specimens are stained with a LIVE/DEAD BacLight bacterial viability kit, CLSM imaging can differentiate between live green bacteria and dead red bacteria. This method can count the bacteria along the dentinal tubules [41] and provide the percentage of dead bacteria at a specific depth, as well as provide information about biofilm structure and organization inside the dentinal tubules following treatment. Determination of colony-forming units is a method with high variability because the use of a logarithmic scale means this method can only estimate the number of bacteria at depths determined by the bur diameter, but cannot provide exact cell numbers [41; 42]. There are a number of studies [43; 44; 45; 46] in which antibacterial assessments were made by flushing the canal with saline and inserting sterile paper points to absorb the contents inside the canal. The CFUs that can be grown on these paper points are then counted. We did not include such studies in our review because this method evaluates the CFU of planktonic bacteria inside the root canal space, and planktonic bacteria do not represent the clinical or in vivo situation where bacteria tend to invade the tubules and to be organized in a biofilm. SEM is a good qualitative way to assess the presence or absence of bacteria and smear layer, but it does not provide a quantitative analysis [23; 24]. Thus, studies that used SEM as the sole method of evaluation also were not included in the present study. The studies that were included in this review are highly heterogeneous in design, with no standardization of the methods used. Each study incubated the bacteria using a different method and for a different period, used variable amounts of irrigants, irrigated the specimens with different size irrigation needles, and stored the teeth in different solutions, which can affect the penetrability and characteristics of the dentin specimens. All these varying parameters made it very difficult to compare the findings, and it was not feasible to conduct a meta-analysis. We suggest creating a standard for future studies designed to assess in vitro bacterial penetration.