Microbiological Methods Used in Endodontics

Microbial Identification by Culture and Molecular Methods

Conventionally, microorganisms from root canals were identified from samples obtained during endodontic treatment procedures via culture techniques that grew bacteria with improved media. Although culture techniques are laborious and require a great deal of knowledge and lab experience, accurate identification of species by means of anaerobic methods, biochemical tests and analysis for antibiotic sensitivity can be efficiently accomplished. Traditional culture-based studies have identified an increased number of anaerobes, which in some cases they totally predominate the microbial results from teeth with primary infections.

More recently, however, molecular techniques have been applied on root canal samples [36]. Molecular techniques do not rely on culture but instead use specific probes to search for bacterial DNA and/or RNA. The principle of molecular techniques is based on targeting highly conserved DNA sequences that different species of microorganisms possess. These conserved genes, mainly the 16S rRNA gene, make molecular identification extremely accurate. A plethora of molecular methods now exist some of which can also quantify different bacterial groups, at least in relative values. Molecular studies have shown an unsuspected wide range of bacteria, including previously uncultured bacteria, for which most laboratories do not search with conventional culture. By using even more advanced methods like pyro- sequencing, it has been possible also to identify a lot more species previously unknown in root canal infections. On the other hand, molecular biology methods are hampered by the fact that they do not distinguish between dead and viable bacteria. Generally, it is believed that molecular biology methods especially in teeth under treatment disclose dead bacteria or even remaining nucleic acids that are of little relevance for the infection (“false positives”). It is also important to point out that the root canal flora is highly variable depending at the stage of infection.

In conclusion, it is important to point out that both techniques are complementary to each other rather than one can substitute the other. Further clinical trials combining both techniques are granted in order to complement their results (Fig. 2.3).

Outline of the methods used for the isolation and identification of root canal bacteria

Fig. 2.3 Outline of the methods used for the isolation and identification of root canal bacteria

 
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