Is it really penetration? Part 2. Locomotion of Enterococcus faecalis cells within dentinal tubules of bovine teeth

Kirsch, Jasmin; Basche, Sabine; Neunzehn, Jörg; Dede, Maria; Dannemann, Martin; Hannig, Christian; Weber, Marie-Theres

doi:10.1007/s00784-019-02865-5

Cited by 8 publications

(11 citation statements)

References 55 publications

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“…The root canal samples with the dentinal tubules were analyzed at 1,000-fold magnification using the light filter for DAPI (BP 365, FT 395, LP 397). The area of ocular grid allowed visualization of the total length of the dentinal tubules (Kirsch et al, 2019).…”

Section: Microscopical Methodsmentioning

confidence: 99%

“…The other half of the root canal was analyzed with scanning electron microscopy (Kirsch et al, 2019). Hereby, the root canal halves were fixed with glutaraldehyde for scanning electron microscopic investigation (SEM).…”

Section: Microscopical Methodsmentioning

confidence: 99%

“…Followed by dehydration in an ascending series of isopropanol and chemical drying through the iterative transfer into hexamethyldisilazane (HMDS), the samples were fixed on SEM stubs and were sputtered with gold-palladium. Scanning electron microscopy was carried out using a Philips ESEM XL 30 in Hi-Vacuum mode by detecting secondary electrons for imaging (Kirsch et al, 2019).…”

Section: Microscopical Methodsmentioning

confidence: 99%

“…A very common method to detect possible residual microorganisms within root canals in in vitro-studies is the collection of contaminated debris as well as biofilm with paper points directly out of the root canal and the analysis via determination of colony-forming units (CFU) (Görduysus et al, 2012;Tan et al, 2015;Tzanetakis et al, 2015). Furthermore, microscopic methods like fluorescent microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), or cultivation are commonly used to evaluate bacterial colonization and biofilm formation (Herzog et al, 2017;Kirsch et al, 2017Kirsch et al, , 2019Mohmmed et al, 2017;Ruiz-Linares et al, 2017;Tsesis et al, 2018). However, these methods are time-consuming and labor-intensive.…”

Section: Introductionmentioning

confidence: 99%

“…faecalis is the most commonly found bacterial species in recurrent root canal infections (Portenier et al, 2003). This is at least partly due to its capability to invade deep into dentinal tubulus (Kirsch et al, 2017(Kirsch et al, , 2019. A major factor for this ability is the expression of adhesins mediating adherence to collagen which is the main organic component of dentin (Love, 2001).…”

Section: Introductionmentioning

confidence: 99%

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Quantification of Bacterial Colonization in Dental Hard Tissues Using Optimized Molecular Biological Methods

et al. 2020

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Bacterial infections of root canals and the surrounding dental hard tissue are still a challenge due to biofilm formation as well as the complex root canal anatomy. However, current methods for analyzing biofilm formation, bacterial colonization of root canals and dental hard tissue [e.g., scanning electron microscopy, confocal laser scanning microscopy (CLSM) or determination of colony forming units (CFU)] are time-consuming and only offer a selective qualitative or semi-quantitative analysis. The aim of the present study is the establishment of optimized molecular biological methods for DNA-isolation and quantification of bacterial colonization via quantitative PCR (qPCR) from dental hard tissue. Root canals of human premolars were colonized with Enterococcus faecalis. For isolation of DNA, teeth were then grinded with a cryo mill. Since the hard tissues dentin and especially enamel belong to the hardest materials in the human organism, the isolation of bacterial DNA from root dentin is very challenging. Therefore, treatment steps for the isolation of DNA from grinded teeth were systematically analyzed to allow improved recovery of bacterial DNA from dental hard tissues. Starting with the disintegration of the peptidoglycan-layer of bacterial cells, different lysozyme solutions were tested for efficacy. Furthermore, incubation times and concentrations of chelating agents such as EDTA were optimized. These solutions are crucial for the disintegration of teeth and hence improve the accessibility of bacterial DNA. The final step was the determination of prior bacterial colonization of each root canal as determined by qPCR and comparing the results to alternative methods such as CFU. As a result of this study, optimized procedures for bacterial DNA-isolation from teeth were established, which result in an increased recovery rate of bacterial DNA. This method allows a non-selective and straightforward procedure to quantify bacterial colonization from dental hard tissue. It can be easily adapted for other study types such as microbiome studies and for comparable tissues like bones.

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Section: Microscopical Methodsmentioning

confidence: 99%

Section: Microscopical Methodsmentioning

confidence: 99%

Section: Microscopical Methodsmentioning

confidence: 99%