Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Trinkl, V.
Establishement of fluorescence in situ hybridization for the microbial analysis of the vermiform appendix
Humanmedizin; [ Diplomarbeit ] Graz Medical University; 2021. pp. 77
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Moissl-Eichinger Christine
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- Abstract:
- Background: The pathogenesis of acute appendicitis, an inflammatory disease of the appendix vermiformis, is still not fully understood. The current state of research suggests that Fusobacteria play a crucial role in the pathological process. Fusobacteria are known to cause oral infections. In endodontic infections methanogenic archaea were detected alongside Fusobacteria, potentially due to a symbiotic relationship between them. It was the aim of this study to establish a Fluorescence in situ hybridization (FISH) protocol on appendix sections and in further consequence, to visualize the spatial organization and composition of bacteria and archaea in the diseased appendix. Methods: 60 appendix samples were gathered from children undergoing appendectomy, due to the clinical assumption of appendicitis, at the pediatric surgery department of the LKHUniversitätsklinikum Graz, to test methods of fixation, embedding, cutting and performing of rRNA-based fluorescence in situ hybridization. Results: The appendix samples were fixed in Carnoy solution, which, compared to ethanol-based fixation on stool samples, showed an equally good preservation of the microbial cellular integrity. Furthermore Carnoy solution preserved the appindeceal mucus layer and tissue in the appendix samples. Samples were embedded in Technovit 8100®, as it does not have to be eluted from the sections prior to staining. In order to ensure the necessary anoxic polymerization conditions, blocks were put in vacuum chambers to harden. A comparison of several slides and drying regimes showed, the best adherence of sections after drying for 3 hours at 40°C on SuperFrost™ slides. In tests on cell suspensions, our permeabilization of the microorganisms with lysozyme and Proteinase K and subsequent hybridization with their respective probe enabled the examination of microbial cells with a confocal laser scanning microscope. However, despite several attempts of optimization, we were finally unable to visualize microorganisms in our appendix samples via FISH. Conclusion: As the visualization of microorganisms in appendix sections via FISH was not successful with the tested methods of fixation, embedding and hybridization, further experiments to optimize the process should take place.