Browsing by Author "Kriem, Lukas Simon"

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Open Access
The use of confocal Raman microscopy for the evaluation of in-vitro biofilm model structures
(2022) Kriem, Lukas Simon; Rupp, Steffen (apl. Prof. Dr.)
In nature, the majority of microorganisms grow and accumulate on surfaces. These microorganisms are in general surrounded by an extracellular matrix, also generating a biofilm. Extensive research has been done to further understand these biofilms, especially those that cause human diseases such as subgingival biofilms where their accumulation on teeth over time can cause gingivitis and periodontitis. While dynamics, formation and composition of these biofilms are well known, techniques for continuously monitoring the formation of subgingival biofilm are limited. In recent years, advancements in the field of optical spectroscopic techniques have provided an alternative for analyzing three-dimensional microbiological structures, in addition to the traditional destructive or biofilm staining techniques. In this work, it was demonstrated that the use of confocal Raman spectroscopy, coupled with multivariate analysis, provides an approach to differentiate common subgingival bacteria. In addition, a workflow was developed that allows for the spatial differentiation of bacteria in an in vitro model simulating a subgingival biofilm, a technique which was also confirmed by mapping a second mixed species in vitro biofilm found on medical devices. The present work demonstrates the use of confocal Raman Microscopy to differentiate common subgingival bacterial species (Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus mutans, Veillonella dispar and Prevotella nigrescens) and including their identification in unknown samples. In a second step, a workflow was established to evaluate and differentiate bacterial species in two dual-species in vitro biofilm models, using confocal Raman microscopy. The first biofilm model comprised of Actinomyces denticolens and Streptococcus oralis was cultured using the ‘Zürich in vitro model’. Candida albicans and Pseudomonas aeruginosa were cultured as a second dual-species biofilm to confirm the established workflow. Both biofilms were then analyzed using confocal Raman Microscopy. Cluster analysis was used to spatially differentiate and map the biofilm models over a specified area. To confirm species clustering within the cultured biofilms, confocal laser scanning microscopy was coupled with fluorescent in-vitro hybridization. Furthermore, dense bacteria interface area samples, as an artificial model of clusters in a biofilm, were used to test the developed multivariate differentiation model. This confirmed model was successfully used to differentiate species in a dual-species biofilm that were additionally compared and confirmed by morphology analysis. The results show that the developed workflow was able to identify main clusters of bacteria based on spectral ‘fingerprint region’ information acquired from confocal Raman microscopy. Using this workflow, it was demonstrated that confocal Raman microscopy can be used to spatially analyze dual-species in vitro biofilms, thus providing an alternative technique to map biofilm models.