A new method for qualitative multi-scale analysis of bacterial biofilms on filamentous fungal colonies using confocal and electron microscopy
2017
Guennoc, Cora Miquel | Rose, Christophe | Guinet, Frédéric | Miquel, Igor | Labbé, Jessy | Deveau, Aurélie | Interactions Arbres-Microorganismes (IAM) ; Institut National de la Recherche Agronomique (INRA)-Université de Lorraine (UL) | Ecologie et Ecophysiologie Forestières [devient SILVA en 2018] (EEF) ; Institut National de la Recherche Agronomique (INRA)-Université de Lorraine (UL) | BioSciences Division [Oak Ridge] ; Oak Ridge National Laboratory [Oak Ridge] (ORNL) ; UT-Battelle, LLC-UT-Battelle, LLC | French National Research Agency through the Laboratory of Excellence ARBRE ANR-11-LABX-0002-01 ; Plant-Microbe Interfaces Scientific Focus Area in the Genomic Science Program ; Office of Biological and Environmental Research in the DOE Office of Science ; United States Department of Energy DE-AC05-00OR22725 | ANR-11-LABX-0002,ARBRE,Recherches Avancées sur l'Arbre et les Ecosytèmes Forestiers(2011)
Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.
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