title: A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy.
abstract: 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.
authors.affiliation: Interactions Arbres - Microorganismes, UMR1136, INRA Université de Lorraine.
authors.forename: Cora
authors.initials: C
authors.lastname: Miquel Guennoc
authors.affiliation: Ecologie et Ecophysiologie Forestières - PTEF, UMR 1137, INRA Université de Lorraine.
authors.forename: Christophe
authors.initials: C
authors.lastname: Rose
authors.affiliation: Interactions Arbres - Microorganismes, UMR1136, INRA Université de Lorraine.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Guinnet
authors.affiliation: Interactions Arbres - Microorganismes, UMR1136, INRA Université de Lorraine.
authors.forename: Igor
authors.initials: I
authors.lastname: Miquel
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory.
authors.forename: Jessy
authors.initials: J
authors.lastname: Labbé
authors.affiliation: Interactions Arbres - Microorganismes, UMR1136, INRA Université de Lorraine; aurelie.deveau@inra.fr.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Deveau
mesh_terms: D001419:Bacteria; D018441:Biofilms / Q000254:growth & development; D005658:Fungi / Q000254:growth & development; D055399:Laccaria / Q000254:growth & development*; D008828:Microbiological Techniques / Q000295:instrumentation / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D011551:Pseudomonas fluorescens / Q000502:physiology*