Miquel Guennoc Cora, Rose Christophe, Guinnet Frédéric, Miquel Igor, Labbé Jessy, Deveau Aurélie
Interactions Arbres - Microorganismes, UMR1136, INRA Université de Lorraine.
Ecologie et Ecophysiologie Forestières - PTEF, UMR 1137, INRA Université de Lorraine.
J Vis Exp. 2017 Jan 25(119):54771. doi: 10.3791/54771.
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.
细菌生物膜经常在真菌表面形成，并可参与众多细菌 - 真菌相互作用过程，如代谢合作、竞争或捕食。生物膜的研究在许多生物学领域都很重要，包括环境科学、食品生产和医学。然而，很少有研究关注此类细菌生物膜，部分原因是研究它们存在困难。文献中描述的大多数生物膜定性和定量分析方法仅适用于在非生物表面或均匀且薄的生物表面（如单层上皮细胞）上形成的生物膜。虽然激光扫描共聚焦显微镜（LSCM）常用于分析原位和体内生物膜，但由于菌丝网络的厚度和三维结构，当应用于真菌菌丝上的细菌生物膜时，这项技术极具挑战性。为克服这一缺点，我们开发了一种将显微镜技术与限制真菌菌落中菌丝层积累的方法相结合的方案。使用这种方法，我们能够通过LSCM和扫描电子显微镜（SEM）在多个尺度上研究真菌菌丝上细菌生物膜的发育。本报告描述了该方案，包括微生物培养、细菌生物膜形成条件、生物膜染色以及LSCM和SEM可视化。
