Cappitelli Francesca, Villa Federica, Polo Andrea
Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università degli Studi di Milano, Milan, Italy,
Methods Mol Biol. 2014;1147:341-66. doi: 10.1007/978-1-4939-0467-9_24.
Actinobacteria, cyanobacteria, algae, and fungi form subaerial biofilm (SAB) that can lead to material deterioration on artistic stone and frescoes. In studying SAB on cultural heritage surfaces, a general approach is to combine microscopy observations and molecular analyses. Sampling of biofilm is performed using specific adhesive tape and sampling of SAB and the substrate with sterile scalpels and chisels. Biofilm observations are carried out using optical and scanning electron microscopy. Specific taxa and EPS in biofilm can be readily visualized by fluorochrome staining and subsequent observation using fluorescence or confocal laser scanning microscopy. The observation of cross sections containing both SAB and the substrate shows if biofilm has developed not only on the surface but also underneath. Following nucleic acid extraction, 16S rRNA gene sequencing is used to identify bacterial taxa, while 18S rRNA gene and internal transcribed spacer (ITS) sequence analysis is used to study eukaryotic groups. In this chapter, we illustrate the protocols related to fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), and denaturing gradient gel electrophoresis (DGGE).
放线菌、蓝细菌、藻类和真菌会形成气生生物膜（SAB），这种生物膜会导致艺术石材和壁画的材料劣化。在研究文化遗产表面的SAB时，一种常用方法是将显微镜观察与分子分析相结合。使用特定胶带对生物膜进行采样，并用无菌手术刀和凿子对SAB和底物进行采样。使用光学显微镜和扫描电子显微镜对生物膜进行观察。通过荧光染料染色并随后使用荧光或共聚焦激光扫描显微镜观察，可以很容易地看到生物膜中的特定分类群和胞外聚合物（EPS）。对包含SAB和底物的横截面进行观察，可显示生物膜是否不仅在表面形成，还在其下方形成。核酸提取后，使用16S rRNA基因测序来鉴定细菌分类群，而使用18S rRNA基因和内转录间隔区（ITS）序列分析来研究真核生物群体。在本章中，我们阐述了与荧光原位杂交（FISH）、扫描电子显微镜（SEM）和变性梯度凝胶电泳（DGGE）相关的实验方案。
