Culture-independent methods to study subaerial biofi lm growing on biodeteriorated surfaces of stone cultural heritage and frescoes

Abstract

Actinobacteria, cyanobacteria, algae, and fungi form subaerial biofi lm (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 biofi lm is performed using specifi c adhesive tape and sampling of SAB and the substrate with sterile scalpels and chisels. Biofi lm observations are carried out using optical and scanning electron microscopy. Specifi c taxa and EPS in biofi lm can be readily visualized by fl uorochrome staining and subsequent observation using fl uorescence or confocal laser scanning microscopy. The observation of cross sections containing both SAB and the substrate shows if biofi lm 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 fl uorescence in situ hybridization (FISH), scanning electron microscopy (SEM), and denaturing gradient gel electrophoresis (DGGE).