Culture-Independent Methods to Study Subaerial Biofilm Growing on Biodeteriorated Surfaces of Stone Cultural Heritage and Frescoes

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)