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

  • Francesca CappitelliEmail author
  • Federica Villa
  • Andrea Polo
Part of the Methods in Molecular Biology book series (MIMB, volume 1147)


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).

Key words

Optical Fluorescence and electron microscopy Fluorescence in situ hybridization (FISH) Scanning electron microscopy (SEM) Denaturing gradient gel electrophoresis (DGGE) Adhesive tape strip Sterile scalpel 


  1. 1.
    ICOMOS International Scientific Committee for Stone (ISCS) (2008) ICOMOS-ISCS Illustrated glossary on stone deterioration patterns. Ateliers 30 Impression, Champigny/Marne, FranceGoogle Scholar
  2. 2.
    Gorbushina AA (2007) Life on the rocks. Environ Microbiol 9:1613–1631PubMedCrossRefGoogle Scholar
  3. 3.
    Urzì C, De Leo F (2001) Sampling with adhesive tape strips: an easy and rapid method to monitor microbial colonization on monument surfaces. J Microbiol Methods 44:1–11PubMedCrossRefGoogle Scholar
  4. 4.
    Polo A, Cappitelli F, Brusetti L et al (2010) Feasibility of removing surface deposits on stone using biological and chemical remediation methods. Microb Ecol 60:1–14PubMedCrossRefGoogle Scholar
  5. 5.
    Polo A, Gulotta D, Santo N et al (2012) Importance of subaerial biofilms and airborne microflora in the deterioration of stonework: a molecular study. Biofouling 28:1093–1106PubMedCrossRefGoogle Scholar
  6. 6.
    Nubel U, Garcia-Pichel F, Muyzer G (1997) PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl Environ Microbiol 63: 3327–3332PubMedCentralPubMedGoogle Scholar
  7. 7.
    White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols, a guide to methods and applications. Academic, San Diego, pp 315–322Google Scholar
  8. 8.
    Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118PubMedCrossRefGoogle Scholar
  9. 9.
    Urzi C (2008) Fluorescent in-situ hybridization (FISH) as molecular tool to study bacteria causing biodeterioration. In: May E, Jones M, Mitchell J (eds) Heritage microbiology and science: microbes, monuments and maritime materials. Royal Society of Chemistry, Cambridge, pp 143–150Google Scholar
  10. 10.
    Amann RI, Binder BJ, Olson RJ et al (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925PubMedCentralPubMedGoogle Scholar
  11. 11.
    Cappitelli F, Principi P, Pedrazzani R et al (2007) Bacterial and fungal deterioration of the Milan Cathedral marble treated with protective synthetic resins. Sci Total Environ 385: 172–181PubMedCrossRefGoogle Scholar
  12. 12.
    Müller E, Drewello U, Drewello R et al (2001) In situ analysis of biofilms on historic window glass using confocal laser scanning microscopy. J Cult Herit 2:31–42CrossRefGoogle Scholar
  13. 13.
    Stahl DA, Amann R (1991) Development and application of nucleic acid probes. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. John Wiley & Sons Ltd, Chichester, pp 205–248Google Scholar
  14. 14.
    Piñar G, Gurtner C, Ramos C et al (2002) Identification of Archaea in deteriorated ancient wall paintings by DGGE and FISH analysis. In: Galan E, Zezza F (eds) Protection and conservation of the cultural heritage of the Mediterranean cities. Balkema, LisseGoogle Scholar
  15. 15.
    Manz W, Amann R, Ludwig W et al (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst Appl Microbiol 15: 593–600CrossRefGoogle Scholar
  16. 16.
    Roller C, Wagner M, Amann R et al (1994) In situ probing of Gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides. Microbiology 140: 2849–2858PubMedCrossRefGoogle Scholar
  17. 17.
    Urzi C, La Cono V, Stackebrandt E (2004) Design and application of two oligonucleotide probes for the identification of Geodermatophilaceae strains using fluorescence in situ hybridization (FISH). Environ Microbiol 6: 78–685Google Scholar
  18. 18.
    Schönhuber W, Zarda B, Eix S et al (1999) In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes. Appl Environ Microbiol 65:1259–1267PubMedCentralPubMedGoogle Scholar
  19. 19.
    Pawley JB (1995) Handbook of biological confocal microscopy, 2nd edn. Springer, New York, pp 453–467CrossRefGoogle Scholar
  20. 20.
    Pawley D, Flinchbaugh J (2006) The current state: progress starts here. Manuf Eng 137:71Google Scholar
  21. 21.
    Gulotta D, Goidanich S, Bertoldi M et al (2012) Gildings and false gildings of the baroque age: characterization and conservation problems. Archaeometry 54:940–954CrossRefGoogle Scholar
  22. 22.
    Cappitelli F, Toniolo L, Sansonetti A et al (2007) Advantages of using microbial technology over traditional chemical technology in removal of black crusts from stone surfaces of historical monuments. Appl Environ Microbiol 17:5671–5675CrossRefGoogle Scholar
  23. 23.
    Cappitelli F, Salvadori O, Albanese D et al (2012) Cyanobacteria cause black staining of the national museum of the American Indian building (Washington, D.C., USA). Biofouling 28:257–266PubMedCrossRefGoogle Scholar
  24. 24.
    Urzì C, La Cono V, De Leo F, Donato P (2003) Fluorescent in situ hybridization (FISH) to study biodeterioration. In: Saiz-Jimenez C (ed) Molecular biology and cultural heritage. Balkema Publishers, Lisse, pp 55–60Google Scholar
  25. 25.
    de Vos MM, Nelis HJ (2003) Detection of Aspergillus fumigatus hyphae by solid phase cytometry. J Microbiol Methods 55:557–564PubMedCrossRefGoogle Scholar
  26. 26.
    Teertstra WR, Lugones LG, Wosten HAB (2004) In situ hybridization in filamentous fungi using peptide nucleic acid probes. Fungal Genet Biol 41:1099–1103PubMedCrossRefGoogle Scholar
  27. 27.
    Prigione V, Marchisio VF (2004) Methods to maximise the staining of fungal propagules with fluorescent dyes. J Microbiol Methods 59:371–379PubMedCrossRefGoogle Scholar
  28. 28.
    Villa F, Cappitelli F, Principi P et al (2009) Permeabilization method for in-situ investigation of fungal conidia on surfaces. Lett Appl Microbiol 48:234–240PubMedCrossRefGoogle Scholar
  29. 29.
    Flemming H-C, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8:623–633PubMedGoogle Scholar
  30. 30.
    Loy A, Maixner F, Wagner M, Horn M (2007) ProbeBase—an online resource for rRNA-targeted oligonucleotide probes: new features 2007. Nucleic Acids Res 35: D800–D804PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Pruesse E, Quast C, Knittel K et al (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35:7188–7196PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Quast C, Pruesse E, Yilmaz P et al (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:590–596CrossRefGoogle Scholar
  33. 33.
    Cutler NA, Oliver AE, Viles HA et al (2013) The characterisation of eukaryotic microbial communities on sandstone buildings in Belfast, UK, using TRFLP and 454 pyrosequencing. Int Biodeterior Biodegr 82:124–133CrossRefGoogle Scholar
  34. 34.
    Giacomucci L, Bertoncello R, Salvadori O et al (2011) Microbial deterioration of artistic tiles from the façade of the Grande Albergo Ausonia & Hungaria (Venice, Italy). Microb Ecol 62:287–298PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Francesca Cappitelli
    • 1
    Email author
  • Federica Villa
    • 1
  • Andrea Polo
    • 1
  1. 1.Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’AmbienteUniversità degli Studi di MilanoMilanItaly

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