Dual phylogenetic staining protocol for simultaneous analysis of yeast and bacteria in artworks


The detection and analysis of metabolically active microorganisms are useful to determine those directly involved in the biodeterioration of cultural heritage (CH). Fluorescence in situ hybridization with oligonucleotide probes targeted at rRNA (RNA-FISH) has demonstrated to be a powerful tool for signaling them. However, more efforts are required for the technique to become a vital tool for the analysis of CH’s microbiological communities. Simultaneous analysis of microorganisms belonging to different kingdoms, by RNA-FISH in-suspension approach, could represent an important progress: it could open the door for the future use of the technique to analyze the microbial communities by flow cytometry, which has shown to be a potent tool in environmental microbiology. Thus, in this work, various already implemented in-suspension RNA-FISH protocols for ex situ analysis of yeast and bacteria were investigated and adapted for allowing the simultaneous detection of these types of microorganisms. A deep investigation of the factors that can affect the results was carried out, focusing particular attention on the selection of the fluorochromes used for labelling the probe set. The resultant protocol, involving the use of EUK516–6-FAM/EUB338–Cy3 probes combination, was validated using artificial consortia and gave positive preliminary results when applied in samples from a real case study: the Paleolithic archaeological site of Escoural Cave (Alentejo, Portugal). This approach represents the first dual-staining RNA-FISH in-suspension protocol developed and applied for the simultaneous investigation of CH biodeteriogenic agents belonging to different kingdoms.

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This work was co-financed by European Union-European Regional Development Fund ALENTEJO 2020 through the project “HIT3CH-HERCULES Interface for Technology Transfer and Teaming in Cultural Heritage” (ALT20-03-0246-FEDER-000004) and by FCT-Fundação para a Ciência e a Tecnologia through the project “MICROTECH-ART-Microorganisms Thriving on and Endamaging Cultural Heritag—an Analytical Rapid Tool-” (PTDC/BBB-IMG/0046/2014). M. González-Pérez acknowledges FCT for the economic support through the post-doctoral grant SFRH/BPD/100754/2014. Authors are also grateful to the Alentejo Regional Directorate for Culture and archaeologist António Carlos Silva for allowing the sampling from Escoural Cave.

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Correspondence to Ana Teresa Caldeira.

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González-Pérez, M., Brinco, C., Vieira, R. et al. Dual phylogenetic staining protocol for simultaneous analysis of yeast and bacteria in artworks. Appl. Phys. A 123, 142 (2017). https://doi.org/10.1007/s00339-016-0725-0

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  • Fluorescence in situ hybridization
  • Biodeterioration
  • Cultural heritage microbiology
  • Phylogenetic staining