**Thanks**

*Advanced Methods and New Materials for Cultural Heritage Preservation*

*sample, (b) biofilm at substrate, and (c–e) fungal hyphae, (magnification 400×).*

The methods and techniques for assessing biofilm characteristic to historical monuments are difficult to standardize because of differences that may interfere (local climate, natural or artificial microclimate, microflora diversity composition, microbial pigment appearance, and the presence of rare or unknown species) and

*In situ observations using the epifluorescence microscopy technique and AO stain: (a) bacteria in suspension* 

Among the easiest to apply and less expensive methods, but with a high degree of relevance, are the microscopy techniques. From the classic optical to the specialized microscope with fluorescence microscopy, to the most advanced microscopy (SEM or SEM-BSE, TEM), all of these have increased the degree of enlargement

The discovery of morphological types and interactions between microorganisms (symbiosis, attachment, and complexity of matrix synthesis) was a feature of superior electron microscopy analysis systems and contributed to increasing the

On the analyzed Fresco area, Matia Loggia as a case study, in situ techniques applied for biodegradation assessment reveal an intense biological activity on the analyzed lithic systems. Following microscopic analyses, the extemporaneous samples highlighted the colonization of the analyzed surfaces with bacterial and fungal hyphae. The intense bacterial activity was noted in all the samples taken from the points located at a distance of 1 m from the base of the walls by the in situ technique. The presence of fungus has been noted in interference areas between different types of

This study was supported by the grant PN-III-P1-1.2-PCCDI-2017-0476,

None of the authors have any competing interests in the manuscript.

Verginica Schröder, Daniela Carutiu Turcanu, Adina Honcea, and Rodica-Mariana Ion equally designed and performed the research. All authors reviewed the

**6. Conclusions**

**Figure 7.**

change the results between samples.

and the identification of structural details, respectively.

level of understanding of the ecology of microbiota systems.

**90**

substrates.

**Acknowledgements**

**Conflict of interest**

**Contributions**

manuscript.

no.51PCCDI/2018, from UEFISCDI-MEN.

The authors express many thanks to PhD Sofia Teodorescu, PhD Ioana-Daniela Dulama, and PhD Alin Ioan Bucurica from Valahia University, ICSTM, for OM and SEM-EDS, measurements.
