a loss of information for future research. There-fore only totally non-invasive techniques were adopted. Different types of swabs and adhesive tapes were employed in sampling fungal or bac-terial elements from paper (Figure 1). Wooden cotton sterile swabs were used to collect biologi-cal particles from the surface of the drawing.
Porous membranes of different materials (nylon, polycarbonate or cellulose nitrate) with a natural electrostatic charge were also used to collect fun-gal aerial hyphae, conidiophora or fruiting struc-tures, together with a few damaged fibres from the substrata. These objects, that can be valuable in diagnostic phases, cling to the charged surface of membranes and can be gently pulled from the mat. Both membranes and swabs can be used for direct observation with an electronic scanning microscope or used to perform molecular analysis.
SEM-EDX technique
Single fibres dust and surface material recovered with membranes or sampled with cotton swabs were analysed using a variable pressure SEM instrument (EVO50, Carl-Zeiss Electron Micros-copy Group) fitted with a detector for electron backscattered diffraction (BSD). Only following an initial observation of the samples using SEM in VP mode at 20 kV were some of the samples coated in gold (using a Baltec Sputter Coater) and then subjected to further analysis in high vacu-um (HV) mode. Sputtering was performed under an Argon gas flow at a working distance of 50 mm at 0.05 mbar, and a current of 40 mA for 60 seconds, so as to create a film of gold of about 15
nm thicknesses. Reference elemental intensities acquired from pure compounds (standards) are commonly used to calibrate SEM-EDX systems. In the case study presented here, conventional ZAF correction integrated into the Oxford INCA 250 microanalysis package was applied to the spec-trum dataset (Oxford Insspec-truments).
Molecular analysis
Porous membranes and cotton swabs were direct-ly used for DNA extraction using the Fast DNA SPIN kit for soil (Bio 101) with modifications.
DNA crude extracts were further used for PCR-DGGE fingerprint analysis of the bacterial 16S rDNA (Schabereiter-Gurtner et al., 2001) and the Internal Transcribed Regions (ITS) (Michaelsen et al., 2006). Clone libraries from these amplified fragments were screened by DGGE and selected clones sequenced (Schabereiter-Gurtner et al., 2001).
Conclusions
In the specific case of certain filamentous fungi, the fruiting body shape, conidia size and orna-mentation can lead to positive identification at least at the genus level. The fungal species Eurotium halophilicum (C.M. Chr., Papav. & C.R.
Benj. 1959) was found in foxing spots especially on the back of the drawing by means of SEM (Zeiss EVO 50- High Vacuum) imaging (Figure 2).
Conidia appeared in different sampling points single or in groups, slightly ovate, echinulate with prominent scars and conidiophores finely covered with a layer of hairy structures (Figure 2). Microscopic features of fungal structures as observed by SEM are consistent with those deter-mined by Christensen et al. (1959) in the original description of E. halophilicum and by Montanari et al. (2012). E. halophilicum is an obligate xerophilic organism with a high tolerance to water stress:
the minimum observed water activity (Aw) for germination and growing is 0,675, the lowest for any Eurotium species. Its occurrence is associated with air-dust (Montanari et al., 2012 and refer-ences therein) or house-dust in association with mites and Aspergillus penicillioides, and storage of dry food. E. halophilicum is reported as associated to foxing spots by Florian and Manning (2000) who published a SEM picture of the fungus without identifying it; with library material by Michaelsen et al. (2010) who found the fungus by DGGE-fingerprinting, without culturing it; and by Montanari et al. (2012) who isolated several
Fig. 1
strains of this species from library materials freshly infected.
This finding is consistent with the hypothesis that absolute tonophilic fungi germinate on pa-per metabolising mainly organic acids, oligosac-charides and proteic compounds. These compo-nents react chemically together on the materials at a low water activity forming brown products and oxidative reactions on paper that result in localised foxing spots. SEM imaging showed also the presence of other fungal species, not only as single spores, but as propagules and small myce-lial masses.
DGGE-fingerprinting, a molecular technique based on direct extraction of DNA from environ-mental samples, allowed the comparison of dif-ferent sampling techniques and DNA extraction protocols enabling the optimization of tools for the analysis of such valuable object. In addition, a complete screening of the biodiversity of the fungal community inhabiting the portrait (on the face and on the reverse side) was obtained and showed the putative differences in microbial composition among different samples indicat-ing, in general, a higher biodiversity as initially suspected. Additional phylogenetic analyses revealed the presence of fungi with well known cellulolytic activities, with potential for the de-struction of the investigated material.
Acknowledgments
The authors would like to thank Dr.
Maria Cristina Misiti, Director of the Istituto Centrale per il Restauro e la Con-servazione del Patrimonio Archivistico e Librario in Rome and the Royal Library in Turin, Italy for the opportunity of studying the precious portrait. G. Piñar and the molecular analyses performed in this study were financed by the Austrian Science Fund (FWF) project “Elise-Richter V194-B20”
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Figure captions
Figure 1. Sampling approach to obtain fungal spores from Leonardo da Vinci’s self portrait. Application of porous membranes (picture by D. Corciulo, ICRC-PAL).
Figure 2. Scanning electron microscopy (SEM) image of conidia belonging to the fungal species E. halophilicum, collected by swab sampling in foxing spots on the back of the drawing. Image obtained with a Zeiss SEM EVO 50 in High Vacuum mode on a gold sputtered sample, oper-ating at an acceleroper-ating voltage of 20 kV equipped with a detector for secondary electrons (SE).
Authors
Guadalupe Piñar | Katja Sterflinger Department of Biotechnology, Vienna In-stitute of Bio Technology (VIBT), Universi-ty of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria guadalupe.pinar@boku.ac.at katja.sterflinger@boku.ac.at Flavia Pinzari
ICRCPAL-Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario, Laboratorio di Biologia, Ministero per i Beni e le Attivita Culturali, Via Milano 76, 00184 Rome, Italy.
flavia.pinzari@beniculturali.it