Paper Conservation: Decisions & Compromises

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 spectrum
dataset (Oxford Instruments).
Fig. 1
a loss of information for future research. Therefore
only totally non-invasive techniques were
adopted. Different types of swabs and adhesive
tapes were employed in sampling fungal or bacterial
elements from paper (Figure 1). Wooden
cotton sterile swabs were used to collect biological
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 fungal
aerial hyphae, conidiophora or fruiting structures,
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 Microscopy
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 vacuum
(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
Molecular analysis
Porous membranes and cotton swabs were directly
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 ornamentation
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 determined
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 (A w
) 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 references
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
ICOM-CC Graphic Documents Working Group Interim Meeting | Vienna 17 – 19 April 2013
106