This article is an excerpt of my thesis in my Masteral of Science Major in Microbiology. The research discusses different problems encountered on paintings and some possible ways to solve these problems. This topic on restoration and conservation of paintings is close to my heart since it has been my interest and at the same time I'm also a practicing art restorer since 2000.

1. Chemical Control of Fungi Infesting Easel Oil Paintings
at the University of Santo Tomas Museum of Arts and
Sciences
Crisencio M. Paner
College of Fine Arts and Design, University of Santo Tomas, Manila,
Philippines
Abstract
Two hundred paintings of Filipino masters, all accessioned at
the UST Museum of Arts & Sciences, were surveyed to determine their
state of preservation. Forty-three (21.5%) were found to be in different
states of deterioration with mold attack as the most common cause.
Fungal infestations were most evident on pigments, wood support and
paper backing. The infested paintings, based on the records at the
museum, are between 50 and 100 years old. Infesting molds were
isolated by swab method and purified through a series of transfer in
plates of Malt extract agar (MEA) with pH 3.5 and incubation
temperature of 28º
C. Forty-eight isolates in six genera as follows were
obtained and identified based on cultural and morphological
characteristics in MEA: Aspergillus and Penicillium (Deuteromycota);
Rhizopus, Mucor and Cunninghamella (Zygomycota); and
Chaetomium (Ascomycota). Aspergillus is the most prevalent with 77%
occurrence in all the paintings sampled. It is followed by Penicillium,
13%, and by Chaetomium, 4%. The lesser isolates each with an
occurrence rate of 2% are Mucor, Cunninghamella, and Rhizopus.
Varnish among the painting materials tested completely
inhibited the growth of the fungi. But the non-pigment components
Linseed oil and canvas fabric yielded colony growth larger than the
control. All 12 paint pigments tested were supported of growth but at

2. different degrees; most utilized was sap green, and the least, burnt
umber.
For the five fungicides assayed in vitro by Disc Diffusion
method and determination of MIC and MLC, most effective overall
against the fungi was Preventol R-80, followed in decreasing efficacy
by Umonium-38, Clotrimazole, boric acid, and Dithane M-45. All
neither dissolved nor reacted with the paint pigments in actual trials
on oil paintings. But Dithane M-45 and boric acid blemished the
paintings with residues after evaporation. Preventol R-80 still worked
better than Umonium-38; it enhanced the clarity and luster of treated
paintings.
Keywords:
Oil painting, restoration, conservation, fungicides, Filipino masters,
University of Santo Tomas, Museum, fungi, molds
Introduction
Paintings are fragile creations susceptible also to different kinds of
deterioration. One of the most common causes of deterioration is
microbial attack, particularly by molds. These microorganisms often
attack and infest paintings when the environmental conditions to which
the art objects are exposed are conducive for their growth and
development. Researchers have shown, for example, that molds rapidly
developed on the paintings when relative humidity was high (around
70%) and temperature increases (Dhawan et al., 1991; Pavlogeogatos,
2003). Moreover, the organic nature of the materials composing a
painting may serve as nutrients for most molds (Agrawal et al., 1989),
just as they do on painted parts of ordinary homes.
Biodeterioration of paintings due to infestation by fungi is a
common problem in most tropical countries like the Philippines where

3. rainy season is longer, temperature is higher (25-310
C), and most of the
time the air is very humid (Tse et al., 2008).
Architect Clarissa Avendaño (pers. comm., 2002), Assistant Director of
the Museum of the University of Santo Tomas, admits that mold
infestation is a problem in the museum especially with their stocked
paintings. There is no air conditioner in the storage room due to budget
limitations. Louvers have been installed on the door of the room so that
air may circulate and partly prevent fluctuation of temperature and
humidity inside. Still, high humidity in the storage room during the rainy
season subjects the collections to fungal growth.
The UST Museum has in its holdings art objects of immeasurable
value to Philippine culture and history. Of the collections, the paintings
are among those most susceptible to deterioration caused by fungi. This
is the first study of its kind focusing on the valuable paintings in the
museum.
It hopes to provide (a) much-needed knowledge on biodeterioration
of paintings in our country, (b) baseline information to restorers and
conservators regarding specific guidelines on the conservation of the
kinds of paintings under study, and (c) background on the fungicides
against specific fungi that attack local paintings.
The study focuses on the problem of fungal infestation of easel oil
paintings at the UST Museum. It aims to:
(1) Isolate and identify the molds responsible for the deterioration of
aforementioned paintings at the Museum;
(2) Determine the degree by which painting components favor the
growth of the isolated fungi;
(3) Evaluate selected fungicides for their inhibitory or eradicative effects
on the isolated fungi; and
(4) Test the solubility of pigments of a mock painting in selected
fungicides.

4. Materials and Methods
Survey of the paintings
The easel paintings used for the study came from the Museum of
the University of Santo Tomas. A total of 200 of these art objects were
surveyed with permission from the Museum Director. Surveying
included careful visual inspection as well as taking photographs of oil
paintings observed to be infested with molds. Other conservation
problems of the paintings in the storage room were also noted.
Isolation of molds
For isolation of molds, a modified method of Dhawan (1983) was
adopted. Sterilized cotton buds moistened with sterile-distilled water
(SDW) were swabbed gently on the affected surface of the painting and
the spores, mycelia, or both collected were aseptically shaken off in a
flask containing 100 mL SDW. Due to the heavy fungal infestation of
the easel painting available for study, the resulting fungal suspensions
were diluted to up to 10-6
likewise with SDW. Aliquots in1-mL volumes
from each dilution level were pour-plated with 9 mL full-strength Malt
Extract Agar (MEA). The pH of the agar medium was adjusted to 3.5
prior to autoclaving to inhibit the expected growth of bacteria
originating from the swabbed paintings. Prepared culture plates were
incubated at room temperature for 3-5 days. All fungal colonies that
developed, except those of Chaetomium, had aerial conidiophores with
conidia or sporangiophores with sporangiospores within sporangia.
Thus, pure cultures were obtained by the rapid spore-touch technique
using a glass needle drawn out under flame from disposable Pasteur
pipettes. Pure cultures were maintained in MEA slants in test tubes.
Identification of molds

5. Each fungal isolate for identification was cultured on MEA agar
blocks on glass slides based on Henrici’s culture technique (Appendix
1). Subsequent sporulating growth was examined with both stereoscopic
and brightfield microscopes. Wet mounts with either plain SDW or
lactophenol were also prepared for detailed microscopic examination of
hyphae and asexual or sexual spores under high-power and oil-
immersion objectives.
Fungal genera were identified using literature on fungal taxonomy
and Mycology (Ainsworth, 1973; Alexander, 1977; Barnett & Hunter,
1972; Ellis, 1976; Garry & Robert, 1979; Bryce, 1971; Raper & Fennel,
1977; Raper & Thom, 1968; Subramanian, 1971; Sutton, 1980).
Utilization of Painting Materials by Fungi
The different fungi were tested for their capacity for growth in
the presence of various painting materials (12 pigments, canvas, linseed
oil, varnish). Triplicate plates of 1.5% agar with 12.5% (w/v) of each
painting material were prepared in advance. Pour-plated MEA (1.5%
agar) with standardized fungal spore suspension in Tween-Water (TW)
(8.75 x 106
/mL) were also prepared. Soon after solidification, agar discs
were bored out using the mouth of sterilized test tubes. One such spore-
seeded agar disc was deposited at the center of the test agar plates with
painting materials. Triplicate control plates without painting material per
test fungus were seeded with MEA discs likewise with immobilized
fungal spores. All plates were incubated at 30ºC for up to 7 days and,
thereafter, mycelial growth diameters in millimeters were taken.
Evaluation of the selected fungicides
The five fungicides under study were obtained from different
sources. Preventol R-80 and Umonium-38 were brought by the author
from Italy. These were given as a gift by his professor in Art
Restoration, Prof. Ma. Pia Nugari of the Istituto Centrale per il Restauro
in Rome, Italy. Professor Nugari explained that Preventol R-80 contains

6. 80% benzalkonium chlroride, while Umonium-38 is composed of 0.32%
benzalkonium chloride and two kinds of alcohols to increase efficacy
(Prof. M.P. Nugari, pers. comm., 2004). Clotrimazole was purchased as
“Canesten (1% of 3 g)” brand from a local drug store. Lastly, Boric acid
and Dithane M-45 were obtained as part of the inventory of the UST
Graduate School Roque Thesis Laboratory.
Disk diffusion assay. This was carried out with MEA, a culture
medium suitable for growing the test organisms. Fresh fungal spore
suspensions in TW (8.75x106
/mL) were pour-plated in triplicate.
Standard 8mm paper discs for antibiotic assay (Becton-Dickinson) were
soaked for a minute in different concentrations of fungicides (1-10% in
SDW) and, after air-drying, deposited carefully at the center of the
solidified plated medium with immobilized fungal spores. For control
plates, discs soaked as long in SDW and subsequent drying were used.
Zones of growth inhibition were measured in millimeter 3 days after
incubation of test and control plates at room temperature.
Minimum Inhibitory and Lethal concentrations. These were done
for fungicides with zones of inhibition in the Disk Diffusion Assay. A
series of tubes containing Mueller-Hinton Broth (MHB) with 1% to 12%
(at 0.1% intervals) of the fungicides was seeded with 1-mL aliquots of
fresh fungal spore suspension (8.75 x 106
/mL). The lowest concentration
of the fungicide resulting in no fungal growth after 3 days of incubation
was taken as the Minimum Inhibitory Concentration (MIC). On the other
hand, the Minimum Lethal Concentration (MLC) was ascertained by
subcultures from the tubes showing no growth into fresh MHB lacking
fungicide. The lowest concentration from which the microorganism did
not recover or grow in the subculture was taken as the MLC of the
fungicide (Prescott et al., 1999).
Effect of Fungicides on Painting
A series of concentrations as follows was prepared for the
fungicides: 0.2%, 0.39%, 0.78%, 1-10%, 12.5%, 25%, 50%, and 100%.

7. Aliquots in 50 µL volumes were carefully deposited using a
Micropipettor on the selected surfaces of two oil paintings. One was a
mock painting prepared by brushing a ready-made canvas ( 8” x 10” )
with black, white, green, blue, orange, yellow, violet and red pigments
premixed with linseed oil as binder and turpentine as thinning agent. The
mock painting was allowed to air-dry for 2 weeks. The other was a 1975
painting on canvas (unvarnished) from a personal collection. Deposited
drops of fungicide were allowed to stand for at least 3 min. After the
exposure period, the remaining droplets were pipetted-off and examined
by digital macro-photography to look for signs of color change or
discoloration to indicate dissolution of the painted pigments. Recovered
droplets were also examined microscopically for the presence of
pigment crystals, precipitates, and other signs of solubility. Reaction of
the paint pigment on canvas to the fungicides was either positive or
negative in comparison with parallel control with only plain SDW in lieu
of fungicide applied to the two test paintings.
Results and Discussion
Majority of some 200 easel oil paintings surveyed at the UST
Museum of Arts and Sciences were found under deterioration due to
infestation by fungi. A total of 48 strains of fungi were isolated and
these are distributed into the genera Aspergillus (77%), Penicillium
(13%), Chaetomium (4%), Rhizopus (2%), Cunninghamella (2%), and
Mucor (2%) (Fig.2). The damages suffered by the paintings range from
surface damage (81%) caused by 40, discolorations (14%) by 6, and
structural (5%) by 4 fungal isolates (Table 1 & Fig. 1).
Except for varnish, all painting materials tested served as nutrient
sources the growth of the fungi (Table 2). Canvas and the pigment
binder gave larger colony diameter than the control medium water agar.
The pigments supportive of fungal growth in decreasing order of
preference by the fungi is as follows: sap green; lemon yellow; ivory
black; ultramarine; phthalo blue; brilliant red; viridian hue; yellow
ochre; medium yellow; crimson; Chinese white; burnt umber.

8. Of the five fungicides tested in vitro by the Disc Diffusion Assay
and MIC-MLC determination (Table 2), Preventol R-80 ranked first,
followed in decreasing order of effectivity by Umonium-38,
Clotrimazole, Dithane M-45, and Boric acid. Tried on actual oil
paintings, only Preventol R-80 and Umonium-38 could be recommended
for restoration of mold-infested art pieces. Both neither dissolved nor
left residue deposits on the paintings, but Preventol R-80 was better
because it imparted increased clarity and lustrous coloration to the
treated paintings (Fig. 3). Dithane M-45 and boric acid, on the other
hand, decreased the aesthetic value of the painting as both left marked
deposits on the paintings.
Conclusion
Just as it is with a malady on a person, the best dictum to counter fungal
infestation of oil paintings is availability of proper facilities for the
maintenance of the essential environmental parameters (e.g.,
temperature and relative humidity) to ward off fungal attack. In the
local tropical scenario, fungal infestation is inevitable, but it can be
minimized. Attempts at restoration of damage paintings require
sufficient knowledge of the paint pigments, the probable infesting molds
in the immediate environment, and the pros and cons in the use of
remedial fungicides.
Acknowledgement
I would like to thank the following for their significant
contributions to the completion of this study: University of Santo
Tomas, particularly Rev. Fr. Rolando V. dela Rosa, O.P.,Rector, for
providing the financial support and Prof. Irineo J. Dogma, Jr. Ph.D., my
thesis adviser, for his unwavering support.

9. About the Author: Prof. Crisencio Paner had undergone training in
Rome, Italy through a scholarship grant given the Italian
government on the “Restoration and Conservation of Arworks
particularly paintings”. He has been teaching at the College of Fine
Arts and Design,University of Santo Tomas Manila for more than 18
years now. He has also been restoring paintings and other artworks
since 2000. His portfolio can be found in his blog,
http://cmpaner.blogspot.com (The Painting Doctor-
Restorer/Conservator). He can be contacted at mobile nos. 0999-
9401794 or at Tel. 02 416-2489)
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List of Tables and Figures: