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Sampling with adhesive tape strips: an easy and rapid method to monitor microbial colonization on monument surfaces
Journal of Microbiological Methods 44 Ž2001. 1–11 www.elsevier.comrlocaterjmicmeth
Sampling with adhesive tape strips: an easy and rapid method to monitor microbial colonization on monument surfaces Clara Urzı`) , Filomena De Leo Department of Microbiological, Genetic and Molecular Sciences, UniÕersity of Messina, Salita Sperone, 31 I-98166 Messina, Italy Received 20 July 2000; received in revised form 12 October 2000; accepted 17 October 2000
Abstract An easy and fast non-destructive method for sampling from monument and art object surfaces is proposed. The results obtained after sampling in regions of black spots and discoloration using adhesive tape strips showed that the method is useful for monitoring microbial colonization as well as for the identification of biodeteriogens. This technique is easy to apply, inexpensive and reproducible. The presence of fungi or algae on stone surfaces can be checked at an early stage of colonization or after cleaning procedures, without damage to the sampled surface. In addition, it is possible to obtain information on the morphology and taxonomy of microorganisms, and their relationships with the colonized material surfaces. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Adhesive tape; Fungi; Algae; Monument surfaces; Non-destructive method
1. Introduction The use of non-destructive techniques to monitor microbial colonization before andror after conservation treatments of monuments andror of artistic objects is often required. Several non-destructive methods have been proposed for direct or indirect evidence of microbial colonization. Needle procedures, agar fingerprinting, measurement of microbial activity, detection of bacteria by fluorescent antibodies, bioluminescent techniques and ATP assays have been described by several Authors ŽEckhardt, 1978; May and Lewis, 1988; Hirsh et al., 1995; Wollenzien et al., 1995;
) Corresponding author. Tel.: q39-090-676-5196; fax: q39090-392-733. E-mail address: [email protected] ŽC. Urzı`..
Ranalli et al., 2000.. However, despite their undeniable utility, these techniques do not give any information about the relationships between the microorganisms and the surface itself. Sterflinger and Krumbein Ž1995. proposed a replica method to check biological colonization of the rock surface and the connection between organisms and stone, through SEM observations. This technique, even though of wide potential application, does not allow cultivation of microflora present and thus does not solve problems related to the taxonomy of the species involved. The use of adhesive tape is a common practice in clinical mycology for the direct observation of Dermatophytes from skin and their further isolation by inoculation of small pieces of the strips on suitable cultural media. Gargani Ž1968. first applied this technique in the field of cultural heritage, for evidence of fungal growth on frescoes after the flood of 1966 in Florence.
0167-7012r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 Ž 0 0 . 0 0 2 2 7 - X
2
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
The aim of this paper is to demonstrate that adhesive tape sampling as a non-destructive technique can be used as a reliable and easy method on stone surfaces to monitor microbial colonization. For this reason the technique was used on different stone surfaces, and the presence of microflora was demonstrated both directly under microscopy, as well as by cultivation on suitable media.
2. Materials and methods 2.1. Sampling and preparation of adhesiÕe strips Sampling with adhesive tape strips ŽFungi tape DID, Milan, Italy. was carried out from rocks and monuments located in different European Countries. Table 1 summarizes the main characteristics of the stones that were sampled. Strips were gently applied to the stone surface and were then immediately placed on sterile glass microscope slides and kept in a box until arrival in laboratory. The adhesive tape strips were then cut in small pieces Žabout 5 = 5 mm. and sorted for microscopical and cultural analysis.
2.2. Microscopical analyses 2.2.1. Light and epifluorescence microscopy Observations for microscopy were carried out without any specific preparation. A drop of sterile water, Amman’s lactophenol solution, or Acridine Orange solution Ž0.1%. was put between a glass slide and the tape Žface down.. A cover glass was placed on the top, in order to keep the tape as flat as possible ŽUrzı` and Albertano, 2001.. Direct observations of samples were carried out using a light microscope equipped with phase contrast ŽDML, Leica. andror a light epifluorescent microscope ŽDMR, Leica. equipped with a mercury lamp Ž50 W. and 450–490 nm excitation filter. 2.2.2. Scanning electron microscopy For SEM observations, a piece of adhesive tape ŽFungi tape DID. was mounted face up on a stab with a biadhesive tape ŽAdhesive Tabs, Electron Microscope Science, Washington, USA., then dehydrated via an ethanol series and covered with carbon powder. Observations were carried out using a SEM, S420 Cambridge equipped for EDX analyses of the Earth Science Department of Messina University.
Table 1 Provenance of the sampled surfaces, reference code, number of samples and type of rock Provenance
Ref. code
No. of samples
Stone
Muro Farnesiano, ŽParma, Italy.
MF
5
Brick and mortars
Scurano’s Pieve ŽParma, Italy.
PV
5
Stone sandstone
Templete Mudejar ŽGuadalupe, Spain.
TM
12
Mortars
Victory Gate ŽGermany.
VG
4
Laas marble
Eureka-Euromarble exposure site in Munich ŽGermany.
Mu¨
8 2 2 2
Carrara marble Pentelic marble Ekeberg marble Laas marble
Eureka-Euromarble exposure site in Messina ŽItaly. Moscow ŽRussia. Arles ŽFrance.
EU
6
Carrara marble
Concert Hall ŽHelsinki, Finland.
CH
3
Carrara marble
Total
49
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
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Fig. 1. Carrara marble taken from the outer facade of the Concert Hall building in Helsinki ŽFinland.. The surface is entirely covered by black structures. Magnification 63 = .
2.3. Cultural analysis Microorganisms that could be cultivated were isolated and identified by inoculation of small pieces of the strips directly into liquid medium BG11 for algae ŽRippka et al., 1979. and in duplicate in solid medium ŽDRBC, King et al., 1979; Urzı` et al., 1992. for fungi. No attempts were made to isolate cultivatable bacteria. Incubation was carried out at 288C for 1 month each in the light and in the dark, respectively. Algae and cyanobacteria were initially determined to be present or absent, and then attributed to morphological groups ŽRippka et al., 1979.. Identification of fungi was mainly carried out on the basis of the macroscopic features of colonies and on the micromorphology of reproductive structures, according to Barnett and Hunter Ž1972.; Fassatiova` Ž1986.; Hoog de Ž1987. and Ellis Ž1971, 1976..
scopic observation. These samples Žthree from Muro Farnesiano, three from Victory Gate and one from Carrara marble block exposed in Munich. were withdrawn from surfaces that had recently been cleaned or were without any evident biological alteration.
3. Results 3.1. Microscopical analysis Among the forty-nine samples examined, only seven samples were microbe free by direct micro-
Fig. 2. Sampling by adhesive tape from the facade of Scurano’s Pieve, showing chains of meristematic fungi. Bar s10 mm. Figs. 2–8 consist of light microscopy observations.
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C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
Fig. 5. Explorative hyphae from a fungal cluster attached to a calcite flake. The sample was taken from the surface of a Carrara marble block in Munich. Bar s 50 mm.
Fig. 3. Sample taken from the surface shown in Fig. 1. Clusters of fungi are seen in chains. Further isolation and identification evidenced that they belong to a strain of Alternaria alternata. Bar s10 mm.
However, forty-two samples, corresponding to either black spots or intercrystalline growth ŽFig. 1. showed colonization by dematiaceous fungi. The fungal colonization was evidenced mainly as presence of chains of melanized cells or black clusters ŽFigs. 2 and 3.. In some cases, germinative or explorative structures derived from single spores or conidia were observed, as a clear demonstration that
Fig. 4. Germinating spore seen under phase contrast microscopy. This structure resembles a conidium of Epicoccum purpurescens. The sample was taken from a a protected part of a Carrara marble item. It has been exposed in Munich, Germany, since 1991 in the frame of Eureka-Euromarble EU496 project. Bar s10 mm.
fungi actively colonize the rock ŽFigs. 4 and 5.. In addition, a close connection between black fungal clusters and stone crystals was observed in most of these samples ŽFig. 6.. In a few samples Ž10r42., photosynthetic organisms were observed by light microscopy. However, some differences were observed in agreement with the colonization pattern: Ža. when phototrophic colonization was already evident by the naked eye Že.g.
Fig. 6. Adhesive tape sampled from an Ekeberg marble block exposed in Munich. Clusters of fungi are clearly visible, closely connected with the marble flakes. In some cases, explorative hyphae or rhizoid structures had formed and created a stronger connection with the marble itself. Bar s 50 mm.
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
in seven samples taken from Templete Mudejar., under the microscope a massive growth in the shape of microbial mat or biofilm was seen ŽFig. 7.; in contrast, in the three samples taken from Carrara and Ekeberg marble, recently exposed to outdoor conditions in Munich, phototrophic microorganisms were occasionally found closely associated with the fungal clusters ŽFig. 8.. Bacteria were directly found only under epifluorescent microscopy in five of the samples examined. SEM observation of adhesive tapes gave good results in agreement with those obtained under light microscopy. Microbial colonization was observed as a biofilm ŽFig. 9., clusters of microorganisms and as small chains of cells ŽFig. 10. whose size and shape was comparable with those of meristematic struc-
5
tures seen under light microscopy ŽFigs. 2 and 3.. The connection between microorganisms and stone was also evident ŽFigs. 11 and 12.. EDX microelements analysis on the samples taken with the adhesive tape, was useful to indicate the presence of inorganic pollutants, such as Ti, S, Fe, observed in the sample shown in Fig. 13, probably due to their release from fuel combustion.
3.2. Analysis of cultures Cultural analysis confirmed that there was colonization, as already observed through microscopy in almost all the positive samples ŽTable 2..
Fig. 7. Algal biofilm and fungal cells visible in the sample taken from the Templete Mudejar in Spain. Biological patina was already visible to the naked eye. Bar s 50 mm.
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C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
Fig. 8. Algal and fungal cluster sampled from the Ekeberg marble in Munich. Algal growth was observed only in sheltered parts Žfolders.. Bar s 50 mm.
Fungi were isolated from all positive samples seen by microscopy. Among the strains isolated the most frequently found were dematiaceous hyphomycetes. These were assigned to the genera Cladosporium, Alternaria, Phoma, Ulocladium. In some cases ubiquitous non-dematiaceous strains of the genera Penicillium, Aspergillus and Fusarium were isolated. Black meristematic fungi were isolated in some samples Ž10r42. and assigned to the genera Coniosporium and Phaeoteca. Black yeasts of the genus Exophiala were rarely isolated Ž1r42.. In addition, cultural analysis showed the presence of fungi in three samples taken from Victory Gate, in which the previous microscopic observations did not show the presence of microorganisms. The strains isolated from these samples belonged to the genera Cladosporium, Mucor and Phoma. Algae and cyanobacteria were isolated only when they were predominant compared to the other microorganisms Že.g. case of the Templete Mudejar samples, Fig. 7.. In these cases, they were described
as unicellular green algae Ž Chlorella-like. and filamentous N2-fixing cyanobacteria of the Nostocaceae group.
4. Discussion The results obtained demonstrated that the sampling method proposed offers the possibility of gaining information on the microbial colonization of material surfaces without being destructive of valuable material. The presence of fungi andror algae, when they occurred, was easy to demonstrate, and thus to ascribe a specific alteration in the surface material with the fungal or algal colonization. In addition, in situ morphology of microorganisms on the colonized surfaces and their relationships to the substratum was shown. A good correspondence between microscopic observations and cultural analysis was also noticed. Cultural analysis allowed isolation and identification of microorganisms, including slow growing meris-
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
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Fig. 9. SEM micrograph of an adhesive tape taken from Muro Farnesiano in Parma, Italy. A microbial biofilm of prokaryotic cells Žcoccoidal cells whose size was below 1 mm. is clearly visible. Figs. 9–13 consist of SEM observations.
tematic black fungi. No significant omission of information with respect to other methods was observed and it was possible to see and isolate fungal species typically found on rock surfaces and considered to be the cause of biodecay ŽWollenzien et al., 1995; Sterflinger and Krumbein, 1997.. It is worth noting that it was possible to isolate and thus also identify black meristematic fungi, which are notoriously very difficult to isolate among fast growing species ŽUrzı` et al., 2000.. The limit of the method, beside the fact that it gives only qualitative information on the microorganisms present, is that it gives an underestimation of the bacterial flora. In fact, no bacteria were seen by light microscopy. This could be due to bacteria being more evident when staining procedures are
used, and also because by seeing them under light microscopy they should be present in the order of at least ; 10 6 cellsrml ŽMadigan et al., 1997..
5. Conclusions Adhesive tape sampling can be widely and safely applied in the field for the conservation of monuments and can be used not only by microbiologists but also by skilled restorers and conservators. The simple technique offers the following advantages v
it is a non-destructive method and can be used for monitoring microbial colonization over a period
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
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Fig. 10. Clusters and small chains of eukaryotic cells, presumably fungi. Sample taken from Scurano’s Pieve. Further isolation attempts showed the presence of black yeast such as Exophiala sp. and of Alternaria spp.
v
v
it is inexpensive and does not need any instrument except a microscope it shows the existing relationships between the
surface and the colonizing microorganisms Žstage of growth., diffusion, correspondence with a particular alteration of the surface
Fig. 11. Small flake taken from the sandstone Portal of Scurano’s Pieve. Attached cluster of cells are clearly visible.
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
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Fig. 12. Enlargement of Fig. 11, in which the close connection between microorganisms and stone is clearly visible.
Fig. 13. EDX analysis of microelements found on the sample shown in Fig. 9. Besides calcium cations, due to the rock composition, the presence of Ti, S and Fe is attributable to residues of combustion fuels Žthe sampled area was localized in proximity to the exit of a car park..
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
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Table 2 Results obtained after the examination of adhesive tapes under microscopy, description of microflora and correspondence with cultural analysis Ref. code
MF PV TM VG Mu¨ Carrara
No. positive
Observations under
Cultural analysesa
samples observed under microscopy
microscopy
F
ArC
2r5 5r5 12r12 1r4
Biofilm Black fungal cells in clusters and chains Algal biofilm and black fungi Brown-black clusters of cells Black fungal clusters closely connected with crystals; in two samples algae were observed
y q q q
y y q y
q
y
q
y
q
y
q q q
y y y
7r8 Clusters of meristematic black fungi Mu¨ Pentelic
2r2 Black fungal chain and clusters closely connected with crystals
Mu¨ Ekeberg
2r2 Black fungal clusters closely connected with crystals
Mu¨ Laas EU CH a
v
v
2r2 6r6 3r3
Black fungal clusters Black fungal chains
F, Fungi; ArC, algaerCyanobacteria; q, positive; y, negative.
it evidences the relationships occurring in the microbial community Že.g. prevailing microorganisms. it allows the identification of the isolated microorganisms.
Acknowledgements This research was partially presented at the 10th Workshop of the Eureka-Euromarble EU 496 project in Stockholm. We like to thank Dr. Maurizio Triscari for his kind support with SEM analysis. The authors acknowledge the financial support of European Community, through contracts No. ENV4-CT980704, No. ENV4-CT98-0707 and EVK4-1999-00061, Consiglio Nazionale delle Ricerche ŽC.N.R.. through contract No. 99.03872.PF36 and ex-MURST 60%.
References Barnett, H.L., Hunter, B.B., 1972. Illustrated Genera of Imperfect Fungi. Burgess, Minneapolis.
Eckhardt, F.E.W., 1978. Microorganisms and weathering of a sandstone monument. In: Krumbein, W.E. ŽEd.., Environmental Biogeochemistry and Geomicrobiology, Vol. 2. Ann. Arbor. Science, pp. 675–686. Ellis, M.B., 1971. Dematiaceous Hyphomycetes. CAB International Mycological Institute, Kew. Ellis, M.B., 1976. More Dematiaceous Hyphomycetes. CAB International Mycological Institute, Kew. Fassatiova, ´ O., 1986. Moulds and filamentous fungi in technical microbiology. In: Bushell, M.E. ŽEd.., Progress in Industrial Microbiology, Vol. 22. Elsevier, Amsterdam. Gargani, G., 1968. Fungus contamination of florence art masterpieces before and after the 1966 disaster. In: Walters, A.H., Elphick, J.J. ŽEds.., Deterioration and Protection of Materials, Vol. 1. Elsevier, Amsterdam, pp. 252–257. Hirsh, P., Eckhardt, F.E.W., Palmer Jr., R.J., 1995. Methods for the study of rock-inhabiting microorganisms — A mini review. J. Microbiol. Methods 23, 143–167. Hoog de, G.S., 1987. Taxonomic aims in the yeast-like fungi. In: Hoog de, G.S., Smith, M.T., Weijman, C.M.A. ŽEds.., Proceedings of an International Symposium on the perspectives of taxonomy, ecology and phylogeny of yeasts and yeast-like fungi. Elsevier, Amsterdam, pp. 13–16. King Jr., A.D., Hocking, A.D., Pitt, J.I., 1979. Dichloran rose bengal medium for enumeration and isolation of moulds from foods. Appl. Environ. Microbiol. 37, 959–964. Madigan, M.T., Martinko, J.M., Parker, J., 1997. Brock, Biology of Microorganisms. 8th edition Prentice Hall, New Jersey. May, E., Lewis, F.J., 1988. Strategies and techniques for the study
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11 of bacterial populations on decaying stonework. In: Domaslowski, W., Ciabach, J. ŽEds.., Proceedings of the 6th International Congress on Deterioration and Conservation of Stone, 2. Nicholas Copernicus University Press, Torun, pp. 59–70. Ranalli, G., Pasini, P., Roda, A., 2000. Rapid diagnosis of microbial growth and biocide treatments on stone materials by bioluminescent low-light imaging technique. In: Fassina, V. ŽEd.., Proceedings of 9th International Congress on Deterioration and Conservation of Stone, 1. Elsevier, Amsterdam, pp. 499–505. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanier, R.Y., 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacterial. J General Microbiology 111, 1–61. Sterflinger, K., Krumbein, W.E., 1995. Precision replicas of microbially contaminated surfaces for optical and SEM analysis. J. Microbiol. Methods 23, 301–308. Sterflinger, K., Krumbein, W.E., 1997. Dematiaceous fungi as a major agent for biopitting on Mediterranean marbles and limestones. Geomicrobiol. J 14, 219–230.
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Urzı, ` C., Lisi, S., Criseo, G., Zagari, M., 1992. Comparazione di terreni per l’enumerazione e l’isolamento di funghi deteriogeni isolati da materiali naturali. Ann. Microbiol. Enzimol. 42, 185–193. Urzı, ` C., De Leo, F., Hoog de, G.S., Sterflinger, K., 2000. Recent advances in the molecular biology and ecophysiology of meristematic stone-inhabiting fungi. In: Ciferri, O., Tiano, P., Mastromei, G. ŽEds.., Of Microbes and Art. The Role of Microbial Communities in the Degradation And. Protection of Cultural Heritage. Kluwer AcademicrPlenum, New York, pp. 3–19. Urzı, ` C., Albertano, P., 2001. Studying phototrophic and heterotrophic microbial communities on stone monuments. In: Doyle, R.J. ŽEd.., Methods in Enzymology, Academic Press, San Diego CA, in press. Wollenzien, U., Hoog de, G.S., Krumbein, W.E., Urzı, ` C., 1995. On the isolation of microcolonial fungi occurring on and in marble and other calcareous rocks. Sci. Total Environ. 167, 287–294.
Sampling with adhesive tape strips: an easy and rapid method to monitor microbial colonization on monument surfaces Clara Urzı`) , Filomena De Leo Department of Microbiological, Genetic and Molecular Sciences, UniÕersity of Messina, Salita Sperone, 31 I-98166 Messina, Italy Received 20 July 2000; received in revised form 12 October 2000; accepted 17 October 2000
Abstract An easy and fast non-destructive method for sampling from monument and art object surfaces is proposed. The results obtained after sampling in regions of black spots and discoloration using adhesive tape strips showed that the method is useful for monitoring microbial colonization as well as for the identification of biodeteriogens. This technique is easy to apply, inexpensive and reproducible. The presence of fungi or algae on stone surfaces can be checked at an early stage of colonization or after cleaning procedures, without damage to the sampled surface. In addition, it is possible to obtain information on the morphology and taxonomy of microorganisms, and their relationships with the colonized material surfaces. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Adhesive tape; Fungi; Algae; Monument surfaces; Non-destructive method
1. Introduction The use of non-destructive techniques to monitor microbial colonization before andror after conservation treatments of monuments andror of artistic objects is often required. Several non-destructive methods have been proposed for direct or indirect evidence of microbial colonization. Needle procedures, agar fingerprinting, measurement of microbial activity, detection of bacteria by fluorescent antibodies, bioluminescent techniques and ATP assays have been described by several Authors ŽEckhardt, 1978; May and Lewis, 1988; Hirsh et al., 1995; Wollenzien et al., 1995;
) Corresponding author. Tel.: q39-090-676-5196; fax: q39090-392-733. E-mail address: [email protected] ŽC. Urzı`..
Ranalli et al., 2000.. However, despite their undeniable utility, these techniques do not give any information about the relationships between the microorganisms and the surface itself. Sterflinger and Krumbein Ž1995. proposed a replica method to check biological colonization of the rock surface and the connection between organisms and stone, through SEM observations. This technique, even though of wide potential application, does not allow cultivation of microflora present and thus does not solve problems related to the taxonomy of the species involved. The use of adhesive tape is a common practice in clinical mycology for the direct observation of Dermatophytes from skin and their further isolation by inoculation of small pieces of the strips on suitable cultural media. Gargani Ž1968. first applied this technique in the field of cultural heritage, for evidence of fungal growth on frescoes after the flood of 1966 in Florence.
0167-7012r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 Ž 0 0 . 0 0 2 2 7 - X
2
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
The aim of this paper is to demonstrate that adhesive tape sampling as a non-destructive technique can be used as a reliable and easy method on stone surfaces to monitor microbial colonization. For this reason the technique was used on different stone surfaces, and the presence of microflora was demonstrated both directly under microscopy, as well as by cultivation on suitable media.
2. Materials and methods 2.1. Sampling and preparation of adhesiÕe strips Sampling with adhesive tape strips ŽFungi tape DID, Milan, Italy. was carried out from rocks and monuments located in different European Countries. Table 1 summarizes the main characteristics of the stones that were sampled. Strips were gently applied to the stone surface and were then immediately placed on sterile glass microscope slides and kept in a box until arrival in laboratory. The adhesive tape strips were then cut in small pieces Žabout 5 = 5 mm. and sorted for microscopical and cultural analysis.
2.2. Microscopical analyses 2.2.1. Light and epifluorescence microscopy Observations for microscopy were carried out without any specific preparation. A drop of sterile water, Amman’s lactophenol solution, or Acridine Orange solution Ž0.1%. was put between a glass slide and the tape Žface down.. A cover glass was placed on the top, in order to keep the tape as flat as possible ŽUrzı` and Albertano, 2001.. Direct observations of samples were carried out using a light microscope equipped with phase contrast ŽDML, Leica. andror a light epifluorescent microscope ŽDMR, Leica. equipped with a mercury lamp Ž50 W. and 450–490 nm excitation filter. 2.2.2. Scanning electron microscopy For SEM observations, a piece of adhesive tape ŽFungi tape DID. was mounted face up on a stab with a biadhesive tape ŽAdhesive Tabs, Electron Microscope Science, Washington, USA., then dehydrated via an ethanol series and covered with carbon powder. Observations were carried out using a SEM, S420 Cambridge equipped for EDX analyses of the Earth Science Department of Messina University.
Table 1 Provenance of the sampled surfaces, reference code, number of samples and type of rock Provenance
Ref. code
No. of samples
Stone
Muro Farnesiano, ŽParma, Italy.
MF
5
Brick and mortars
Scurano’s Pieve ŽParma, Italy.
PV
5
Stone sandstone
Templete Mudejar ŽGuadalupe, Spain.
TM
12
Mortars
Victory Gate ŽGermany.
VG
4
Laas marble
Eureka-Euromarble exposure site in Munich ŽGermany.
Mu¨
8 2 2 2
Carrara marble Pentelic marble Ekeberg marble Laas marble
Eureka-Euromarble exposure site in Messina ŽItaly. Moscow ŽRussia. Arles ŽFrance.
EU
6
Carrara marble
Concert Hall ŽHelsinki, Finland.
CH
3
Carrara marble
Total
49
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
3
Fig. 1. Carrara marble taken from the outer facade of the Concert Hall building in Helsinki ŽFinland.. The surface is entirely covered by black structures. Magnification 63 = .
2.3. Cultural analysis Microorganisms that could be cultivated were isolated and identified by inoculation of small pieces of the strips directly into liquid medium BG11 for algae ŽRippka et al., 1979. and in duplicate in solid medium ŽDRBC, King et al., 1979; Urzı` et al., 1992. for fungi. No attempts were made to isolate cultivatable bacteria. Incubation was carried out at 288C for 1 month each in the light and in the dark, respectively. Algae and cyanobacteria were initially determined to be present or absent, and then attributed to morphological groups ŽRippka et al., 1979.. Identification of fungi was mainly carried out on the basis of the macroscopic features of colonies and on the micromorphology of reproductive structures, according to Barnett and Hunter Ž1972.; Fassatiova` Ž1986.; Hoog de Ž1987. and Ellis Ž1971, 1976..
scopic observation. These samples Žthree from Muro Farnesiano, three from Victory Gate and one from Carrara marble block exposed in Munich. were withdrawn from surfaces that had recently been cleaned or were without any evident biological alteration.
3. Results 3.1. Microscopical analysis Among the forty-nine samples examined, only seven samples were microbe free by direct micro-
Fig. 2. Sampling by adhesive tape from the facade of Scurano’s Pieve, showing chains of meristematic fungi. Bar s10 mm. Figs. 2–8 consist of light microscopy observations.
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C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
Fig. 5. Explorative hyphae from a fungal cluster attached to a calcite flake. The sample was taken from the surface of a Carrara marble block in Munich. Bar s 50 mm.
Fig. 3. Sample taken from the surface shown in Fig. 1. Clusters of fungi are seen in chains. Further isolation and identification evidenced that they belong to a strain of Alternaria alternata. Bar s10 mm.
However, forty-two samples, corresponding to either black spots or intercrystalline growth ŽFig. 1. showed colonization by dematiaceous fungi. The fungal colonization was evidenced mainly as presence of chains of melanized cells or black clusters ŽFigs. 2 and 3.. In some cases, germinative or explorative structures derived from single spores or conidia were observed, as a clear demonstration that
Fig. 4. Germinating spore seen under phase contrast microscopy. This structure resembles a conidium of Epicoccum purpurescens. The sample was taken from a a protected part of a Carrara marble item. It has been exposed in Munich, Germany, since 1991 in the frame of Eureka-Euromarble EU496 project. Bar s10 mm.
fungi actively colonize the rock ŽFigs. 4 and 5.. In addition, a close connection between black fungal clusters and stone crystals was observed in most of these samples ŽFig. 6.. In a few samples Ž10r42., photosynthetic organisms were observed by light microscopy. However, some differences were observed in agreement with the colonization pattern: Ža. when phototrophic colonization was already evident by the naked eye Že.g.
Fig. 6. Adhesive tape sampled from an Ekeberg marble block exposed in Munich. Clusters of fungi are clearly visible, closely connected with the marble flakes. In some cases, explorative hyphae or rhizoid structures had formed and created a stronger connection with the marble itself. Bar s 50 mm.
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
in seven samples taken from Templete Mudejar., under the microscope a massive growth in the shape of microbial mat or biofilm was seen ŽFig. 7.; in contrast, in the three samples taken from Carrara and Ekeberg marble, recently exposed to outdoor conditions in Munich, phototrophic microorganisms were occasionally found closely associated with the fungal clusters ŽFig. 8.. Bacteria were directly found only under epifluorescent microscopy in five of the samples examined. SEM observation of adhesive tapes gave good results in agreement with those obtained under light microscopy. Microbial colonization was observed as a biofilm ŽFig. 9., clusters of microorganisms and as small chains of cells ŽFig. 10. whose size and shape was comparable with those of meristematic struc-
5
tures seen under light microscopy ŽFigs. 2 and 3.. The connection between microorganisms and stone was also evident ŽFigs. 11 and 12.. EDX microelements analysis on the samples taken with the adhesive tape, was useful to indicate the presence of inorganic pollutants, such as Ti, S, Fe, observed in the sample shown in Fig. 13, probably due to their release from fuel combustion.
3.2. Analysis of cultures Cultural analysis confirmed that there was colonization, as already observed through microscopy in almost all the positive samples ŽTable 2..
Fig. 7. Algal biofilm and fungal cells visible in the sample taken from the Templete Mudejar in Spain. Biological patina was already visible to the naked eye. Bar s 50 mm.
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C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
Fig. 8. Algal and fungal cluster sampled from the Ekeberg marble in Munich. Algal growth was observed only in sheltered parts Žfolders.. Bar s 50 mm.
Fungi were isolated from all positive samples seen by microscopy. Among the strains isolated the most frequently found were dematiaceous hyphomycetes. These were assigned to the genera Cladosporium, Alternaria, Phoma, Ulocladium. In some cases ubiquitous non-dematiaceous strains of the genera Penicillium, Aspergillus and Fusarium were isolated. Black meristematic fungi were isolated in some samples Ž10r42. and assigned to the genera Coniosporium and Phaeoteca. Black yeasts of the genus Exophiala were rarely isolated Ž1r42.. In addition, cultural analysis showed the presence of fungi in three samples taken from Victory Gate, in which the previous microscopic observations did not show the presence of microorganisms. The strains isolated from these samples belonged to the genera Cladosporium, Mucor and Phoma. Algae and cyanobacteria were isolated only when they were predominant compared to the other microorganisms Že.g. case of the Templete Mudejar samples, Fig. 7.. In these cases, they were described
as unicellular green algae Ž Chlorella-like. and filamentous N2-fixing cyanobacteria of the Nostocaceae group.
4. Discussion The results obtained demonstrated that the sampling method proposed offers the possibility of gaining information on the microbial colonization of material surfaces without being destructive of valuable material. The presence of fungi andror algae, when they occurred, was easy to demonstrate, and thus to ascribe a specific alteration in the surface material with the fungal or algal colonization. In addition, in situ morphology of microorganisms on the colonized surfaces and their relationships to the substratum was shown. A good correspondence between microscopic observations and cultural analysis was also noticed. Cultural analysis allowed isolation and identification of microorganisms, including slow growing meris-
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
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Fig. 9. SEM micrograph of an adhesive tape taken from Muro Farnesiano in Parma, Italy. A microbial biofilm of prokaryotic cells Žcoccoidal cells whose size was below 1 mm. is clearly visible. Figs. 9–13 consist of SEM observations.
tematic black fungi. No significant omission of information with respect to other methods was observed and it was possible to see and isolate fungal species typically found on rock surfaces and considered to be the cause of biodecay ŽWollenzien et al., 1995; Sterflinger and Krumbein, 1997.. It is worth noting that it was possible to isolate and thus also identify black meristematic fungi, which are notoriously very difficult to isolate among fast growing species ŽUrzı` et al., 2000.. The limit of the method, beside the fact that it gives only qualitative information on the microorganisms present, is that it gives an underestimation of the bacterial flora. In fact, no bacteria were seen by light microscopy. This could be due to bacteria being more evident when staining procedures are
used, and also because by seeing them under light microscopy they should be present in the order of at least ; 10 6 cellsrml ŽMadigan et al., 1997..
5. Conclusions Adhesive tape sampling can be widely and safely applied in the field for the conservation of monuments and can be used not only by microbiologists but also by skilled restorers and conservators. The simple technique offers the following advantages v
it is a non-destructive method and can be used for monitoring microbial colonization over a period
C. Urzı, ` F. De Leo r Journal of Microbiological Methods 44 (2001) 1–11
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Fig. 10. Clusters and small chains of eukaryotic cells, presumably fungi. Sample taken from Scurano’s Pieve. Further isolation attempts showed the presence of black yeast such as Exophiala sp. and of Alternaria spp.
v
v
it is inexpensive and does not need any instrument except a microscope it shows the existing relationships between the
surface and the colonizing microorganisms Žstage of growth., diffusion, correspondence with a particular alteration of the surface
Fig. 11. Small flake taken from the sandstone Portal of Scurano’s Pieve. Attached cluster of cells are clearly visible.
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Fig. 12. Enlargement of Fig. 11, in which the close connection between microorganisms and stone is clearly visible.
Fig. 13. EDX analysis of microelements found on the sample shown in Fig. 9. Besides calcium cations, due to the rock composition, the presence of Ti, S and Fe is attributable to residues of combustion fuels Žthe sampled area was localized in proximity to the exit of a car park..
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Table 2 Results obtained after the examination of adhesive tapes under microscopy, description of microflora and correspondence with cultural analysis Ref. code
MF PV TM VG Mu¨ Carrara
No. positive
Observations under
Cultural analysesa
samples observed under microscopy
microscopy
F
ArC
2r5 5r5 12r12 1r4
Biofilm Black fungal cells in clusters and chains Algal biofilm and black fungi Brown-black clusters of cells Black fungal clusters closely connected with crystals; in two samples algae were observed
y q q q
y y q y
q
y
q
y
q
y
q q q
y y y
7r8 Clusters of meristematic black fungi Mu¨ Pentelic
2r2 Black fungal chain and clusters closely connected with crystals
Mu¨ Ekeberg
2r2 Black fungal clusters closely connected with crystals
Mu¨ Laas EU CH a
v
v
2r2 6r6 3r3
Black fungal clusters Black fungal chains
F, Fungi; ArC, algaerCyanobacteria; q, positive; y, negative.
it evidences the relationships occurring in the microbial community Že.g. prevailing microorganisms. it allows the identification of the isolated microorganisms.
Acknowledgements This research was partially presented at the 10th Workshop of the Eureka-Euromarble EU 496 project in Stockholm. We like to thank Dr. Maurizio Triscari for his kind support with SEM analysis. The authors acknowledge the financial support of European Community, through contracts No. ENV4-CT980704, No. ENV4-CT98-0707 and EVK4-1999-00061, Consiglio Nazionale delle Ricerche ŽC.N.R.. through contract No. 99.03872.PF36 and ex-MURST 60%.
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