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ARTICLE IN PRESS C. Abrusci et al. / International Biodeterioration & Biodegradation 56 (2005) 58–68 59 conservation and preservation of 430,000 roll-cans that constitute an important and irreplaceable historical and cultural heritage. The image stability depends on both intrinsic factors, e.g. the nature of the polymer base, and extrinsic factors, e.g. environmental conditions. Archivists have encountered the problem of film deterioration caused by inappropriate storage conditions of temperature and moisture.Many efforts have focused on the decrease of the physical damage caused by the ‘‘vinegar syndrome’’ in cellulose acetate-based films (Catalina and Del Amo, 1999) and the fading of colour dyes.In contrast, there is a lack of knowledge about the contamination by bacteria and fungi in cinematographic archives and its influence on film stability.Studies on microbial contamination of cinematographic films stored in archives are scant and have been reviewed by Abrusci et al. (2004a).The main work has been done by Opela (1992) and involved the analysis of cellular concentration and the identification of the principal fungal contaminants in the air, on floors, in cans and on cinematographic films in two different archives of the Slovak Republic.As in other studies of indoor habitats, the results indicated that certain environmental conditions, such as, air flow, temperature, movement of personnel, etc., had a crucial effect on microbial concentrations in the diverse places analyzed.The fungal biodiversity was higher in the air than in the surface of the films, biodiversity being limited in archives, with filamentous species in the genera Penicillium and Aspergillus being the predominant micromycetes (Opela, 1992). Aspergillus and Penicillium have been reported as the most frequently found micromycetes in indoor air and buildings from cold and temperate climate regions (Frisvad and Gravesen, 1994; Hyva¨ rinen et al., 2002).These fungi are considered to be primary colonizers, since they are capable of growing at a w o0:8, although many species have the optimum for growth at a w values close to 1 (Nielsen, 2003).Other genera of fungi (Alternaria, Cladosporium, Phoma, etc.) are secondary colonizers, because they require a w values at least between 0.8 and 0.9 (Nielsen, 2003).The presence of spores and/or vegetative cells of microorganisms on the surface of materials indicate the possibility of future biodegradation or biodeterioration.However, colonization or microbial growth on a material almost always results in biodeterioration of the material.There are different ways in which microorganisms can compromise the structure and function of polymeric materials (Flemming, 1998).For instance, they can produce pigmentation or degrade of one or more compounds.In other cases, they can hydrate, and as well as causing fouling, they may penetrate into the polymeric material.These mechanisms of biodeterioration occur when microorganisms are metabolically active and growing, but although the environment contains an extremely rich variety of microorganisms, usually only a few of the microorganisms isolated from a substrate are responsible for damaging it. In cinematographic films, the organic components mentioned previously can be considered as potential carbon sources for growth of microorganisms, if environmental conditions are adequate.Gelatin, the binder of the photographic emulsion, is the most biodegradable of the substrates.It is composed of high molecular weight polypeptides derived from collagen, and the most widely used photographic gelatin is type-B, obtained by basic pre-treatment of bones (Abrusci et al., 2004b).Many prokaryotic and eukaryotic microorganisms exhibit proteolytic capacity, so they are able to degrade gelatin. Stickley (1986) studied by viscometry the biodegradation of 5% aqueous gelatin solutions at 37 1C using single strains of Bacillus and Pseudomonas. In both cases, reduced viscosity was detected after 24 h. In a more detailed study, Abrusci et al.(2004c) viscosimetrically analysed the biodegradation of type-B gelatin (Bloom 225) by bacteria isolated from cinematographic films, and noted that temperature is a very important factor affecting rate of biodegradation (Abrusci et al., 2004c). As for film base materials, a large number of studies have been carried out on biodegradation of cellulose acetates (CA) by both bacteria and fungi.The degree of hydroxyl substitution (acetylation) has been found to be a factor with a strong influence on biodegradation, those CA with a higher degree of acetylation being more resistant to microbial attack (Buchanan et al., 1993; Nelson et al., 1993; Sakai et al., 1996).According to Sakai et al.(1996), CA biodegradation is mediated by the cooperative action of esterase(s), lipase(s) and cellulase(s).These studies were carried out adding pure cultures of identified species (Samios et al., 1997; Nelson et al., 1993) or using mixed populations such as those included in compost (Gu et al., 1993b).The cellulose triacetate used in the photographic industry has a degree of substitution of approx.2.7.Hence, its resistance to biodegradation should be much higher than the other components of the film.In fact, it has been reported that microorganisms attack the organic part of the film emulsion especially at R.H. 460% ða w 40:6Þ (Gordon, 1983).In tropical climates fungal growth is a special problem, but it is also possible to detect these troublesome growths during the summer in temperate zones with high relative humidity. Owing to the risk of biodeterioration of cinematographic film in the Spanish archives, we isolated and identified bacteria and fungi from selected samples of cinematographic films collected in different archives. The identification studies in organisms from black and white cinematographic films are presented here, along with an analysis of the ability of these isolates to hydrolyse the gelatinous component of films.
60 ARTICLE IN PRESS C. Abrusci et al. / International Biodeterioration & Biodegradation 56 (2005) 58–68 2. Materials and methods 2.1. Cinematographic film samples Film samples from collections at Barcelona, Madrid and Gran Canaria archives were supplied by Filmoteca Espan˜ ola.The sampling and transport of the samples were carried out under aseptic conditions.The films supplied appeared to be in good condition. 2.2. Isolation of microorganisms from cinematographic films The isolation procedure for all samples was performed in a laminar-flow chamber and manual operations were carried out using sterile disposable gloves. From each cinematographic sample, two or three pieces were placed on trypticase-soya-agar (TSA), for heterotrophic bacteria, and Sabouraud-glucose-agar (SAB) supplemented with choramphenicol for fungi.Petri dishes were incubated at 30 1C for 1–2 weeks.Cultures were observed daily under a stereoscopic microscope to check the presence of bacterial colonies and/or fungal mycelium (Fig.1). 2.3. Characterization of bacteria Each bacterial strain isolated from the film samples was cultured on TSA either at 30 or 37 1C for 48 h. Replicates were made and preserved at 4 1C or 80 1C using TSB liquid medium supplemented with glycerol. Gram- and simple staining were performed using exponential TSA cultures, and oxidase and catalase tests made.Acid production from glucose and sucrose on OF basal medium, growth on Simmons citrate agar, and starch and gelatin hydrolysis, were tested.In all Grampositive (and some Gram-negative strains), the ability to form spores was tested by examining 24-, 48- and 72-h, and 1-week cultures on TSA, as well as the Schaeffer- Fulton specific stain (Forbes et al., 2002).Morphological characters were examined under differential interference contrast (Nomarski) and phase contrast using a Zeiss Laboval 4 optical microscope. Fig.1.One-week Petri dish cultures at 30 1C showing on left microfungi P. chrysogenum (upper) and A. alternata (lower), and on right B. subtilis developing from fragments of film. After the preliminary morphological and physiological characterization as used by Oppong et al.(2000), bacterial strains were identified employing commercial identification kits.Depending on the bacterial nature, different commercial assays were employed such us API 20 NE, API 20E, API 50 CHB and API STAPH.The preparation and inoculation procedures followed the recommendations of the manufacturer (BioMerieux Espan˜ a S.A.), with the strains being incubated at 30 1C for 24 h.The numeric profiles obtained were compared with a bacterial database using the commercial software APILAB from BioMerieux.Simultaneously, additional diagnostic tests were made with some bacterial strains, such as growth on McConkey or mannitol salt agar and coagulase and lysostaphin tests. 2.4. Isolation and characterization of fungi For isolation of micromycetes, film samples were incubated on potato-dextrose-agar (PDA) at 28 1C or 30 1C for 1–2 weeks.Replicates were preserved at 4 1Cor 80 1C in PD broth medium supplemented with 10% glycerol. The following media were used for characterization: (a) malt extract agar (MEA) composed of malt extract (Difco) 20 g, peptone 1 g, glucose 20 g, agar 20 g, distilled water 1 L; (b) Czapek yeast extract agar (CYA) composed of K 2 HPO 4 1 g, Czapek concentrate 10 ml, yeast extract (Difco) 5 g, Sucrose 30 or 200 g, agar 15 g, distilled water 1 L; (c) 25% glycerol nitrate agar (G25N) composed of K 2 HPO 4 0.75 g, Czapek concentrate 7.5 ml, yeast extract 3.7 g, glycerol, analytical grade 250 g, agar 12 g, distilled water 750 ml; (d) potato-carrotagar (PCA) composed of shredded potato 20 g, shredded carrot 20 g, agar 20 g, distilled water 1 L; and (e) PDA (Difco).Czapek concentrate contained: NaNO 3 30 g, KCl 5 g, MgSO 4 .7H 2 O 5 g, FeSO 4 .7H 2 O 0.1 g ZnSO 4 .7 H 2 O 0.1 g, CuSO 4 .5H 2 O 0.05 g, distilled water 100 ml. Seven-day cultures on MEA, CYA (with either 3% or 20% sucrose), and G25N were used for identification of Aspergillus and Penicillium species, based on the procedures described by Pitt (2000) and Klich (2001). The other strains were cultured on PDA and PCA for 21 days at 22 1C and 80% R.H. and then subjected to cycles of NUV wavelength light exposure-darkness (12 h each) at room temperature for at least 14 days to stimulate sporulation.Morphological analysis of the isolates was carried out with a Leitz Diaplan microscope, equipped with differential interference contrast optics.The fungal isolates were identified to species (Aspergillus and Penicillium) or generic level. The yeast isolate was identified on the basis of metabolic/physiological features using the API 20C- AUX kit (BioMerieux Espan˜ a, S.A.), and simple crystal violet staining was used for observing cell size and shape.
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