Ethanol as an Antifungal Treatment for Silver Gelatin Prints: Implementation Methods Evaluation

1 Introduction

For photographic collections, fungal development is a recurring problem. Most photographs are easily used as a substrate for fungal growth because they are made of proteinaceous materials such as gelatin or albumen, and polysaccharides such as cellulose; both material groups also being hygroscopic materials. Fungal development can result in the loss of parts or the totality of the photographic image by hydrolysis of the substrate (paper and colloid). The prevention of such damages can be done with a strict control of the storage climate to avoid fungal growth; however what are the options once the fungi have developed?

In France several antifungal treatments are used. For example, treatments using ethylene oxide or gamma ray have been the subject of various research projects and are used to treat fungi in archival collections.

Ethylene oxide fumigation is very efficient in killing fungi, insects, and bacteria (Flieder and Capderou 1999, 144–151); however it can cause damage to photographic prints. The gelatin peptic chains are broken, thus resulting in a high viscosity loss (Tomsŏvá et al. 2016). As well, cellulosic materials are more hydrophilic after treatment, resulting in a higher sensitivity to further fungal attacks (Jacek 2004; Valentin 1986; Nittérus 2000a, 22–40). Ethylene oxide is also a very toxic compound: mutagen and carcinogenic for humans, it is forbidden to use in North America and several European countries (Trehorel 1988).

The dose of gamma rays needed to inactivate fungi is not consensual, and ranges from 4.5 kGy to 18 kGy (Flieder and Capderou 1999; Pavon Flores 1975). Gamma rays also cause damage to photographic gelatin, such as viscosity loss after an exposure of 2.5 kGy of radiation (Tomsŏvá et al. 2016) and increase of the print’s density after an exposure of 90 kGy of radiation (Adamo et al. 2012). Even if this dose is much higher than the one needed to kill the fungi, it is important to keep in mind that exposure to gamma rays is cumulative. Thus if a photograph is treated several times in its life, the 90 kGy dose will be attained at some point. The radiation also degrades the photographic paper support which presents damage similar to the one caused by ageing (weakening and yellowing), resulting from cellulose depolymerisation and oxidization (Butterfield 1987; Nittérus 2000a, 25–40).

These two treatments are costly and more adapted to large collections. So, what are the available options for treating single items or a small collection of photographs? Today, photograph conservators use pure ethanol or a deionised water–pure ethanol mixture of different ratios applied on the surface of the prints with a cotton swab. This method, while widely used, has not been tested and its fungicidal effects have not been studied. There is some research on the use of alcohols as a fungicide for paper objects. The use of a water-ethanol mixture with a 30:70 (v/v) ratio is the most recommended (Nittérus 2000b, 101–105; Jacek 2004; Meier 2006; Sequeira et al. 2016), however authors disagree on its most effective implementation method between spraying, bathing, and vapour fumigating (Nittérus 2000b, 101–105; Bacílková 2006; Meier 2006).

In this study, we intended to clarify which implementation method of water-ethanol (30:70 v/v) mixture inactivates fungal growth on silver gelatin prints. Three implementation methods of this mixture were tested. In order to check if the cotton-swab application, corresponding to a short contact time with mechanical action, was efficient or not, we chose to test a short direct contact between the mixture and the photograph, with and without mechanical action, to determine the influence of this factor. The use of the mixture as vapour, previously tested, was chosen as our third implementation method because of the treatment possibilities it opens, such as treating photographs whose surfaces are too damaged for contact, and treating several photographs at the same time. Those three methods are not representative of today’s practice in conservation labs but correspond to three levels of intervention on the prints: without contact, with contact, and with contact and mechanical action.

Various fungi have been identified on silver gelatin prints; however, as the sampling is done with swabs, the identified species may not be the ones responsible for the prints’ deterioration (Sclocchi et al. 2013). For this study, four fungal species were selected within the most common encountered species on photographs (Lucas 2016, 289–294): Alternaria alternata, Aspergillus niger, Chaetomium globosum, and Penicillium brevicompactum.

2 Material and methods

2.6 Evaluation of fungal inactivation

Once treated, the sample was placed with the emulsion down on M₂Lev medium (for 1 L of water: 20 g malt, 3 g yeast extract Biomerieux^®, 15 g agar Biomerieux^®), and then incubated at 25 °C.

The colony diameter was observed visually after seven days. Its diameter was assessed qualitatively in comparison to the diameter of the untreated control samples. The samples were marked as “++” when the colony diameter was similar or larger than the control, “+” when inferior than the control, or “-” when no growth was visible (Figure 1).

Figure 1:

Example of colony diameter for Aspergillus niger.

When no growth was observed after seven days of incubation, the samples were incubated for an additional seven days and the colony diameter was observed again.

3 Results

4 Discussion

One of the major interests of the present study was the antifungal treatment of artificially contaminated silver gelatin prints. The development of all the tested species within a week at 25 °C demonstrated that rapid decontamination (within 48 hours), or at least preservation, of photographs after flooding is required to avoid fungal growth.

Fungal species are, in general, cellulolytic. This was shown for C. globosum and A. niger (Lakshmikant 1990), and P. brevicompactum and A. alternata (Méheust 2012), but these species were also potentially capable of degrading gelatin. P. brevicompactum (Bingley and Verran 2013), A. alternata (Abrusci et al. 2006), and some isolates of C. globosum (Lech 2016) proved to degrade gelatin. We did not find any reference on the existence of gelatinase positive isolates among A. niger because this species is recognized as a high amylase producer; however, many other Aspergilli such as A. versicolor (Abrusci et al. 2005), A. ustus, and A. nidulans (Abrusci et al. 2007) were found gelatinase positive. It is suggested that all the species tested in the present study were cellulolytic, but it was not determined whether they were also gelatinase positive.

The protocol allowed determination of fungal inactivation by ethanol vapours. Fungi are strictly aerobic organisms, however they can grow with oxygen levels as low as approximately 2 % depending on the species (Nguyen Van Long and Dantigny 2016). Control samples demonstrated that the species grew at the surface of the agar medium, even if the sample was turned upside down, with the print over the mycelium. Therefore negative experiments that did not exhibit growth were not due to oxygen limitation but to fungal inactivation. Prior to ethanol treatments, the samples were entirely covered with mycelium and conidia. It can be expected that treatments that proved effective in the present study against heavy inoculum will be even more effective against lighter inoculum.

The obtained results depend on the different tested strains. Indeed each strain has a different growth speed and sensitivity to external agents. For instance, C. globosum’s ascospores are contained into perithecias, which makes the spores more resistant to external agents. In this experiment, A. niger and C. globosum showed more resilience to every tested treatment compared to the two other tested strains.

The vapour implementation showed the best results with the inactivation of all strains at fourteen incubation days (25 °C) after two hours of treatment.

The samples treated by direct contact with the water-ethanol mixture showed consistent results within the two batches, with and without mechanical removal.

The mechanical removal of spores, by swiping the microfiber cloth on the sample surface, did not influence fungal growth in this experiment. Indeed, the results from this batch of samples are similar to the batch without cloth swiping. The spores’ removal was partial, and the remaining spores resumed growth as the water-ethanol contact did not inactivate them; however, another study has shown that the mechanical removal of fungal spores, by reducing the number of spores on the surface, reduces the overall fungal activity on heritage objects (Prevet 2016).

For vapour implementation, the experiments that were negative after one week of incubation were re-incubated at the same conditions for another week. They remained negative, thus suggesting that, in these conditions, all the mycelium and all the conidia were inactivated. A diameter of the colony similar or larger than the control did not mean that no fungal fragments of the mycelium or conidia were inactivated. Parts of the mycelium and a fraction of the conidia were inactivated, but not enough to prevent growth and germination in many parts of the print. For more drastic treatments, more sections of the mycelium and a more important fraction of the conidia were inactivated. Therefore, it was hypothesized that viable cells were not present in all places of the print. Accordingly, a delay was observed for the growth outside the print, and eventually the diameter of the colony was less than the control. The observation of colonies not growing all around the print but appearing at first only on one edge of the print strengthened this hypothesis.

5 Conclusion

The present work was undertaken to clarify which implementation method of a water-ethanol (30:70 v/v) mixture inactivates fungal growth on silver gelatin prints.

The efficiency of the fungal growth inactivation depends on the temperature and on the time of contact between the water-ethanol mixture and the fungus. In a heritage context, working at high temperature (over 20 °C) is not possible because it would cause damage to the photograph’s constituents. Consequently, contact time is the relevant parameter. Our results show that the longer times tested with vapour implementation successfully inactivated fungal growth. The four tested strains were inactivated after only two hours of exposure to water-ethanol vapours; however, the direct contact tests did not inactivate fungi as the contact times, of between 30 seconds and 8 minutes, were too short.

The vapour treatment shows promising use in heritage conservation as it could be used to treat storage spaces or larger volumes of photographic prints because ethanol vapour can reach any remote place. The lower flammability limit for ethanol is 3.3 kPa (Anonymous 2003) and it can be obtained at 25 °C with an ethanol-water mixture close to 70:30 (v/v). For safety reasons, it is suggested to use an ethanol-water mixture close to 40:60 (v/v) but to extend the ethanol vapour application from 2 to 24 h (Dao et al. 2010).

In conclusion, before being used on photographs this treatment’s long term effects on silver gelatin prints and other photographic techniques must be evaluated further. Some studies have already been performed regarding this matter. Firstly, the water-ethanol mixture was shown not to damage the paper (Sequeira et al. 2016; Weiß 2006); however, butanol vapours lead to a slight modification in photographic gelatin structure resulting in a decrease in viscosity (Tomsŏvá et al. 2016). Complementary tests on the long-term effect of a water-ethanol (30:70 v/v) mixture on photographic materials should be performed.