Introduction

From the invention of photography in the 1840s to the emergence of digital techniques in the early 21st century1, photography has played a vital role in recording major historical events, social development, and revolution. During the development of photography, numerous back-and-white (B&W) photographic negative films have been produced, and preserved in archives, museums, and various cultural heritage institutions.

A typical B&W photographic negative film has a multi-layered structure, including a protective layer, a photographic emulsion layer, a polymeric support, and a back-coating layer (as shown in Fig. 1)2. The protective layer, coated on the photographic emulsion layer, is composed of approximately 1 μm crosslinked gelatin, which is used to protect the photographic images from being damaged during development and conservation3. The photographic emulsion layer, with a thickness of 15–20 μm, is the most essential part of the B&W photographic negative film. In its unexposed state, this layer consists of photographic gelatin and silver halide. Upon exposure and development, the silver halide transforms into silver particles, while the unexposed silver halide is removed using a fixing bath. The polymeric support, with a thickness of about 120 μm, consists of plasticized transparent plastic, such as cellulose nitrate, cellulose triacetate, or polyester, to enhance the mechanical strength of the photographic emulsion4. The back-coating layer, composed of gelatin and about 1 μm thick, is applied to the opposite side of the polymeric support to prevent curling and protect against fluctuations in temperature and humidity during development5. Based on the raw materials used in the B&W photographic negative film, the two sides of the film contain a large amount of gelatin, a natural protein derived from collagen of animal skin tissue or bone6,7,8,9.

Fig. 1
figure 1

Schematic diagram illustrating the structure of a black-and-white (B&W) photographic negative film.

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During the photographic process, lighter objects can reflect more visible light, leading to the formation of more silver particles from the decomposition of silver halide at the corresponding positions on the negative film10. This results in lower light transmittance and the generation of high-density areas, i.e., the dark and black areas on the negative film. In contrast, darker objects reflect less visible light, resulting in fewer silver particles forming at the corresponding positions, higher light transmittance, and the formation of low-density (light) areas on the negative film11,12. Consequently, the image recorded on the B&W photographic negative film is an inverse representation of the gray scale of the actual object, known as a negative image. Then photographic processing converts the latent image into a visible one, makes the image permanent, and renders it insensitive to further exposure to light13,14,15,16.

In the process of printing photos from the B&W photographic negative film, the film is illuminated using visible light, projecting the recorded image onto the silver-salt photographic paper14. Then, the silver halide on the sensitive silver-salt photographic paper decomposes to form a latent image, and the sensitive silver-salt photographic paper is then developed, rinsed, and processed to create a stable and visible image10. During the development of B&W photos, the visible light transmittance is high in the low-density areas of the negative film, causing more silver halide to decompose at the corresponding position of the photosensitive silver-salt paper, accompanied by the high level of image grey scale10,14. Conversely, in the high-density areas of the negative film, the silver particles absorb visible light, leading to low light transmittance. This further results in less silver halide decomposition at the corresponding points on the photographic paper and lower gray levels in the final image. This process produces an image with a gray scale that is the inverse of both the negative film and the original photographed object, resulting in a positive image.

As mentioned above, gelatine is the primary component of photographic films, as present in the protective layer, photographic emulsion layer, and back-coating layer. Under unfavorable environmental conditions, such as high temperature and relative humidity—particularly when relative humidity exceeds 60%—photographic negative films become highly susceptible to microbial contamination, a common issue for B&W photographic negatives17,18,19,20,21. Microbial growth and reproduction on the surface of photographic films could cause the biodegradation of gelatine, leading to damage to the images recorded on the films22,23,24,25,26. It is reported that microbial colonization on the back-coating layer tends to cause more severe biodeterioration compared to the emulsion layer, as the silver particles in the emulsion inhibit microbial growth26,27,28,29. When microorganisms grow on the photographic emulsion layer, they can degrade image-forming materials, resulting in serious damage and potential image loss. When it grows on the surface of the back-coating side of the B&W photographic films, it degrades the gelatin in the back-coating layer, forms a rough surface, and obscures images on the negative films.

The microorganism-contaminated B&W photographic negative film (Fig. 2a), dating back to the 1970s, had been stored in an inadequate conservation environment with high temperature and high humidity, resulting in contamination of the back-coating surface. Preliminary experiments identified the microorganisms as Penicillium, Aspergillus, Chaetomium sp., etc.30 As shown in Fig. 2a, the microbial growth formed a grayish-white surface, covering almost the entire surface of the negative film. This contamination resulted in a developed image with reduced clarity using the contaminated B&W negative film, as shown in Fig. 2b, where irregular gray-white patches are evident (Fig. 2b and c). As reported in previous studies, obscured images and image loss caused by biodeterioration are well acknowledged on films18,21,24, while how the presence of mold and mycelia affect image quality for the developed photo is unclear under different circumstances, i.e., for films with high- and low- density of Ag particles. This study aims to investigate the changes in surface roughness, visible light reflectance, and visible light transmittance of B&W photographic negative films with high and low density of silver particles contaminated by microorganisms, in order to provide scientific evidence and data support for better understanding and conservation for such objects.

Fig. 2: Contaminated B&W photographic films and developed images.
figure 2

Microbial contamination of B&W photographic negative film and its impact on recorded image, (a) the back-coating surface of a contaminated photographic negative film with microbial growth; (b) an image developed from the microorganisms contaminated B&W photographic negative film; (c) a magnified view (4 ×) labeled in the red line in Fig. 2b.

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Experiment

Three types of samples used in this study

Three types of the B&W photographic negative films were used for characterization and analysis in this study, i.e., the historical B&W photographic negative film, and two model samples including the unexposed B&W photographic negative film (representing the model film with low-density of Ag particles) and the exposed B&W photographic negative film (representing the model film with high-density of Ag particles) before and after stimulated microorganism contamination. The historical B&W photographic negative film, dating back to the 1970s used in this study, is from the collection of the Engineering Research Center of Historical-Cultural Heritage Restoration and Conservation, and exhibited severe microbial contamination on its back-coating layer.

The contemporary B&W photographic negative film (produced in 2021, 60 mm wide, Shanghai Chengjian Industry Limited Co.) was exposed to natural light for 10 s and subsequently immersed in the developing solution for 20 min to reduce the AgX to Ag. Then, the film was washed with deionized water more than three times to remove any residual developer to stop the development process. The film was then immersed in the fixing solution for 20 min to remove the remaining AgX, and was washed again with deionized water three times to form a homogeneous and stable negative film with high density. To prepare a model of microorganism-contaminated B&W photographic negative film, the lens wiping paper (Fushun City Minzheng Filter Paper Factory with a thickness of 27 μm), was covered on to the surface of the back-coating layer. This model sample was labeled as the model microorganism-contaminated high-density film sample.

The unexposed B&W photographic negative film was soaked in the fixing solution for 20 min in the darkroom to remove the AgX in the negative film. It was then removed and rinsed with deionized water three times to wash away the residual fixing solution, resulting in a homogeneous and stable low-density negative film. Finally, the lens wiping paper was covered on the surface of the back-coating layer of the negative film to simulate the microorganism-contaminated low-density B&W photographic negative film.

Scanning electrode microscopy (SEM)

A tungsten filament scanning electron microscope (Hitachi SU3500, Tokyo, Japan) was used to examine the surface morphology of the historical B&W photographic negative film with microorganism contamination and the contemporary negative film without microorganism contamination. The observations were conducted at an accelerating voltage of 10.0 kV, before which the surface of all samples was coated with a layer of gold to enhance conductivity and image quality.

Measurement of sample surface roughness

The 2D and 3D surface roughness and morphology of the film samples were characterized using the Keyence VK-X250 shape measurement laser microscopy system for the three types of samples, i.e., the historical B&W photographic negative film, and the other two model samples, including the unexposed B&W photographic negative film, i.e., model low-density film and the exposed B&W photographic negative film, i.e., model high-density film, before and after stimulated microorganism contamination. The measurement data were then processed using the VK-H1XMC multi-file analysis software to obtain the surface roughness parameters, in accordance with the standard ISO 25718-231. The four calculated parameters include the arithmetical mean height of the scale-limited surface (Sa), maximum height of the scale-limited surface (Sz), arithmetic mean peak curvature (Spc), and developed interfacial area ratio of the scale-limited surface (Sdr) using the following equations:

$$\begin{array}{c}\begin{array}{c}{Sa}=\frac{1}{A}{\displaystyle\iint }_{\check{A}}^{z\left(x,y\right){dxdy}}\\ {sz}={sp}+{sv}\\ {spc}=-\frac{1}{2}\frac{1}{n}\mathop{\sum }\limits_{k=1}^{n}\left(\frac{{\partial }^{2}z(x,y)}{\partial {x}^{2}}+\frac{{\partial }^{2}z(x,y)}{\partial {y}^{2}}\right)\end{array}\\ {sdr}=\frac{1}{A}\left[{\displaystyle\iint }_{\widetilde{A}}\left(\sqrt{\left[1+{\left(\frac{\partial z(x,y)}{\partial x}\right)}^{2}+{\left(\frac{\partial z(x,y)}{\partial x}\right)}^{2}\right]-1}\right){dxdy}\right]\end{array}$$

In the above-mentioned equations, symbol A represents the numerical value of the evaluation area, and the symbol à represents the ___domain (of integration or definition); z(x, y), which signed normal distance from the reference surface to the scale-limited surface; Sp is the largest peak height value of the scale-limited surface and Sv is the largest pit depth value of the scale-limited surface.

Optical density (OD)

The relationship between optical density (OD) and transmittance (T) is as follows:The optical density was determined for the model high- and low-density B&W photographic negative films before and after treatment of stimulated microorganisms contamination, using the X-rite 341C Portable Transmission densitometer following the standard32. The relationship between optical density (OD) and transmittance (T) is as follows:

$${OD}={\mathrm{lg}}\frac{1}{T}\,{or}\,T=\frac{1}{{10}^{{OD}}}$$

Five points were measured for each sample and the average was calculated.

Visible light reflectance and transmittance

Perkin Elmer Lambda 950 ultraviolet near-infrared spectrophotometer was employed to explore the visible light transmittance and visible light reflectance of model high- and low-density B&W photographic negative films before and after treatment of stimulated microorganism contamination, in the range of 350–800 nm. Three randomly selected areas of each sample were measured.

Results and discussions

Surface properties of the microorganisms-contaminated B&W photographic negative film

The surface morphology of an uncontaminated contemporary B&W photographic negative film and a microorganism-contaminated historical B&W photographic negative film was examined using scanning electron microscopy (SEM), as shown in Fig. 3a and d. The surface of the contemporary film appears smooth and free from microorganisms, while the historical film displays a visible layer of mycelia on its surface. Further analysis of the 2D and 3D surface roughness was conducted using a laser microscopy system to characterize both the uncontaminated and contaminated films.

Fig. 3: Surface morphology of the B&W photographic films before and after contamination.
figure 3

(a) SEM image of the contemporary B&W photographic negative film without microorganism contamination; (b) 2D surface morphology of uncontaminated contemporary B&D photographic negative film; (c) 3D surface morphology of uncontaminated contemporary B&D photographic negative film; (d) SEM image of the historical B&W photographic negative film contaminated by microorganisms; (e) 2D surface morphology of the historical B&W photographic negative film contaminated by microorganisms; (f) 3D surface morphology of the historical B&W photographic negative film contaminated by microorganisms.

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As shown in Fig. 3, the 2D (Fig. 3b and e) and 3D (Fig. 3c and f) surface morphologies reveal notable differences between the two samples. In Fig. 3b and c, the surface of the uncontaminated contemporary film is smooth and clean. In contrast, Fig. 3e and f shows the presence of abundant mycelia on the surface of the contaminated historical film, with the 3D topography indicating that the mycelium occupies positions below the reference surface (average height of the surface plane). The mycelial structures on the contaminated film surface exhibit a concave shape, with heights ranging from −0.4 μm to 0.5 μm. The overall height of the negative film, including the microorganisms, spans from −10 μm to 20 μm, with negative values indicating depths below the reference surface. These observations suggest that microbial activity has led to the biodegradation of gelatin on the film surface, contributing to increased surface roughness.

Quantitative roughness parameters further highlight these changes. For the contemporary negative film, the arithmetical mean height (Sa), maximum height (Sz), and arithmetic mean peak curvature (Sdr) were 0.04 μm, 0.87 μm, and 0, respectively. In contrast, these parameters increased significantly for the contaminated film, with values of 1.61 μm, 29.2 μm, and 0.27, respectively. The arithmetic mean peak curvature (Spc) also increased dramatically from 145 mm–¹ for the contemporary film to 1244 mm–¹ for the contaminated film, indicating a much sharper surface profile. These changes in mean height and peak curvature result in an increased developed interfacial area ratio for the microorganism-contaminated film, providing further evidence of the impact of microbial growth on the film’s surface properties. This increase is primarily attributed to the heightened surface roughness caused by microbial contamination, which promotes diffuse reflection of visible light on the film surface25.

A comparison between the uncontaminated contemporary and microorganism-contaminated B&W photographic negative films shows a significant increase in visible light reflectance on the surface of the contaminated film (Fig. 4). This increase is primarily attributed to the heightened surface roughness caused by microbial contamination, which promotes diffuse reflection of visible light on the film surface. This result indicates that microbial contamination significantly elevates the surface roughness of B&W photographic negative films, causing the surface area to deviate from the main interface and creating a sharp, irregular topography. During image development on photographic paper, visible light illuminating the uneven, contaminated surface diffuses, resulting in increased reflectance. In light of this result, model high-density and low-density film samples were therefore prepared by covering the lens wiping paper on the film, to stimulate the rough surface of the contaminated films. It is worth noting that the transmittance was not explored for the uncontaminated contemporary and microorganism-contaminated B&W photographic negative films, as transmittance properties are significantly influenced by the density of Ag particles within the films, making direct comparison less relevant to this study.

Fig. 4: Light reflectance of the films with and without microorganism contamination.
figure 4

Visible light reflectance on the surface of the contemporary B&W photographic negative film (labeled with blue line) and the B&W photographic negative film with microorganism contamination (labeled with red line).

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Characterization of the model samples

Preparation of model samples of microorganisms-contaminated B&W photographic negative film

For the historical B&W photographic negative film, the uneven distribution of silver particles, combined with the presence of microorganisms, contributes to the surface inhomogeneity. In order to achieve good homogeneity of stimulated mold distribution for model film samples and therefore obtain representative, repeatable, and comparable data, model film samples were prepared by placing a layer of lens wiping paper on the surface of contemporary films, to increase the roughness of film surface, as both mycelia on the contaminated film surface and fibers in the lens wiping paper are filamentary, and fibers in the lens wiping paper are more homogeneously distributed. The model negative films and corresponding developed images are shown in Fig. 5. It is evident that covering the negative film surface with lens wiping paper significantly reduces the gray level of the developed image, resulting in obscured images similar to those found in microbially contaminated films (as shown in Fig. 2).

Fig. 5: Contemporary B&W photographic before and after stimulated microorganism contamination.
figure 5

(a) The contemporary B&W photographic negative film without microorganism contamination; (b) the developed image using the B&W photographic negative film shown in Fig. 5a; (c) the model B&W photographic negative film covered with the lens wiping paper to stimulate homogeneous microorganism contamination; (d) the developed image using the model B&W photographic negative film showing in Fig. 5c.

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This method was further used to create both high- and low-density model-contaminated films, as illustrated in Fig. 6. The high-density model film sample was prepared by exposing contemporary film to natural light for 10 seconds (Fig. 6a), resulting in a surface with densely distributed Ag particles. The high-density film sample was covered with the lens wiping paper to prepare the model-contaminated high-density film sample (Fig. 6b). The model low-density film sample was the unexposed contemporary film (Fig. 6c), and this low-density film sample was covered with the lens wiping paper to prepare the model contaminated low-density film sample (Fig. 6d).

Fig. 6: The high-density negative film before and after stimulated contamination treatment.
figure 6

(a) The exposed B&W photographic negative film with high-density Ag particles; (b) the B&W photographic negative film after exposure with high-density Ag particles covered with the lens wiping paper; (c) the unexposed B&W photographic negative film denoted as low-density film sample; (d) the unexposed B&W photographic negative film covered with the lens wiping paper.

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Figure 7 shows the changes in the surface morphology and roughness parameters of the model microorganism-contaminated B&W photographic negative film before (Fig. 7a and b) and after (Fig. 7c and d) stimulated microorganism contamination. It can be seen that the surface morphology and roughness parameters of the model contaminated samples were similar to that of microorganisms contaminated historical photographic negative film, after the lens wiping paper was placed onto the surface of the B&W photographic negative film. Then, this method was used for the preparation of model microorganism-contaminated B&W photographic negative film in the subsequent experiments.

Fig. 7: 2D and 3D surface morphology of the contaminated film and a model sample.
figure 7

(a) 2D and (b) 3D morphology of microorganism-uncontaminated B&W photographic negative film; (c) 2D and (d) 3D morphology of model sample microorganism-contaminated B&W photographic negative film.

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Optical density of the model negative film

As shown in Table 1, optical density (OD) measurements were performed for both model high-density and low-density film samples before and after simulated microorganism contamination. After covering the film surface with lens-wiping paper, the OD of the high-density film sample decreased from 2.42 to 2.15 (n = 5), while the OD of the low-density film sample dropped from 0.15 to 0.05 (n = 5). It is worth noting that the coefficient of variation of OD was below 5% for both high- and low-density film samples covered with the lens wiping paper, indicating that the simulated contaminated model samples present good homogeneity. Additionally, the transmittance of the high-density sample decreased by 0.33%, while the low-density sample showed a significant decrease of 18.34% after being covered with the lens wiping paper. The marked reduction in visible light transmittance for the model low-density sample, compared to the high-density sample, is attributed to the absence of Ag particles in the unexposed low-density film. In contrast, the high-density film, containing densely packed Ag particles, already displayed low transmittance prior to being covered with lens-wiping paper.

Table 1 Optical density (OD) of model high- and low-density B&W photographic negative films before and after stimulated microorganism contamination
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Reflectance and transmittance of the model samples before and after treatment of stimulated microorganism contamination

As shown in Fig. 8a, the visible light reflectance of the model high-density B&W photographic negative film samples initially ranges from ~5% to 10%. After being treated with simulated microorganism contamination, this reflectance increases significantly to ~37%, indicating the formation of a rougher film surface. Additionally, the transmittance of the model high-density film decreased only by ~0.3% following this treatment. In contrast, as shown in Fig. 8b, the transmittance of the model high-density film decreased by only ~0.3% after the simulated contamination, suggesting minimal change compared to the pronounced increase in reflectance. These particles absorb most incident light, thereby restricting light penetration and minimizing the impact of surface roughness on transmittance.

Fig. 8: Light reflectance and transmittance of model high-density films before and after stimulated contamination.
figure 8

Surface visible light (a) reflectance and (b) transmittance of the model high-density B&W photographic negative film samples before (labeled as blue line) and after (labeled as red line) stimulated microorganism contamination for three randomly selected spots. Surface visible light (c) reflectance and (d) transmittance of the model low-density B&W photographic negative film samples before (labeled as blue line) and after (labeled as red line) stimulated microorganism contamination for three randomly selected spots.

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In Fig. 8c, the reflectance of model low-density B&W photographic negative film increased from approximately 8% to 35% after treatment with stimulated microorganism contamination. This change is nearly identical to the reflectance change observed in the model high-density film samples, indicating that reflectance performance is primarily influenced by the surface morphology of the films. This is due to that reflectance performance mainly depends on the surface morphology of the films. The transmittance of the model low-density film sample decreased by ~30% after the treatment of stimulated microorganism contamination. Due to the inherently low silver particle content in low-density film samples, the transmittance is much higher than that of the model high-density film samples, as shown in Fig. 9d. After the treatment of the stimulated microorganism contamination, the diffuse reflection is more pronounced, leading to a significant decrease in transmittance for the model low-density film samples.

Fig. 9: Mechanism of developing contaminated photos.
figure 9

Schematic diagram of the effect of microorganisms on the developed photos using the B&W photographic negative films: (a) the development process and mechanism of B&W photos using the uncontaminated B&W photographic negative film; (b) the development process and mechanism of B&W photos using the B&W photographic negative film with microorganism contamination.

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During the development of photos using negative films, high-density films produce whitish images when exposed to visible light10,11,12,13,14. This occurs because the high concentration of Ag particles in high-density films blocks most light from passing through, resulting in reduced exposure on the corresponding photographic paper. After the high-density film samples are treated with simulated microorganism contamination, the developed images continue to appear whitish, with small color changes before and after treatment due to the already low light transmittance. In contrast, untreated low-density films allow most of the light to penetrate during irradiation, resulting in darker-developed photos18. Following simulated microorganism contamination, the developed photos appear much lighter and whiter. This change is attributed to the rougher surface caused by the layer of stimulated microorganisms, which increases diffuse reflection and prevents more light from passing through. Consequently, the color variation between developed photos using high-density films is minimal, while the color difference between developed photos using low-density films becomes significantly more pronounced after contamination treatment.

Mechanism

When microorganisms grow in the low-density areas of a B&W photographic negative film, they consume the gelatin on the film surface, producing extensive mycelia that significantly increase the surface roughness17,18,19,20,21,22,23,24. This increased roughness results in greater diffuse reflection of visible light during the photo development process. Additionally, microorganisms absorb some of the visible light, causing a significant decrease in light transmittance. Therefore, the silver halide on the photographic paper is not adequately exposed or decomposed to form the latent image. This leads to a reduced presence of black silver particles on photographic paper and the formation of several white patches in areas that should appear black, severely impacting the integrity and clarity of the developed images.

In the high-density areas of the B&W photographic negative film, microbial growth also leads to increased surface roughness and diffuse reflection, with some absorption of visible light. However, due to the inherently low transmittance in high-density areas—caused by the high concentration of Ag particles—severe contamination does not significantly affect light transmittance. Therefore, during the development process, the effect on recorded images in high-density areas is less pronounced, and the overall impact on image quality remains minimal.

Conclusion

In this study, the effect of microbial contamination on the B&W photographic negative films and the developed photos was explored by investigation of surface roughness, reflectance, and transmittance of the films using historical film sample, model high- and low-density film samples before and after the treatment of stimulated microorganism contamination. The results indicate that the surface of B&W photographic negative film becomes uneven after contamination by microorganisms, leading to a significant increase in surface roughness and visible light reflectance. In high-density areas, where the initial visible light transmittance is low, the change in transmittance remains minimal. However, in low-density regions, the increased surface reflectance results in a significant decrease in transmittance, which largely affects the gray level of developed B&W photos. These findings demonstrate that the diffuse reflection caused by the rough surface of microorganism-contaminated negative films contributes to image blurring and a reduction in the overall quality of both the films and their developed photos. Based on these findings, it is proposed that restoring the original appearance of the recorded image could be achieved by developing methods to eliminate the rough surface and reduce the reflection and absorption of visible light on microorganism-contaminated photographic negative films. This will be an area of focus for future work.