Nutrient agar plates

Escherichia coli strain NCTC 9001 were cultured on nutrient agar plates (Lab M, UK) at 37 °C for 24 h. Using a sterile metallic loop, colonies of E. coli were aseptically transferred into 50 mL of sterile nutrient broth (Lab M, Manchester, UK) and incubated for 24 h at 37 °C. The bacterial suspension was transferred into two sterile universal bottles and centrifuged at 1721× g for 10 min. The supernatant was discarded and the cells were recovered into 10 mL sterile water. An optical density (OD_{540 nm}) of 1.0 ± 0.1 determined that the bacterial cell concentration in the suspension added to the polymeric surfaces was in the order of ca. 5.0 × 10⁸ CFU/mL. The number of cells that were removed from the surface was established by performing serial dilutions and examining the plates after incubation. Sterile horse blood (TCS Biosciences, Buckingham, UK) was used in the conditioning film assays and was diluted using sterile dH₂O to 10% (v/v). Blood and cell samples were mixed in a 1:1 ratio before application to the surfaces, ensuring that the same number of bacteria was applied to each polymer sample.

Five historical paper documents were offered for analysis by the Hellenic General State Archives (Athens, Greece). Four out of five documents dated back to 19th century (1840–1843), while the fifth one dated back to 20th century (1919). The samples were macroscopically examined and selected for further analysis, based on macroscopic patterns of biodeterioration such as discoloration, permanent staining, structural damage, and musty odor (Figure 1). Two sampling strategies were employed. In the first one, samples were collected from documents (surface area circa 30 cm²) demonstrating macroscopic biodeterioration patterns using sterile cotton swabs, whereas in the second one, sterile scalpels were used to remove small fragments (circa 0.5 cm²) directly from a heavily biodegraded area of documents. Both cotton swabs and paper fragments were kept at 4°C, in sterile vials till use. Cotton swabs were then used to inoculate Malt Yeast Extract agar plates (Lab M, UK) containing streptomycin (500 μg/ml) (Serva, Germany) for fungal or Nutrient Agar plates (Lab M, UK) for bacterial isolation. Agar plates were incubated at 30°C up to 7 days or up to 3 days, respectively. All fungal and bacterial isolates were kept at −80°C as glycerol stocks.