Malt extract agar

Hundred microliters (100 mL) of each stock culture were added to labelled tubes containing 5 mL of Nutrient Broth (Oxoid, UK). The bacteria cultures and C. albicans, were incubated at 32.5 ± 2.5 °C for 24 h whiles A. niger was incubated at 22.5 ± 2.5 °C for 48 h. A loopful of each tube containing the bacteria/fungi were then streaked unto sterile plates of nutrient agar and malt extract agar (Oxoid, UK), respectively. The bacteria and C. albicans inoculated plates were incubated at 32.5 ± 2.5 °C for 24 h while A. niger was incubated at 22.5 ± 2.5 °C for 48 h. Colonies of the bacteria and the yeast were introduced into tubes containing 5 mL of Nutrient Broth using a sterile inoculating loop and incubated at 32.5 ± 2.5 °C (bacteria and C. albicans) or 22.5 ± 2.5 °C (A. niger) for 3 h. The inoculums were adjusted to 10⁸ CFU/mL and 10⁸ spores/mL before use.

Egg infection model was performed as described previously [40 (link)]. Eggs were incubated in an incubator with 60% humidity for 10 days at 37°C. Each A. fumigatus strain was grown on malt extract agar (Oxoid) with 5 mg/L doxycycline for 7 days and the conidia were harvested freshly on the day for infection. Each egg was inoculated with 1000 conidia in 100 μL PBS with 5 mg/L doxycycline (corresponding to the volume of the egg) and 20 eggs were inoculated for one strain. Doxycycline was not added to the PBS solution in the Tet-somA (Off state) and the complemented strain. The embryonic death was determined by the loss of movement. Survival data were plotted by Kaplan–Meier curves and statistically analyzed by log rank test using Graph Pad Prism 5.00 (GraphPad Software).

Sample from the inky growth of the observed black spots was obtained from Khufu’s pyramid ascending passage that slopes to the grand gallery of the corridor ascending toward King Khufu’s chamber, an interior site with no or very low light at the corridor entrance (Figure 1). A crust was collected in a sterile bag for culture analysis on Dichloran Rose Bengal Chloramphenicol (DRBC) agar medium (#CM0727, Oxoid, USA) and incubated at 25°C for 2 weeks to 1 month. All individual morphotypes were transferred to Petri dishes containing malt extract agar (MEA, Oxoid, Basingstoke, United Kingdom) and incubated at 25°C to differentiate separate fungal colonies; the dominance of appearance among the morphotypes was with a black yeast morphotype, which was used for morphological and molecular analyses.

Lactobacillus plantarum UFG 121 was available at the Laboratory of Industrial Microbiology of the University of Foggia (Foggia, Italy) and routinely grown in de Man Rogosa Sharpe (MRS) broth (Oxoid, Basingstoke, UK) at 30 °C for 24 h.
Six filamentous fungi, namely Penicillium roqueforti CECT 20508, Penicillium expansum CECT 2278, Penicillium chrysogenum CECT 2669, Aspergillus niger CECT 2805, Aspergillus flavus CECT 20802, and Fusarium culmorum CECT 2148 were provided by the Spanish Type Culture Collection (CECT, Paterna, Spain). Fungal cultures were plated on malt extract agar (Oxoid) and incubated at 24 °C for 5 days.
The commercial fresh yeast Saccharomyces cerevisiae “Lievital” (Lessafre, Marcq-en-Baroeul, France) was resuspended in sterile saline solution, streaked on plates of yeast extract, peptone, dextrose (YPD, Oxoid), and incubated at 30 °C for 48 h. Then, a single colony was resuspended in YPD broth and incubated at 30 °C.

To identify the microbial strains responsible for biodeterioration, fifteen samples were collected before the spray treatment. Particularly, five cotton swabs (Boettger, Paul Boettger GmbH & Co. KG, Bodenmais, Germany) and five fungi-tapes (Scientific Device, Glenview, IL, USA) were collected on the back of the artwork (Figure 2b), while only five cotton swabs were collected on the front. All samples were picked up in correspondence with the area potentially characterised by biodeteriogens’ attack (Figure 2c) and were sent to the microbiology laboratory. Afterwards, samples were seeded on the following nutrient agar: Muller Hilton agar, Malt Extract agar, and PDA (all by Oxoid, Basingstoke, UK) and incubated for 7 days at 30 °C. At the end of the incubation time, the recognition of the fungal strains was carried out using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) Bruker Daltonics (Bruker, Bremen, Germany).

All fungal reference strains and clinical isolates were grown on malt extract agar (Oxoid, Wesel,
Germany) at 35°C for 5 days. An agar block of approximately 1 cm² in
size was transferred to a 1.5-mL reaction tube and homogenized with a micropestle. The grounded
mycelium–agar mixture was incubated at 56°C for 60 minutes with
500 μL of extraction buffer 18 (link) containing
0.2 M Tris–HCl, 0.5 M sodium chloride, 0.01 M EDTA (pH 8.0), 1%
SDS and 1 mg/mL proteinase K. After the addition of an equal volume of phenol (Roti-Phenol,
Roth, Germany), the mixture was vigorously shaken and centrifuged at
20 000 g for 3 min. The supernatant containing the DNA was
transferred to a new reaction tube and mixed with an equal volume of chloroform. After
centrifugation at 20 000 g for 3 min, 0.2 volume of 5 M
ammonium acetate and 0.7 volume of 2-propanol were added to the supernatant. The solution was
incubated at room temperature for 10 min and then centrifuged at
20 000 g for 10 min. The resulting pellet of DNA was washed
with 500 μL of 70% ethanol, centrifuged at
20 000 g for 2 min, and air-dried. The dry pellet was
re-suspended in 100 μL of Tris-EDTA (TE) buffer (pH 8.0) and stored
at 4°C until use. DNA concentration was measured with a Nanodrop 1000 spectrophotometer
(Thermo Scientific, Dreieich, Germany).