Touching the (almost) untouchable: a minimally invasive workflow for microbiological and biomolecular analyses of cultural heritage objects

Abstract

Microbiological and biomolecular approaches to cultural heritage research have expanded the established research horizon from the prevalent focus on the cultural objects' conservation and human health protection to the relatively recent applications to provenance inquiry and assessment of environmental impacts in a global context of a changing climate. Standard microbiology and molecular biology methods developed for other materials, specimens, and contexts could, in principle, be applied to cultural heritage research. However, given certain characteristics common to several heritage objects-such as uniqueness, fragility, high value, and restricted access, tailored approaches are required. In addition, samples of heritage objects may yield low microbial biomass, rendering them highly susceptible to cross-contamination. Therefore, dedicated methodology addressing these limitations and operational hurdles is needed. Here, we review the main experimental challenges and propose a standardized workflow to study the microbiome of cultural heritage objects, illustrated by the exploration of bacterial taxa. The methodology was developed targeting the challenging side of the spectrum of cultural heritage objects, such as the delicate written record, while retaining flexibility to adapt and/or upscale it to heritage artifacts of a more robust constitution or larger dimensions. We hope this tailored review and workflow will facilitate the interdisciplinary inquiry and interactions among the cultural heritage research community.

Keywords: 16S rRNA gene; archaeological artifacts; biocodicology; cultural heritage; medieval manuscripts; microbial signatures; microbiome; minimally invasive.

Figure 1

Schematic representation of an interdisciplinary framework to studying the microbiome of cultural heritage objects. Photo credit: UBL Ms 11 and Ms 12, courtesy of Manuscript Center and University Library Leipzig, Germany.

Figure 2

(A) Representative experimental setup for sampling a written cultural heritage object (Ms 12, a parchment codex) on-site (at University Library Leipzig, UBL) under aseptic conditions, using the swabbing method. (B) Scanning electron micrograph showing a swab used for sampling the parchment codex (UBL, Ms 12). The arrow points to a possible endospore structure nested in the fibers of the swab head, surrounded by unidentified debris from the sampled object. Photo credit: (A) C. Flocco, Leibniz Institute DSMZ, in accordance with UBL. (B) C. Flocco, Leibniz Institute DSMZ and Manfred Rohde, Helmholtz Center for Infection Research (HZI).

Figure 3

Comparison of the performance of the sampling methods on solid cultivation medium. (A) Agar plate with the nitrocellulose membrane used for sampling, showing continuous growth along the micro-dent in the solid cultivation medium formed around the membrane border. (B) Agar plate showing well-defined and spaced microbial colonies obtained by direct spread plating with the swab used for sampling. Both samples were taken from comparative areas of a parchment codex (UBL, Ms12) and cultivated on trypticase soy yeast medium at room temperature (~ 22°C). Photo credit: C. Flocco, Leibniz Institute DSMZ.

Figure 4

Comparison of the sampling performance of different types of swabs, with double-stranded DNA (dsDNA) concentration as endpoint. One folium of a parchment codex (Ms12, 91r) was sampled in parallel with a set of different swabs, distinguishing between text and marginal areas with signs of use (each 100 cm² surface), and subjected to nucleic acids extraction (QIAmp^® DNA Micro Kit, Qiagen) and quantification (Quant-iT™ PicoGreen^® dsDNA Kit, Invitrogen). For comparison purposes, all samples were taken from the same folium to avoid fluctuations bound to a potential different usage of the codex's folia. Given the folium size constraints, one sample per experimental situation or control was taken, for which two technical replicates were generated for nucleic acids quantification. The combination of cotton swab and margin area was not produced due to the size limitation of the object. The controls consisted of swabs of each type without sample, which were extracted as the ones with sample, and also of extraction reactions without swab or sample, as control for extraction kit reagents. Abbreviations: material of the swab head (C) cotton, (V) viscose, and (F) flocked nylon.

Figure 5

Distribution of microbial isolates retrieved from a parchment codex (Ms 12) used for the pilot study, phylum and genus level (16s rRNA gene-based classification, standard Sanger method).

Figure 6

Quantification, integrity, and sizing of the nucleic acids extracts obtained through the methodology optimization process. Selected representative samples were analyzed with the ultrasensitive Femto Pulse System (Agilent). (A) Schematic representation of the experimental design for the optimization of the methodology for nucleic acids extraction. (B) DNA extract profile obtained with the QIAmp^® DNA Micro Kit (Qiagen), by direct extraction of the swab head and collection of one aliquot of nucleic acids extract. (C) DNA extract profile obtained DNeasy PowerBiofilm kit (Qiagen) by direct extraction of the swab head and collection of one aliquot of nucleic acids extract.

Figure 7

Schematic representation of the main modules of a standardized workflow, from heritage object to data, for microbiological and biomolecular approaches to cultural heritage research.