# Black Fungi on Stone-Built Heritage: Current Knowledge and Future Outlook
## 4 Multistep Analyses to Study Black Fungi from Stone Monuments
### Methods of Isolation and Characterization  
The sampling is critical. As reported in the previous paragraph, due to the value of the artifacts, non-invasive sampling methods have been developed over the years and are now widely used. Examples are a needle to take samples from black spots or cavities, scalpel or lancet to scrape fungi from the surface, and adhesive tape sample, useful both  
Figure 5: Microscopic observation of the endolithic behaviour of meristematic black fungi. (**a**) Settled on a thin section of Carrara marble sacrophagus flake showing the meristematic chains deepening inside the marble, magnification 400X; (**b**) SEM image showing MCF into the marble with dissolution pattern following the same shape of meristematic cells.  
for microscopy and cultural and molecular analysis as it provides a mirror image of the stone colonization [92; 93; 22].  
Microscopic examination of the samples is the most common practice to obtain evidence of black fungi directly from stone samples. In fact, due to their size, morphology, and pigmentation, black fungi can be directly visualized under optical microscope without a specific preparation. Even with a good light microscope (LM) or, better, with a scanning electron microscope (SEM), detailed information is obtained. Microscopy is useful for the direct visualization of the fungi in the stone sample, for determining what types of relationship they establish with stone material and with other types of microorganisms, and also for addressing the next step of analyses. Unfortunately, fluorescence microscopy (FM) cannot help in detecting and studying black fungi due to the presence of melanin that masks the fluorochrome fluorescence.  
Still, cultural analyses remain the best way to study this group of fungi.  
Black fungi, and in particular MCF, possess a poor ability to compete with fast growing fungi, being characterized by a slow growth rate, and very often require more than 1 month of incubation before visible colonies are seen. These reasons explain why they are difficult to isolate and maintain in culture [94]. Nevertheless, selective cultural media that inhibit the growth of bacteria and of fast-growing fungi are successfully employed both for qualitative and quantitative cultural analyses [22; 95].  
These culture techniques have the advantage of allowing the whole characterization of the isolates by microscopical, biochemical, physiological, and molecular analysis; these latter are indispensable for the identification of MCF that do not have recognizable morphological traits. Multilocus sequencing typing (mlst) is routinely carried out to resolve their taxonomic and phylogenetic position [55; 56] by Blast search homology (Basic Local Alignment Search Tools) available online. However, many nucleotide sequences in the Genkank nucleotide database are not updated in the \"definition\". Therefore, although this approach is within the reach of all laboratories, it requires a deep knowledge of the literature and a curated nucleotide database.  
A deeper genetic characterization of the isolates can be obtained by whole genome analyses but, to date, only four genomes belonging to three species of black fungi (_Conisoparium apollinis_ CBS 100218, _Knufa petricola_ MA5789, _K. petricola_ MA5790, and _Aeminium ludgeri_ DSM 106916) isolated from stone monuments are available in the NCBI database ([[https://www.ncbi.nlm.nih.gov/genome/](https://www.ncbi.nlm.nih.gov/genome/)]([https://www.ncbi.nlm.nih.gov/genome/](https://www.ncbi.nlm.nih.gov/genome/)) accessed on 15 February 2022).  
Cultural techniques also allow us to investigate the biodeteriorative abilities of the isolated species by setting up laboratory experiments that simulate their settlement, colonization, and biodeterioration pattern [96; 97].