## 3 Microbiome Analysis by Culture-Independent Methods  
Knowledge of the microorganisms inhabiting cultural heritage and their involvement in the material deterioration is basic for conservators to formulate actions to preserve and protect cultural heritage. For decades, culture-dependent approaches have been applied to isolate and identity any culturable microorganisms from the microbiome inhabiting cultural heritage [60; 61; 62; 71; 72], but this approach has many shortcomings. Many naturally-occurring microorganisms that can be cultured in laboratory media are selected by the medium composition and also the culturing conditions [73], not the dominant members in the niche or in situ conditions, so the subsequent results of the  
Figure 3: The adhesive sampling device in a slightly different configuration from that shown in Figure 2 used for sampling the surface colonizers on sandstone wall of Angkor temple in Cambodia and then transferred into a sterile plastic bag ready for preservation and transportation.  
physiology and biochemistry of these microorganisms have very limited relevance or value to the whole community composition and the biochemical processed involved. In addition, microorganisms cultured under laboratory conditions experience much higher concentrations of nutrients than the ambient environmental conditions on the cultural heritage, and this critical factor has been largely ignored in practice (Figure 4). The microbiome on sandstone is dominated by halophile and microorganisms are capable of adaptation to the local conditions and also enter a physiological state, called viable but non-culturable (VNC) state under adverse condition of nutrient limitation and low temperature [74; 75]. Under natural condition, a complex microbial community is the normal state rather than the pure culture of a single species in artificial medium. The halophilic condition on sandstone selects relevant microorganisms and similarly antibiotic-resistant populations can be developed after such chemicals have been applied for a while to alter the indigenous microbiome.  
Culturing of microorganisms is a critically-important issue in environmental microbiology and it prevents researchers gaining further detailed insights into the biochemical function from members of a complex microbial community through isolation and pure culture techniques [73]. On the other hand, the development and application of DNA-based molecular methods in conservation sciences overcomes the culture-dependent limitations to reveal the microbial diversity and composition of the whole microbiome with greater depth on cultural heritage of any material type and at any time. Earlier molecular techniques used in conservation sciences include the genetic fingerprinting analyses such as DGE/temperature gradient gel electrophoresis (TGGE), amplified ribosomal DNA restriction analysis (ARDRA), single-strand conformation polymorphism (SSCP), terminal restriction fragment length polymorphism (t-RFLR), and automated ribosomal intergenic spacer analysis (ARISA) after PCR [33]. For instance, a previous study investigated the abundance and diversity of microbial communities thriving on damaged mural paintings in Portugal, by combining culture-dependent methods and culture-independent methods, such as DGE. However, it was found that the coupling of culture-dependent methods and DGE still did not yield enough information to understand the abundance and the diversity of microorganisms present on the wall paintings [76]. Because of the methodological limitation and sensitivity of these genetic fingerprinting analyses, they failed to identify different members of a particular microbial group taxonomically to a satisfactory resolution with confidence.  
Figure 4: A schematic diagram showing the general framework in microbiological analyses of cultural properties and protection.