# Contributions of DNA Sequencing Technologies to the Integrative Monitoring of Karstic Caves
## 2 Cultivation Methods, the First Assessments of Cave Microbial Diversity  
Whatever the ecosystem, a microbial diversity assessment was historically based on classical microbial ecology techniques such as laboratory cultivation, often coupled with microscopy (Table 1). The cultivation of microorganisms appears a simple and cheap method to isolate microbial members from diverse ecosystems. However, the success of microbial cultivation is highly dependent on many factors, such as the ability to reproduce ideal microbial life conditions [16; 17]. The difficulty also lies in avoiding nutritional shock or inadequate substrate concentrations that might accelerate cellular death and/or inhibit the growth of microorganisms [17]. Thus, as cultivation conditions do not always match in situ conditions, many microorganisms will not be cultured, and the microbial diversity documented might not reflect the in situ diversity, implying biases in many studies. Indeed, microbiologists estimate that at the most 1 to 10% of microorganisms can be cultured, and comparisons between cultivation and molecular methods showed that only a minority of bacterial phyla identified through direct PCR amplification of 16S rRNA genes had yielded culturable representatives [18]. Most of the time, the isolation of cave microorganisms has been performed in a two-step screening process [19]. The first step consists of an incubation period often using a nutrient-rich medium (i.e., CN agar or CN gellan gum [20]) that allows the isolation of strains presenting different morphological characteristics. Because of the oligotrophic status of caves, this procedure can be adapted by using culture media with low nutrient amounts (i.e., Tryptic Soy Agar and Reasoner's 2A Agar (R2A) [20]) or diluted media. Using cultivation on seven different media, i.e., Glycerol-Asparagine Agar (GAA), Peptone Yeast Extract-Brain Heart Infusion (PY-BHI) agar, 1000-fold-diluted Tryptic Soy Agar (TSA), Starch-Casein Agar (SCA), Malt Yeast extract Agar (MYA), Soil Extract Agar (SEA), and Tap Water Agar (TWA), all supplemented with the fungal inhibitor nystatin, [PERSON] et al. (2014) identified microorganisms from the genera _Lysobacter_, _Pseudomonas_, _Spingomonas_, _Bosea_, etc., in the Pajsarjeva jama Cave in Slovenia [21]. While all seven growth media allowed microbial isolation, microorganisms were most abundant on low-nutrient TWA. By using low-nutrient medium 0.1\(\times\) R2A instead of high-nutrient media CN agar or CN gellan gum, [PERSON] and [PERSON] (2019) showed that a gold mine located in South Dakota hosted irregular thin and iridescent biofilms, similar to silver biofilms described in limestone caves [22; 23]. They evidenced the dominance of _Alphaproteobacteria_ (67% and 42% of isolates on high- and low-nutrient media, respectively), _Actinobacteria_ (24% and 47%), and, to a lesser extent, _Bacillus_ (4% and 2%). _Betaproteobacteria_ were only present on high-nutrient media (5% of isolates) and _Gammaproteobacteria_ on low-nutrient media (9% of isolates). Other studies used Potato Dextrose Agar (PDA) to promote sporulation and pigment production of fungi often retrieved from caves (e.g., _Alternaria alternata_, _Cladosporium cladosporioides_, _Aspergillus niger_, etc.) [8; 24; 25]. Previous work identified yeast-like fungi in Harmanecka Cave (Slovakia) through cultivation on Sabouraud agar or Czapek-Dox medium, and the density of airborne fungi was 810 \(\pm\) 124 CFU per m\({}^{3}\) air outside vs. 129 \(\pm\) 17 CFU per m\({}^{3}\) of air inside the cave [25]. These examples show the importance of the media chosen to isolate microorganisms. In addition, overgrowth of fast-growing microorganisms (r-strategists) and short incubation times limit the recovery of ecologically valuable slow-growing microorganisms (K-strategists) [17]. Previous work showed that cave _Cyanobacteria_, usually slow growers in these ecosystems, require from