# Methods for Studying Bacterial-Fungal Interactions in the Microenvironments of Soil
## 3 Get Your Hands Dirty: Methods for Studying BFI
### What Are They Doing? Investigating the Molecular Interactions in BFI
Sample preparation is also a fundamental step. Samples should be mounted on a conductive surface and be extremely flat, although a notable exception was given by [PERSON] et al. [189]. Although experiments with these new modern techniques are on the rise, stable isotopes feed to a biological sample is still the most widespread technique to follow metabolic activities and interactions of bacterial-fungal communities in their natural environments. The literature available on stable isotope probing (SIP) is extensive and detailed descriptions can be found elsewhere (e.g., [141; 190; 191]). An incredible advantage of isotope labelling is that the substrate of interest can be directly fed in any artificial set-up chosen to simulate the (micro)-habitats of the organisms of interest (e.g., nature, microcosms, artificial matrixes). For example, different \({}^{13}\)C-marked carbon sources were supplied in microcosms with soil material to monitor respiration rates and C utilization by bacteria and fungi [192]. Using airtight containers, [PERSON] et al. [193] discovered that the nitrogen enriched material in the fungus garden of soil leaf-cutter ants was not derived by their extensive foraging activity, but was instead fixed by N\({}_{2}\)-fixing bacteria hosted by the fungus itself. Another neat example that shows the intricate behaviour of fungi and bacteria is the study of [PERSON] et al. [53] where, by tracing \({}^{13}\)C-substrate, the researchers found out that the ascomycete fungus _Morchella cassipes_ first farms _Pseudomonas putida_ bacteria and then use them as carbon source. The precision and sensitivity of the instruments for the downstream analysis will eventually limit the experimental design. In fact, a common problematic intrinsic to all labelling experiments is the insufficient incorporation rate for detection. This can be avoided by long incorporation time, but in turn it increases the risk of cross-feeding i.e., contaminations [139]. Independently from the isotopically labelled substrates applied (\({}^{13}\)C, \({}^{15}\)N, \({}^{32}\)P, \({}^{2}\)H) and the technology used to detect the isotope incorporation (e.g., mass spectrometer, isotope ratio mass spectrometer, Raman microspectrometry, SIMS) [190], the monitoring of uptake and transfer of metabolites has to always be linked to the identification of the involved microorganisms. This can be achieved by a variety of different methods applied after isotope incorporation. Soil microorganisms can be discriminated based on taxa-specific biomolecules by phosphor lipid fatty acid analysis (PFLA); sterol analysis (e.g., using ergosterol as a biomarker for fungi) DNA-, RNA-, or protein-based methods; by FISH-microautoradiography [194,195]; or isotopic rRNA-arrays [196]. Molecular approaches have also been used for tracing back a specific taxon with labelled DNA, including PCR amplification as well as sequencing [197, 198, 199]. It is also possible to directly clone isotope-labelled DNA and then sequence it [200]. An exciting alternative to these classical taxa-discriminating approaches combines taxa-specific target probes (e.g., FISH, CARD-FISH, DFA) and stable isotope probing (SIP) with the simultaneous detection of atoms produced by nanoSIMS. If based on creative experimental designs, the combination of these techniques can substantially help for linking identity to functions by tracking the uptake and transfer of isotopically labelled compounds in environmental samples where the individual taxa cannot be isolated from one another. A satisfactory overview of these combination methods is given by [PERSON] et al. [201]. Briefly, a microbial community that was grown with metabolic-labelled tracers (e.g., \({}^{13}\)C or \({}^{15}\)N) is fixed, hybridized with phylogenetic probes, and visualized by fluorescence microscopy to obtain a picture of the distribution of specific taxa in space. Then, the sample is analysed by nanoSIMS [155] which maps the existing molecules in the sample by detecting and identifying them from their specific locations.