# Methods for Studying Bacterial-Fungal Interactions in the Microenvironments of Soil
## 3 Get Your Hands Dirty: Methods for Studying BFI
### Reconstructing the Spatial Heterogeneity of Soil
Other sophisticated methods for in situ cultivation are iChip [92] and EcoChip2 [93]. They enable parallel cultivation and isolation of up to now uncultivated microbial species, and can elucidate the dependency of some organisms to one another, due to in situ growth factors, that cannot be simulated under standard laboratory conditions. A common concern for all these types of in situ cultivation is the flow of water. Fungal hyphae are well-stabilized in the soil matrix, but bacteria can easily be transported or washed in and out of the in-growth containers by percolating water. This might ultimately bias BFI analyses. On the other hand, it has been shown that water often follows preferential pre-existing flow lines in the soil [94]. In addition, bacteria can tightly adhere to exogenous material, form biofilms, or be safely sheltered in micro-pores [26]. Ultimately, the real extent to which water can affect the BFI networks is still unknown. A number of new manufacturing approaches have been developed to simulate the soil architecture, but with the advantages of reproducibility, accessibility, and laboratory manipulation. They are usually inoculated with defined fungal and bacteria isolates. As it was aforementioned, synthetic materials, such as clean, spherical 500-\(\upmu\)m (or bigger) glass beads [95; 96; 97; 98]; crushed silica gel particles (irregularly shaped grains) [99; 100]; polystyrene beads [101]; or ground pellets of Nafion [102; 103; 104; 105], have been developed and widely applied as artificial porous media [106] to avoid the limitations given by the opacity of natural materials. Although soil is tremendously more heterogeneous (smaller pore size) and chemically complex, their low reactivity and their optical refractive index, similar to water, allows for the combination of co-cultivation with different visualization techniques (e.g., brightfield and fluorescence imaging, low-field magnetic resonance system, and X-ray CT), as discussed in the next section. Another interesting method for simulating soil microstructure is 3-D printing. Soil-like structures of nylon 12 or resin with parafilm wax [107; 108] can be used to study the exploration and interaction strategies of different microbial inoculates, and may give future insight on BFI in these microhabitats. Some general precautions and limitations should be considered when setting up a microcosm experiment with synthetic soil manufacturing. The material chosen should be: biocompatible or at least not toxic to the organisms of interests, congruent with downstream imaging techniques, and an appropriate proxy for the environment of interest [3]. Microfluidic arrays can be deployed when more \"personalized\" structural constrains and dynamical chemical gradients are needed. The literature on this topic is extensive and more detailed information can be found in several reviews (e.g., [109; 110; 111; 112; 113]). Microfluidic platforms allow for the precisely organization and monitoring of small heterogeneous microbial populations [114; 115] in a three-dimensional geometry [116]. They can be constructed in different dimensions (in volumes as small as ~100 f [117]), materials (e.g., transparent polydimethylsiloxane (PDMS) [116; 118; 119], hydrogels, proteins crosslinked by multiphoton lithography [120], lipid-silica containers [121; 122]), or shapes (e.g., soil micromodels [123; 124], arenas [125], channels [126], mazes [127], or single droplets [128]). Some of the microfabricated biomaterials used for constructing a microfluidic system can be responsive to external stimuli, hence acting as both physical barriers, as well as an additional function for active control, manipulation, and observation of the microbes in real time. Some examples of dynamic parameters that can be tuned are pH, temperature, osmolarity, light intensity, as well as the geometry and the size of micro-chambers.