# 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  
In an ideal word, one would monitor in real time the dynamics of microbial interactions at the soil microscale, retrieving information about microbial taxonomy, distribution, and behaviour as well as about dependencies of these traits from the physicochemical properties of their micro-niches, directly in their natural microenvironment. Needless to say, this is far from the modern reality as it has proven impossible to quantify and analyse the interaction of microorganisms _in actual soil_. [PERSON]'s co-culture experiments [78] are today a standard procedure for experimentally investigating microbial interactions. With inventive approaches (e.g., [79; 80; 81; 82; 83; 84; 85]), agar-based co-culture experiments re-create simplified communities in a controlled environment and thus provide ideal conditions to test ecological concepts concerning community stability and dynamics. However, a clear disadvantage of classical solid nutrient medium is the absence of habitat heterogeneity. Artificial matrixes have been created that try to simulate as much spatial complexity in which BFI exists as possible but, at the same time, ease downstream analysis. Such soil matrices can often be sterilized, can be buried in soil within in-growth containers under natural conditions for a defined period of time, and then can be retrieved and transported to the laboratory with minimal disturbance. The applied in-growth method enables in situ cultivation of the local microbial communities [86; 87] with the assumption that their interaction strategies are retained. Different designs of in-growth containers can be distinguished, such as nylon mesh bags with a mesh size >50 \(\upmu\)m [88], or cartridge [87]. Depending on the downstream analysis to follow, they can be filled with different types of pre-fabricated natural material, such as macro-aggregates of soil fragments (5 mm-sized) [41], coarse sand particles [88; 89], or mixtures of different crushed minerals [87]. These materials offer good field-representative soil aggregates that can be used also for in vitro experiments (e.g., [90]). However, they have the disadvantage of being opaque, such as real soil. This limits the range of applications to which they can be implemented, and thus are often substituted with artificial materials, as it will be described further down below. [PERSON] and colleagues [50], and more recently [PERSON] and colleagues [91], developed the fungal highway column in order to detect bacterial dispersal by fungal hyphae in a forest soil. The fungal highway column consists of a small tube in which one medium \begin{table}
\begin{tabular}{l l l l} \hline \hline BIB-SEM & Broad ion-beam & GFP & Green fluorescent protein \\ BioLP & Biological laser printing & HISH & Halogen _in situ_ hybridization \\ CARD-FISH & Catalysed reporter deposition & IMS & Imaging mass spectrometry \\ CLSM & Confocal laser scanning microscope & MALDI-IMS & Matrix-assisted laser desorption-ionization \\ DESI-IMS & Desorption electrospray ionization & OCT & Optical coherence tomography \\ DFA & Direct fluorescent antibody & PTR-MS & Proton transfer reaction \\ EL-FISH & Elemental labelling & SECM & Scanning electrochemical microscope \\ FACS & Fluorescence-activated cell sorter & SEM & Scanning electron microscope \\ FADS & Fluorescence-activated droplet sorter & (nano)/SIMS & Secondary ion mass spectrometry \\ FIB-SEM & Focused ion-beam & SIP & Stable isotope probing \\ FISH & Fluorescence _in situ_ hybridization & SR-CT & Synchrotron computer tomography \\ FFT & Fluorescence probe techniques & TEM & Transmission electron microscope \\ GC-MS & Gas chromatography & X-ray CT & X-ray computer tomography \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Abbreviations of technique’s names discussed in the review. section is in contact with the soil, while the second one, which is physically separated from the first, can only be colonized by bacteria when they are transported into it via fungal in-growth.