# Methods for Studying Bacterial-Fungal Interactions in the Microenvironments of Soil
## 3 Get Your Hands Dirty: Methods for Studying BFI
### Playing Hide and Seek with Bacteria and Fungi: Unravel Their Physical Interactions  
In mixed culture of bacteria and fungi growing in a matrix with defined topographical and chemical features providing the microorganisms with the opportunity to spatially organize, a set of methodologies can be applied to visualize, identify, and monitor their interaction. Basic techniques for visualization of selected microbes in a matrix include non-specific stains, such as live-dead staining [148], stains with calcofluor white M2R [149, 150], or DAPI [151]. These allow to spatially visualize and quantify bacteria and fungi in close proximity with minimal effort [44], although taxonomical information is not provided. Hence, more advanced fluorescence-conjugated techniques are permanently being developed. They can be based on fluorescent probes targeting distinct short nucleotide sequences (fluorescence probe technique (FPT)), or on direct fluorescent antibodies targeting a specific protein domain (direct fluorescent antibody (DFA)) [139]. Fluorescence in situ hybridization (FISH) is a type of FPT that combines the phylogenetic identification of the taxon targeted on a single cell basis (e.g., prokaryotic cells) with the visualization of their distribution in situ (e.g., [152, 153, 154]). The technique is a pillar in molecular biology, and countless applications and specific developments exists (see for example the reviewing book by [PERSON] and colleagues just published [155]). As it will be described in the next section, FISH as well as DFA are widely used in combination with many classical or modern applications in the recognition of taxa involved in fungal and bacterial molecular interplay. When dealing with a very bright background of fluorescing soil constituents, FISH probing can be combined with tyramide signal amplification. This technique is called catalysed reporter deposition (CARD)-FISH, and is very useful for obtaining higher signal intensities and reduced background interference [156, 157, 158]. The combination of CARD-FISH with confocal laser scanning microscopes (CLSM) allows quantification, localization, and visualization of microbial cells with even more depth-resolution. Transmission and scanning electron microscopes (TEM and SEM, respectively) can also be combined with cellular dyes in order to obtain higher resolutions for both qualitative and quantitative assessment of the targeted organisms. For example, a CARD-based approach using gold nanoparticles (GOLD-FISH) can be used when dealing with very small portions of soil [159]. Fixation techniques for whole soil blocks with special chemicals can be used in order to study the distribution of microorganisms in their specific microenvironments. Blocksof soils are first impregnated with resin. Very thin layers of material can then be serially removed either by a traditional microtome, or by broad or focused ion-beam scanning electron microscopy (BIB- or FIB-SEM) [150]. Alternately, based on the same principle, thin sections of fixed soil can be cut with a diamond-tipped blade and mounted on a microscope slide. If microbes have been previously stained, the soil organisms of interest can be specifically observed [160]. By merging the different layers, three-dimensional images can then be reconstructed from a series of z-dependent two-dimension images with special software and statistical interpolation techniques [161, 160, 162]. Stains and dyes are not without limitations, however. They often unspecifically bind to soil components (including organic matter), thus increasing the background noise that, together with auto-fluorescent objects naturally occurring in the sample, make the recognition of cells in a soil matrix challenging. This is especially true for bacterial and archaeal cells in respect to fungal hyphae, whose physical arrangement and spatial distribution are relatively more straightforward to identify.