Acidified Blue Ink-staining Procedure for the Observation of Fungal Structures Inside Roots of Two Disparate Plant Lineages.

Identifying microscopic mycorrhizal fungal structures in roots, i.e., hyphae, vesicles and arbuscules, requires root staining procedures that are often time consuming and involves chemicals known to present health risks from exposure. By modifying established protocols, our root staining method stains roots using a safe ink- and vinegar-based staining solution, followed by a 2-16 h-long de-staining period. The entire procedure can be completed in less than 6 h (plus up to 16 h de-staining overnight) and roots are suitable for semi-permanent and permanent slide mounting for light microscopy. We tested our method on hundreds of wild-sourced roots from two different plant species: Lycopodiella inundata, a herbaceous clubmoss with tough water-resistant roots, and Sambucus nigra, a temperate woody shrub. Both plants associate with endomycorrhizae, L. inundata predominantly with Mucoromycotina fine root endophytes (MucFRE) and S. nigra with Glomeromycota arbuscular mycorrhizal fungi (AMF). Here we describe a simple, efficient, repeatable and safe method to detect the presence of fungal structures using light microscopy.