MODIFICATIONS TO CLEARING METHODS USED IN COMBINATION WITH VITAL STAINING OF ROOTS COLONIZED WITH VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI

Leek, maize, and pigmented soybean roots colonized by vesicular-arbusc ular mycorrhizal (VAM) fungi were assessed for succinate dehydrogenase (SDH) activity using the nitro blue tetrazolium chloride (NBT)-succin ate method. NBT-succinate-reacted roots, cleared in a 55-degrees-C dry ing oven in 5% (w/v) KOH for 24 h or longer and observed as whole moun ts, revealed signs of intraradical VAM fungus colonization more clearl y than roots cleared by the standard 20% (w/v) boiling chloral hydrate method. Combined clearing of NBT-succinate-reacted roots using boilin g chloral hydrate followed by clearing in 5% KOH at 55-degrees-C for p rolonged periods also improved the visualization of intraradical funga l structures. Bleaching of NBT-succinate-reacted roots using the stand ard NH3-H2O2 method removed pigmentation from roots and did not alter the viability indicator, formazan. Pigmented, field-collected soybean roots were successfully cleared and bleached to reveal signs of viable and nonviable intraradical fungal structures. Counterstaining of NBT- succinate-reacted roots with acid fuchsin clearly revealed both viable and nonviable intraradical fungal structures. The NBT-succinate solut ion infiltrated all intraradical fungal structures after 24 h; formaza n products were observed at similar concentrations in viable structure s after 24, 36, and 48 h.