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Modifications to clearing methods used in combination with vital staining of roots colonized with vesicular-arbuscular mycorrhizal fungi

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Abstract

Leek, maize, and pigmented soybean roots colonized by vesicular-arbuscular mycorrhizal (VAM) fungi were assessed for succinate dehydrogenase (SDH) activity using the nitro blue tetrazolium chloride (NBT)-succinate method. NBT-succinate-reacted roots, cleared in a 55° C drying oven in 5% (w/v) KOH for 24 h or longer and observed as whole mounts, revealed signs of intraradical VAM fungus colonization more clearly than roots cleared by the standard 20% (w/v) boiling chloral hydrate method. Combined clearing of NBT-succinate-reacted roots using boiling chloral hydrate followed by clearing in 5% KOH at 55° C for prolonged periods also improved the visualization of intraradical fungal structures. Bleaching of NBT-succinate-reacted roots using the standard 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 solution infiltrated all intraradical fungal structures after 24 h; formazan products were observed at similar concentrations in viable structures after 24, 36, and 48 h.

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Schaffer, G.F., Peterson, R.L. Modifications to clearing methods used in combination with vital staining of roots colonized with vesicular-arbuscular mycorrhizal fungi. Mycorrhiza 4, 29–35 (1993). https://doi.org/10.1007/BF00203248

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Key words

  • Vesicular-arbuscular mycorrhizae
  • Viability
  • Nitro blue tetrazolium chloride
  • Succinate dehydrogenase
  • Formazan