Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection

During studies on the effect of vesicular-arbuscular (VA) mycorrhiza, caused by species of Endogene, on plant growth, we needed to estimate the amount of mycorrhizal infection in the root cortex. Such infections in gramineous roots stain fairly clearly with standard procedures such as simmering in o·I % trypan blue in lactophenol (Hayman, 1970), or cotton blue and Sudan IV in lactophenol (Nicolson, 1959), and in some dicotyledonous roots by boiling for 10 min in 0'0 I % acid fuchsin in saturated chloral hydrate (Gerdemann, 1955), or standing cold overnight in any of these stains. Infection in onion, our main test plant (Hayman & Mosse, 1970), is difficult to estimate using these methods because, although external hyphae and entry points are strongly stained, internal hyphae, arbuscules and vesicles are usually only well defined near the cut ends of the root segments. To improve stain penetration and clearing in whole mycorrhizal roots of onion and other host plants, and in roots infected by other fungi, we developed...two procedures, which give deeply stained fungal structures which show distinctly against the outlines of the cells in the cortex of intact roots.