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

Abstract

(Eddy) The procedure has been developed for use on roots. Procedure for staining : materials can be fixed in FAA (13 ml formalin, , 5 ml glacial acetic acid and 200 ml 50 % ethanol). Then materials are heated in 10 % KOH for about 1 hour at 90 c. This removes the host cytoplasm and most of the nuclei. Materials were then rinsed in water and acidified with dilute HCL. Staining was done by simmering for 5 min. in 0.05 % trypan blue in lactophenol and the excess staining is removed by clear lactophenol. For roots which are thicker than 2 mm a longer period (more than one hour) of treatment with KOH at 90 c was recommended. Because of the KOH treatment the staining of intracellular fungal tissue would be easier and no cytoplasm would block the visibility of the fungus. For pigmented root materials the treatment with KOH was extended, after which the materials were washed with fresh KOH. Then the materials were immersed in an alkaline solution of hydrogen peroxide (about 10 at 20 c until bleached.