Paraffin embedding allows easy preparation of a series of sections, making it suitable to monitor the development of hyphae and fungal-related structures in infected plant material. This method generally requires the removal of paraffin before the sectioned material can be stained with dyes in aqueous or alcoholic solutions. This process takes longer and consumes a large quantity of solvent. In the present study, we tested several dyes for differential or uniform staining of sections of paraffin-embedded barley leaves infected with Rhynchosporium secalis (Oudem.) J.J. Davis without removal of paraffin.

Differential staining with periodic acid-Schiff (PAS) reagent and fast green resulted in a red to purple color for the vascular tissues, epidermis and cell walls, and green for the mesophyll cells, whereas hyphae and spores of R. secalis were stained red to purple. The optimum contrast was achieved by overstaining in decolorized basic fuchsin, and then by counterstaining in fast green, with the length of staining time being determined by monitoring the color density. Although plant tissues, hyphae and spores were uniformly stained with crystal violet, with toluidine blue O or aniline blue, there was little difficulty in identifying subcuticular hyphae and spores, as these structures are usually on or near the surface of host tissues. Among dyes, toluidine blue O and crystal violet appear to be better than aniline blue in terms of the short period of staining required to obtain optimum colour density. When fungal structures were in the mesophyll as a result of heavy infection, PAS reagent and fast green were superior in differentiating hyphae and spores from host tissues.

Staining of embedded sections before paraffin removal permits simple processing of material and facilitates examination of fungal infection in plant tissues. In addition to saving time and solvents, the risk of tissue damage is reduced. Successful staining of sections from infected barley tissue suggests that staining before paraffin removal may be useful for study of other host-pathogen systems.