Mainly specimens of Strobilurus and Mycena species were used to test the effects of diverse implementations of the rehydration method to prepare fungal specimens for scanning electron microscopy (SEM). Strobilurus spp. have a hymeniderm consisting of pear-shaped, thin-walled cells and dermatocystidia which are thin-walled and fusiform. Pleuro- and cheilocystidia with species specific morphology are found on the hymenophores (Rexer and Kost 1989a, 1989b, Qin et al. 2018). Most species of Mycena have an epicutis of thin radiating hyphae with wart- or fingerlike projections (diverticulate hyphae). Hyphae of the same type were also found on the surface of the stipe, often terminating in characteristic cells. On the hymenophore, cheilocystidia are always present but pleurocystida are only found in some species (Rexer 1994, Aronsen and Læssøe 2016).
In the Strobilurus specimen prepared without AOT treatment (control), the cells of the hymeniderm were deformed, with sunken apexes and lateral walls seen as rings as seen by SEM (Fig. 1a). In a specimen of S. esculentus treated with 1 % AOT without ultrasound and without vacuum treatment, single cells are slightly inflated (Fig. 1b), but the overall result is similar to the result of the control (Fig. 1a). An increase of the AOT concentration to 3 % led to a minor improvement, as seen for the hymenium of S. esculentus (Fig. 1c). Although the solubility of AOT decreases strongly with concentrations larger than 6%, solutions with up to 10 % AOT were tested. With 10 % AOT, the diverticulate hyphae of the epicutis of Mycena meliigena and their terminal cells were insufficiently rehydrated and showed wrinkled surfaces (Fig. 1f). Cconcentrations of AOT between 3 % and 6 % (Fig. 1c, Fig. 1d) led to better shapes of the cells as compared to the control, but the results were not satisfactory. Exposure times of up to 60 hours in AOT were tested, but the quality of the result did not increase compared to 24 hours of soaking.
Therefore, we applied ultrasound and vacuum treatments to the specimens in AOT solutions. This procedure significantly enhanced the rehydration effect of AOT. Fragments of specimens of S. esculentus were put in 3.5 % aqueous solution of AOT and were first treated for 1 min with ultrasound and then incubated for 24 hours in a vacuum desiccator. As a result, the more or less capitate, thin-walled apices of the pleurocystidia of S. esculentus were not sunken (as seen in Fig. 1c) but re-inflated as observed in fresh material (Fig. 2c). The amorphous or crystalline substances which are located at the tips of pleurocystidia of Strobilurus spp. were mostly washed away during the preparation so that the surface of the cell wall was visible (Fig. 2e).
The application of ultrasound for three minutes to material of M. meliigena caused artefacts. Hyphae of the stipitipellis and their terminal cells showed holes of variable sizes (Fig. 1e). Such holes were never seen in specimens treated with ultrasound for only one minute (Fig. 2d). Treatments with 3 % AOT (Fig. 1h) or 5 % AOT (Fig. 1i) resulted in minor differences. However, hyphae deeper in the tissue were better rehydrated with 3 % AOT.
The effects of the optimum treatment parameters resulting from these observations were tested on further species as illustrated in Fig. 2. Strobilurus stephanocystis presented perfectly convex cells of the pileipellis (Fig. 2a) and characteristic pleurocystidia (Fig. 2b). In this case, the amorphic exudate at the tips of the cystidia was not washed off during preparation. In M. rosella as well as S. tenacellus, thin-walled and fusiform pleurocystidia are formed. In both species, most pleurocystidia were inflated as seen by SEM. However, in each specimen a few collapsed cells were obvious (Fig. 2e, f).
The basidiospores of species in various genera like Entoloma, Mycena, Russula and Strobilurus, presented their typical shapes (Fig. 1g-i, 2b, 2g-i). E. conferendum has cruciform-stellate basidiospores which were almost completely rehydrated (Fig. 2g). The hexagonal to octagonal basidiospores with rounded angles of E. undatum (Noordeloos 1987), however, showed slightly wrinkled surfaces (Fig. 2h). The broadly ellipsoid basidiospores of R. ochroleuca (Romagnesi 1996) were completely inflated and their warts and delicate line connections were perfectly visible (Fig. 2i).
These results were compared to structures observed on directly fixed fresh material. Immediately after being collected, fresh material of Mycena cf. abramsii (Rexer 1994, Aronsen and Læssøe 2016) was fixed in glutaraldehyde. The hyphae of the epicutis were densely covered with warts or short outgrowths and slightly embedded in a matrix. These hyphae were well preserved (Fig. 3a), but the matrix masked the morphology of the hyphae to some extent. The cells of the hymenium, however, were less well preserved. Many basidiospores and all the basidia were shrunken or collapsed, only the characteristic cheilocystidia were well preserved (Fig. 3b). These results were similar to results obtained from rehydrated specimens treated with AOT only (Fig. 1g-i).