Cryo-Scanning Electron Microscopy of Mushroom Tissue. A. BOEKESTEIN, K. HULSTEIJN,. L. HESEN,* L.J.L.D. VAN GRIENSVEN*. Technical and Physical ...
SCANNING Vol. 15, 171-173 (1993) OFAMS, Inc.
Received January 19, 1993
Applications
Cryo-Scanning Electron Microscopy of Mushroom Tissue A. BOEKESTEIN, K. HULSTEIJN, L. HESEN,*L.J.L.D. VANGRIENSVEN*
Technical and Physical Engineering Research Service (TFDL-DLO), Wageningen. *Mushroom Experimental Station, Horst, The Netherlands
Key words: cryo-SEM, mushroom tissue, cryofixation, hyphae The study of the ultrastructure of biologic material with scanning electron microscopy (SEM) provides information on fundamental tissue characteristics. This technique is especially important, in the study of bacterial and fungal infections of the white button mushroom, Agaricus bisporiis, and in postharvest changes of fruit bodies. Using SEM and transmission electron microscopy (TEM), Wood et al. (1985) described the development of the primordium of A. bisporus to the mature fruit body. Jasinski et al. ( 1984) used different electron microscopy techniques to show the intracellular damage of mushroom tissue caused by heat treatment. Atkey and Nichols (1983) used cryoSEM to demonstrate the ultrastructural damage effected during handling of mushrooms. In many cases chemical fixation followed by critical point drying has been used for SEM specimen preparation. These treatments may cause ultrastructural alterations and considerable shrinkage in fresh tissue (Bastacky et al. 1985, Falk 1980). The extent of some artefacts in electron microscopic images can only be estimated in comparison with other preparation techniques. Application of cryomethods in electron microscopy to study frozen biologic ultrastructure is an appro-
Address for reprints: Abraham Boekestein RIKILT-DLO P.O.B. 230 NL-6700 AE Wageningen The Netherlands
priate alternative to chemical fixation. However, one has to be careful not to introduce new cryospecific artefacts (Read et al. 1983, Steinbrecht and Zierold 1987). The cryo-SEM technique has been used for fungi before (AllanWojtas and Yang 1987, Beckett and Porter 1982). Also, for the localization of small amounts of water or for the observation of slime layers with a high water content, this method has proved to be very useful (Pearce and Beckett 1985, Van Doorn et al. 1990). The cryoapproach is based on physical fixation of biologic tissues by contact with a cryogenic medium, such as nitrogen slush. If carried out properly, water is frozen so fast that ice crystals will be very small or invisible in the most superficial layer of the specimen of interest. Hence the ultrastructure will not be damaged significantly (Robards and Sleytr 1985). This holds true even if no cryoprotectants are added and makes this approach suitable for study of native undisturbed biologic structures. To assess the effect on an ultrastructural level, we have compared fresh white, button-mushroom tissue prepared by the cryoapproach with chemically fixed mushroom tissue. Mushrooms of the species Agaricus bisporus (Lange) Imbach strain Horst@UI were grown at 22°C and 85% R.H. on commercial compost at the Mushroom Experimental Station at Horst. Mature mushrooms were harvested 2 h prior to processing for electron microscopy. Two different preparation methods were carried out in two replicates. Pieces of tissue (6-8 mm) were dissected with a fresh razor blade. Samples were taken from both the cap surface and its inner tissue. Pieces of tissue were prepared according to the methods described by Falk (1980) and Jasinski et al. (1984). Tissues were fixed for 2 h at room temperature. Specimens were mounted on specimen stubs with Leit-C paste (conductive carbon cement) and sputtered with gold. Pieces of mushroom tissue of the same samples were also mounted on EMSCOPE specimen holders with Tissue-Tek and Leitsilber-200 and cryofixed in nitrogen slush at approximately 60 K in the EMSCOPE SP2000A cryosystem.
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Special care was taken to prevent ice formation by using a cryotransfer device with a moderate vacuum. The device was used to transfer the specimens to the electron microscope. Scanning electron microscopy was performed using the JEOL 35C microscope, equipped with a cold stage to keep the frozen specimens at a temperature of 110 K. For cryoSEM a cryotrap (part of the EMSCOPE system) was used to prevent or diminish contamination of the specimen surface. The accelerating voltage used was 15 kV. In Figures I and 2. the effects of the preparation methods on the observed ultrastructure are compared. Hyphae of the cryofixed specimens are seen to be smoother than the chemically fixed specimens. The hyphae in the cryofixed specimens are more turgid. and the chemically fixed specimens look somewhat crumpled. Hyphal dimensions have not changed appreciably compared with cryofixed specimens taken from the mushroom cap. In chemically
fixed specimens, interhyphal spacings look somewhat smaller. Comparing cap-surface and inner-cap material, we found the hyphae to be significantly thicker in the inner cap, which is in agreement with observations of Craig et al. ( 1979), who analyzed hyphal dimensions in the stipe. In inner-cap material. hyphae could be as thick as approximately 20 pn whereas at the surface of the cap, hyphae were mostly about 5 pn thick. In cryofixed material, frozen pure water in interhyphal spacings can be distinguished from frozen cytoplasm, with its solutes leaked out of damaged hyphae by the appearance of dried solute material. This material is incompletely preserved by chemical fixation. We conclude from our study that cryo-SEM preserves water in mushroom tissue and that there is less collapse and distortion of hyphae than with conventional SEM techniques that involve chemical fixation.
FIG.1 Scanning electron micrograph of a mushroom-cap surface. Bar = 10 pm. ( a ) chemically fixed: (b) cryofixed
Fic,. 2 Scanning electron micrographs of inner-cap material. Bar = 1 0 pn. ( a ) chemically fixed: (b) cryofixed.
A. Boekestein et nl.: Cryo-SEM of mushroom tissue
References Allan-Wojtas P, Yang AF: Solutions to difficulties encountered in low-temperature SEM of some frozen hydrated specimens: Examination of Penicilliunz nulgiownse cultures. J Electron Microsc T e c h 6, 325-333 (1987) Atkey PT. Nichols R: Surface structure of Agaricus bisporus by scanning electron microscopy. Mushroom J 127. 334-335 (1983) Bastacky J, Hayes TL, Gelinas R: Quantification of shrinkage during preparation for scanning electron microscopy: Human lung. Scanning 7, 134-140 (1985) Beckett A, Porter R: Urornvces viciue-fnbae on Eciafuba: Scanning electron microscopy of frozen-hydrated material. Protoplusma I 11,28-37 (1982) Craig GD. Newsam RJ. Gull K. Wood DA: An ultrastructural and autoradiographic study of stipe elongation in Agaricus bisporus. Protoplnsina 98, 15-29 (1979) Falk RH: Preparation of plant tissues of SEM. Scarinirzg Electron Microscopy 11.79-87 ( 1980)
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