Low-temperature scanning electron microscopy of biological specimens

Notes and brief articles acids (G ent ile, 1954; Kamoen, 1969), and it is possible that these can pass through the membrane pores and attack the gelatine matrix of the emulsion. Materials from fungal spores are known to solubilize copper prior to its uptake by the spore (Cochrane, 1958). The factor responsible for reduction of the cations to metallic silver does not pass through the membrane and is active only at the surface of the germ-tubes. As might be expected, it is produced only by the germ-tube, and not by the relati vely inert conidial wall. Further investigations showed that these spurious silver grains did not develop if the development time was reduced from 3 min to 2 min . This is a commonly used method of avoiding background grains in autoradiographs, and is based on the fact that the speck of silver (the latent image) from which a visible silver grain is produced during development is usually smaller in the case of a background latent image than with a latent image produced by a (i-particle from the radioactive specimen (Rogers, 1979). Hence a background latent image requires a longer development time to become large enough to be visible than does a latent image produced by a (i-particle. Thus the specks of metallic silver around the fungal germ-tube would only need to be very small , provided that they were surrounded by further silver bromide to permit build-up of the visible silver grain during development. A tendency to accumulate heav y metals is well known in fungi; indeed, the uptake of copper is the reason for the toxicity of surface-acting fungicides of the Bordeaux type. Spores of some fungi have been shown to accumulate silver from fine suspensions of AgBr and AgCl , and from AgNO a

solution (M iller, McCallan & Weed, 1953 ; Miller & McCallan, 1957). Brown & Smith (1976) observed silver granules in the walls of Cryptococcus exposed to AgNO a solution. Silver is generally the most fungitoxic of the metallic cations (Cochrane, 1958) although its cost has generally precluded its use as a fungicide. This work was supported by a grant from the N.E.R.C ., which enabled M.C.E. to be employed as a Post Doctoral Research Fellow. REFERENCES

BROWN, T . A. & SMITH, D. G. (1976). The effects of silver n itrate on the growth and ultrastructure of the yeast Cryptococcus albidus. Microbios Letters 3, 155-162. COCHRANE, V. W. (1958). Physiology of Fungi. New York: Wiley . EDWARDS, M. C . -& BLAKEMAN, J. P. (1983). An autoradiographic method for determining nutrient competition between leaf epiphytes and plant pathogens. Journal of Microscopy (in press.) GENTILE, A. C. (1954). Carbohydrate metabolism and oxalic acid synthesis by Botrytis cinerea. Plant Phys iology 29, 257-261. KAMoEN, O. (1969). Acid production in plants upon attack by Botrytis cinerea. Mededelingen R ijksfaculteit Landbouunuetenschappen, Gent 34, 944--948. MILLER, L. P. & McCALLAN, S. E. A. (1957). Toxic action of metal ions to fungus spores. Journal of Agricultural and Food Chemistry S, 116-122. MILLER, L. P ., MCCALLAN, S. E. A. & WEED, R. M. (1953). Rate of uptake and toxic dose on a spore weight basis of various fungicides . Contribut ionsfrom the Boyc e Thompson Institute 17, 173-195. ROGERS, A. W . (1979). Techniquesof Autoradiography, 3rd ed . Amsterdam : Else vier /North-Holland Biomedical Press.

LOW-TEMPERATURE SCANNING ELECTRON MICROSCOPY OF BIOLOGICAL SPECIMENS BY D. JONES*, W. J. MCHARDY AND J. M. TAIT

Departments of Microbiology* and Mineral Soils, The Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen, AB9 2QJ, Scotland A technique is described for the examination of fungi in the scanning electron microscope using frozen-hydrated specimens. The procedure eliminates the shrinkage observed in freeze-dried and critical-point dried fungi. Although the advantages of being able to examine frozen biological material in the SEM have been recognized for some time (Echlin, Paden, Dronzek & Wayte, 1970; Echlin & Moreton, 1973) it is only recently that most of the technical difficulties have been overcome (Robards & Crosby, 1979) and commercial systems, compatible with most scanning electron microscope s, have become readily Tran s. Br. my col. Soc. 82 ( 1) (1984).

available. This note presents some preliminary observations made with one such system (EM technology - Hexland Ltd) attached to a Cambridge Instruments S4 stereoscan. Where possible, a comparison is made between cryo-fixed specimens (frozen-hydrated) and others that have been freeze-and critical point-dried (Jones, 1978). With the Hexland apparatus a sample can be

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Notes and brief articles

Figs 1-4. Penicillium oxalicum, Fig.

Trans . Br. mycol. Soc. 82 (1) (1984).

1.

Critical point-dried. Figs 2-4. Frozen-hydrated.

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166

R

Figs 5-8. Phragmidium mucronatum teliospores on leaf of cultivated rose. Fig. 5. Freeze-dried. Figs 6-8. Frozen-hydrated.

Trans. Br. mycol. Soc. 82 (1) (1984).

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167

Figs 9-14. Schizosaccharomycespombe. Figs 9-10. Freeze-dried. Figs 11-12. Critical point-dried. Figs 13-14. Frozen-hydrated.

Trans. Br. mycol. Soc. 8z (1) (1984).

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Fig s 15-17. Schizosaccharomyces pombe. Frozen-hydrated.

rapidly frozen in nitrogen slush at -210°C and transferred under vacuum, thus minimizing frosting, to a pre-chamber stage cooled with liquid nitrogen and with facilities for specimen fracture and coating. From this pre-chamber stage the specimen may be transferred to the cold stage in the microscope itself. Both stages have in-built heaters and the temperature of either is controllable down to about -180°. An independently cooled decontaminator is sited just above the specimen. Exact procedures vary according to the type of specimen being studied but a sequence of steps we have found useful is as follows. The frozen specimen is examined uncoated at a low accelerating Trans. Br . mycol. Soc. 8z (1) (1984).

voltage (to kV) while warming from -150° to - 80°, by which latter temperature any superficial ice evaporates (to condense on the anticontaminator). The microscope stage is then recooled to - 150° before the specimen is transferred to the pre-chamber where it is sputter coated with gold j finally it is returned to the microscope stage, still at -150°, and photographed. Sporulating cultures of Penicillium oxalicum Currie & Thorn, IMI 112755, a rhizosphere fungus, were cultured on Oxoid Czapek-Dox agar from which blocks were cut, approximately 1 x 2 mm across and 2 mm depth, for freezing. Considerable linear shrinkage of the spores and

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Notes and brief articles

Figs 18-19. Pinus contorra var. contorca. Cross-fractured frozen-hydrated leaf. Trans. Br. mycol. Soc. 82 (1) (1984).

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17°

phialides occurred in critical point-dried material (Fig. 1) when compared to the cryo-fixed specimens (Figs 2-4). These observations are in keeping with those of Beckett, Porter & Read (1981) who used another commercially available apparatus - the EMscope Sputter Cryo system. Shrinkage was not evident when spores of Phragmidium mucronatum (Pers.) Schlecht. on rose leaves were examined in the freeze-dried (Fig. 5), critical point-dried (not shown) and frozen-hydrated state (Figs 6-8) and this is likely to be due to the very much thicker cell walls of this fungus. Features of the spore surfaces are more clearly defined in the cryo-fixed specimens. Considerable distortion of cells occurred on freeze-drying cells of Schizosaccharomyces pombe Lindner, IMI 39117, grown on Oxoid malt extract agar (Figs 9-10). Less distortion but a general wrinkling of the cell surface occurred in cells which were critical point-dried (Figs 11-12). The division scars are very pronounced in these specimens. The overall dimensions of frozen-hydrated cells (Figs 13-14) are generally greater than those of freezedried and critical point-dried specimens and the surfaces are much smoother, although some variation in morphology occurred in specimens from different cultures. Some cracking of the surface was occasionally encountered (Figs 15-16) and the width of the division scars was different. Sometimes particles of material occurred on the surface of cells which could be ice or, more likely, cell exudates (Figs 15-17).

A cross-fractured frozen-hydrated pine leaf (Pinus contorta var. contorta) (Figs 18-19) revealed many features of internal structure in a very clear way. The technique could be of value in studying plant tissues invaded by fungi. Cryo-fixation avoids artifacts caused by chemical fixatives and dehydration and also enables tissues to be probed for elemental analysis. Other specimens including insects, protozoa and bacteria have been successfully cryo-fixed using the Hexland apparatus in our laboratory. REFERENCES

BECKETT, A., PORTER, R. & READ, N. D. (1981). Low temperature scanning electron microscopy of fungal material. Journal of Microscopy us, 193-199. JONES, D. (1978). Scanning electron microscopy of Spongospora subterranea. Transactions of the British Mycological Society 70, 292-293. ECHLIN, P. & MORETON, R. (1973). The preparation, coating and examination of frozen biological materials in the SEM. In Scanning Electron Microscopy, pp. 325-332. Chicago: lIT Research Institute. ECHLIN, P., PADEN, R., DRONZEK, B, & WAYTE, R. (1970). Scanning electron microscopy of labile biological material maintained under controlled conditions. In Scanning Electron Microscopy, pp. 49-56. Chicago: lIT Research Institute. ROBARDS, A. W. & CROSBY, P. (1979). A comprehensive freezing, fracturing and coating system for low temperature scanning electron microscopy. In Scanning Electron Microscopy, vol. 2, pp. 325-344. SEM Inc., AMF O'Hare, Ill., U.S.A.

CERATOBASIDIUM CEREALE SP.NOV., THE TELEOMORPH OF RHIZOCTONIA CEREALIS BY D. I. L. MURRAY

C.S.I.R.O. Division of Forest Research, P.O. Box 4008, Canberra, A.C.T. 2600, Australia AND L. L. BURPEE

Department of Environmental Biology, University of Guelph, Guelph, Ontario NIG 2WI, Canada The anamorphic stage of two isolates of Corticium gramineum was identified as belonging in Ceratobasidium hyphal anastomosis group CAG 1 and was found to fit the species concept of Rhizoetonia cerealis, the cause of sharp eyespot of cereals. One isolate of Co. gramineum and a CAG 1 tester strain of R. cerealis were induced to form basidia on agar. The teleomorph is described as a new species, Ceratobasidium cereale. Co. gramineum is regarded as a nomen dubium. Within the form genus Rhizoetonia DC., many fungi possessing predominantly binucleate somatic hyphal cells have been characterised, in the absence of a sexual stage, as belonging to one of seven hyphal anastomosis groups designated CAG 1-7 (Burpee, Sanders, Cole & Sherwood, 1980). Trans. Br. my col. Soc. 82 (1) (1984).

Although Ceratobasidium cornigerum (Bourd.) Rogers has been identified as the teleomorph of certain isolates in CAG2, which includes the important plant pathogens R. fragariae Hussain & McKeen and R. endophytica Saksena & Vaartaja (Burpee et al., 1980), a basidial stage has yet to be

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