scholarly journals A new drying method of biological specimens for scanning electron microscopy: The t-butyl alcohol freeze-drying method.

1988 ◽  
Vol 51 (1) ◽  
pp. 53-59 ◽  
Author(s):  
Hitoshi OSATAKE ◽  
Takao INOUÉ
Author(s):  
Linda M. Sicko ◽  
Thomas E. Jensen

The use of critical point drying is rapidly becoming a popular method of preparing biological samples for scanning electron microscopy. The procedure is rapid, and produces consistent results with a variety of samples. The preservation of surface details is much greater than that of air drying, and the procedure is less complicated than that of freeze drying. This paper will present results comparing conventional air-drying of plant specimens to critical point drying, both of fixed and unfixed material. The preservation of delicate structures which are easily damaged in processing and the use of filter paper as a vehicle for drying will be discussed.


Author(s):  
P. A. Madden ◽  
W. R. Anderson

The intestinal roundworm of swine is pinkish in color and about the diameter of a lead pencil. Adult worms, taken from parasitized swine, frequently were observed with macroscopic lesions on their cuticule. Those possessing such lesions were rinsed in distilled water, and cylindrical segments of the affected areas were removed. Some of the segments were fixed in buffered formalin before freeze-drying; others were freeze-dried immediately. Initially, specimens were quenched in liquid freon followed by immersion in liquid nitrogen. They were then placed in ampuoles in a freezer at −45C and sublimated by vacuum until dry. After the specimens appeared dry, the freezer was allowed to come to room temperature slowly while the vacuum was maintained. The dried specimens were attached to metal pegs with conductive silver paint and placed in a vacuum evaporator on a rotating tilting stage. They were then coated by evaporating an alloy of 20% palladium and 80% gold to a thickness of approximately 300 A°. The specimens were examined by secondary electron emmission in a scanning electron microscope.


Author(s):  
Arthur L. Cohen ◽  
Gerald E. Garner

The surface forms and structures of animal cells have been strikingly preserved for scanning electron microscopy by freeze-drying and by critical point drying both by the method with CO2 used as the transitional fluid and the later procedure which uses a fluorocarbon (Freon 13) as a medium for the transition from the liquid to the gaseous environment. Freeze-drying is often prolonged (5-12 hours as compared with an hour or less by the critical point method) and in our experience with mold cultures on agar, the substrate shrivels and cracks and hyphal filaments are distorted.Despite, and possibly because of a flexible but inelastic cell wall, plant cells often show greater distortion than do animal cells after evaporative drying or replacement dehydration for mixrotechnical work. The animal cell membrane can contract more or less uniformly on drying - as shown by the numerous micrographs of well-preserved erythrocytes, while plant cell walls often crumple. The many scanning electron micrographs of partially collapsed pollen grains bear witness to this fact.


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