9-1-98

0.1mm or less in size and very delicate. Fixation and dehydration are not

a problem. The problems come in the final drying and mounting for

viewing.

I have tried drying by gradually replacing the 100% ETOH with Freon

113. However, the crystals float making them very hard to keep track

of....also they tend to attach to the walls of the small tube and dry there. I

need to get them onto something that can then be put into the SEM.

I normally will have only 2-3 of these crystals so cannot afford to

have any lost in the process. They are very hard to see so I hesitate

trying to put them onto filter paper...I am afraid they will get lost in the

fibers.

I would appreciate any suggestions for ways to handle these guys.

Debby Sherman, Manager Phone: 765-494-6666

Microscopy Center in Agriculture FAX: 765-494-5896

Dept. of Botany & Plant Pathology E-mail: sherman@btny.purdue.edu

Purdue University

1057 Whistler Building

West Lafayette, IN 47907-1057

methods often are well preserved by a solvent drying

method:

Line a glass Petrie dish with a double-layer of filter

paper.

Saturate the paper with chloroform (somebody ought to try

how other solvents perform).

Place a microscope slide onto the filter paper to keep the

specimen off the paper.

Sit the dehydrated, wet (absolute ethanol) specimen, which

may be on a coverslip, a piece of mica etc onto the slide.

Cover the Petrie dish and refrigerate for two days.

Don't open Petrie dish until it has warmed to at least room

temperature (incubator?)

Proceed with sputter coating . . .

The method relies on very slow removal of the solvent

saturated atmosphere within the dish and this lengthy time

allows most of the remaining, trapped water molecules to

leave the specimen without high pressures that distorts and

shrinks specimens.

I have used this method with microscopic nematodes; its not

always perfect but for these critters its about the best

means available. Certainly, its easy and avoids losing

specimens.

Cheers

Jim Darley

ProSciTech Microscopy

PLUS

PO Box 111, Thuringowa QLD 4817 Australia

Phone +61 7 4774 0370 Fax: +61 7 4789 2313

Great microscopy catalogue, 500 Links, MSDS, User Notes

**************************** www.proscitech.com.au

*****

are very smooth and I have used them successfully to entrap samples for

SEM. There are are a variety of pore sizes available. I know they are

available from SPI Supplies--probably other suppliers carry them also.

Larry D. Ackerman

Lily & Yuh Nung Jan Laboratories

Howard Hughes Medical Institute

UCSF, Box 0725, Rm U226

533 Parnassus Ave.

San Francisco, CA 94143

(415) 476-8751 FAX (415) 476-5774

mishot@itsa.ucsf.edu

How about cryo-SEM technique? The virus crystals in suspension are

absorbed on substrata, fast frozen, partial freeze-dried, cryo-coated with

a thin layer of metal, then viewed in a cryo-SEM. I have used this

protocol for many years to look at individual viral particle by our

high-resolution cryo-SEM.

Ya Chen

Ya Chen

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