JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 3:377-378 (1986)

PROCESSING SMALL BIOLOGICAL SPECIMENS FOR TRANSMISSION ELECTRON MICROSCOPY

Henry Hong and John R. Barta Department of Zoology, University o/ Toronto, Toronto, Ontario. Canada M5S IAl

Processing small biological samples for electron microscopy is often a problem because the specimens are either too small to handle individually or the sample will not form a discrete pellet when concentrated by centrifugation. The technique outlined here is a simple procedure for processing small specimens without loss, using readily available and inexpensive materials. Specimens are treated in suspension until the pellet is formed just prior to embedding, thus ensuring optimal fiation, dehydration and infiltration.

Specimens were sequentially fixed in buffered glutaraldehyde and osmium tetroxide, dehydrated through ascending grades of ethanol and infiltrated with Spurr's (1969) low viscosity epoxy resin in 1.5 ml polypropylene micro test tubes. The tubes were centrifuged between each treatment in order to concentrate the specimens and to allow the supernatant to be drawn off. Addition of the next reagent resuspended the specimens. Approximately nine centimeters of polyethylene (catheter) tubing (inner diam. = 0.058 cm, outer diam. = 0.097 cm) was attached to the tip of a 21 gauge needle, fitted onto a 1 cc syringe. After centrifuging the specimen suspension in the micro test tube, the sample was drawn into the polyethylene tubing filling one third of its length followed by an equal volume of air using the syringe to control suction (Fig. I). The dipped end of the tubing was wiped to remove resin coating the exterior. The tubing was detached from the needle (Fig. 2a) and the undipped end was introduced into a snug fitting micro-hematocrit capillary tube (length = 75 mm, internal diam. = 1.2 mm, wall thickness = 0.20 mm). The opposite end of the glass capillary tube was flamed briefly but not long enough for the glass to soften (Fig. 2b). The polyethylene tubing was then pushed further into the capillary tube until contact was made with the heated portion, causing the polyethylene to melt and form a cylindrical plug with a smooth surface for particles to be centrifuged against (Fig. 2c). The undipped end was inserted into the capillary tube because it had not made contact with resin which may interfere with heat sealing of the tube. The polyethylene tubing was rotated to free the plug from the glass, and inserted so that the plug was flush with the glass tip. The tubing in the capillary was spun in a micro-capillary centrifuge to pellet the sample, and transferred to a 70'C oven for eight hours of polymerization with the tubes held vertically (Fig. 3). After the samples cooled, the tubing was removed from the capillary and the polyethylene tube was split with a razor blade. The extracted rod of epoxy resin containing pelleted specimens was reembedded in a BEEM@ capsule or flat embedding mold (Fig. 4), trimmed and sectioned in the usual manner.

This technique has been used to process small numbers of nematode larvae (newborns of Trichinellu spiralis), the protozoan Babesiosoma stableri (parasitizing frog erythrocytes), and the free- living ciliate Colpidium colpoda.

Acknowledgments

Specimens were kindly donated by John Barta, Nancy Rawlinson and Floyd M. Musculus. Equipment and facilities provided by Drs. Ken Wright and Sherwin Desser were appreciated. Special thanks to Dr. Ken Wright for reviewing the manuscript.

6' BEEM is a registered trademark of Better Equipment for Electron Microscopy Inc

Received December 2,1985; accepted February 6,1986.

0 1986 ALAN R. LISS, INC.

378 H. HONG AND J.R. BARTA

Figure Legend

Figure 1 . Collection of sample from resin. Figure 2. Preparation of sample for centrifugation. Figure 3. Polymerization of sample. Figure 4. Reembedding of sample.

Reference

Spurr, A.K. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastr. Res. 26: 31