Journal of Microscop?, Vol. 130, Pt 1, Apr i l 1983, pp. 79-84 Keceie,ed 17 M a r c h 1982; accepted 12 September 1982

A routine flat embedding method for electron microscopy of microorganisms allowing selection and precisely orientated sectioning of single cells by light microscopy

b y OLIVIER L. R E Y M O N D and J E R E M Y D. PICKETT-HEAPS*, Laboratoire de Microbiologie Ginhale, Dipartment de Biologie Vige'tale, Universite' de GenBve, C H - 121 1 GenBve 4, Switzerland, and *Department of Molecular Cellular and Developmental Biology, Biosciences Building, Univer- sip of Colorado, Boulder, Colorado 80309, U.S.A.

ICE Y w O R D S . Flat embedding, scarce microorganisms, cultivated microorganisms, selection, orientation, light microscopy, electron microscopy, perforated block-holder.

S U M M A R Y A simple method is described to embed material in resin, in the form of microscope slides,

to observe it with high resolution light microscopy, to select, orient and section it for TEM. This method can be applied to many kinds of material but is particularly useful for the study of rare or tiny plant or animal microorganisms from field or culture. A diamond scriber, translucent hydrosoluble resin release agent, translucent and smooth resin stubs and a longitudinally perforated block-holder for ultramicrotome are the specific tools of this method.

I N T R O D U C T I O N An embedment of biological material that permits high resolution examination and selection

of individual cells by light microscopy, prior to their being sectioned, is very useful to many biologists. In achieving this end, various problems have to be overcome due to the small size, shape, and orientation of the individual cells in many samples.

Flat embedding methods have been previously described by Bloom (1960), Robins & Gonatas (1964), Aziz & Davies (1968), Chang & Tanaka (1970), Chang (1971), Richters & Valentin (1973), Moore (1975), Sigee (1976), Schibler & Pickett-Heaps (1980) and Duchateau et al. (1980). Special problems, however, are encountered in the selection of very rare and tiny algae, in nanoplankton for example, and unfortunately among the previously published methods some could only be used partially, while the others could not be adapted to our requirements.

The resulting procedure described here is simple and suitable for handling spherical, filamentous or flat material such as bacteria, protozoa, fungi, algae and moss from field or culture.

M E T H O D S List of technical material required

1. Standard microscope slides (26 x 76 mm) with one frosted end. 2. Liquid, translucent, hydrosoluble resin release agent.* 3. Diamond scriber, to fit in the objective turret of the microscope.t 4. Translucent, quick-setting glue, for example cyanoacrylate-ester glue which is very

* Release agent for flat embedding method: Electron Microscopy Sciences (in U.S.A.), Box 251, Ft. Washington, PA 19034; Elmis International (in Europe), Holzgasse 8A, CH-8942 Oberrieden, Switzerland.

3- Diamond scriber: Leitz Wetzlar GmbH, Art. Nr. 2513442, D-6330 Wetzlar, West Germany. 1983 The Royal Microscopical Society

79 6

80 Olivier L. Reymond andJeremy D. Pickett-Heaps

reliable when allowed to set overnight. Research workers who routinely and repeatedly use cyanoacrylics should be warned that these compounds are highly toxic. *

5. Cylindrical resin stubs, with both sides parallel and perpendicular to the main axis. Recommended size, about 15 mm long and 7 mm wide. For example, a modified BEEM capsule resin stub.

6. Standard block-holder for ultramicrotome (for cylindrical stubs).? 7. Usual technical material for microscopy and ultramicrotomy.

Preparation of the technical material 1. Preparation of microscope slides, used as flat moulds for stubs and embedding of material.

Microscope slides are dipped vertically into the liquid release agent to the level of the frosted end. They are then withdrawn and dried vertically for 2 h at room temperature. They are put into a slide box or a staining dish and dried overnight in an oven at 313-343 K. Incomplete drying may prevent subsequent polymerization of the liquid resin.

/‘ -p T W

B

B

Fig. 1. A drop of liquid resin is placed on the flat rough surface (A) of a resin cylindrical stub. The stub is turned upside down (left arrow) on to a microscope slide covered with release agent (read: preparation of technical material, para. 1). After hardening the stub is twisted apart from the slide. The ‘A’ side is now perfectly smooth and must be carefully washed with water to remove the release agent. Smoothing of ‘B’ side is not necessary.

In the following step, the resin square containing the circle area with the biological material on the upper side (read procedure para. 7 and 8) is held with fine tweezers (TW) and carefully washed with water to remove all traces of release agent. I t is then glued on to the smooth side (A) of the stub.

2. Preparation qf stubs (Fig. 1, para. 1). To allow illumination from underneath and precise observation of the embedded and mounted material with the light microscope, the stubs de- scribed in the list of material must possess one very smooth surface. To achieve this smooth surface, follow the directions given in the legend to Fig. 1, para. 1.

3. Preparation of the block-holder (Fig. 4). The block-holder must be perforated longitudin- ally to allow illumination of the pyramid from below during observation with the light micro- scope. The block-holder may also be adapted to fit under the light microscope, but this can usually be achieved without damaging its rigidity.

* Cyanoacrylate-ester glue may be obtained in drugstores or hardware shops under different trade names.

t Block-Holder for Reichert’s ultramicrotomes are convenient and can be easily modified. C . Reichefl. Hernalser Hauptstrasse 219, A-1 170 Wien, Austria.

Flat embedding 81

procedure 1. The biological material, already fured, dehydrated and embedded in liquid resin (Spurr’s

or Epon, for example) is placed on the surface of one microscope slide previously covered with the release agent. A second identical slide is lowered so that its frosted end is opposite the frosted end of the first. The slides must overlap, but not fully cover each other. Since the frosted ends are not covered with release agent, they must not be in contact with resin (Fig. 2).

Fig. 2. Flat embedding. Biological material embedded in lquid resin (BM+R) is placed on a microscope slide as described in ‘Preparation of technical material, para. 1’. A similarly prepared slide is inverted over the first (arrows). This sandwich is then placed in an oven for polymerization. Frosted ends must stay uncovered and free of resin.

If the biological material is thick or cannot stand mechanical pressure, spacers can be used to maintain the slides at the appropriate distance apart.

2. The resin is then polymerized as normal. During polymerization, the two slides must be kept horizontal to prevent any gliding movement of the upper slide.

3. When the resin is hard, excess resin is trimmed from around the slides with a razor blade. Then the slides are separated by grasping their free frosted ends and twisting them slightly.

Incomplete polymerization or poor trimming of the excess resin can hinder easy separation. 4. The layer of resin which remains on the surface of one of the two slides is a thin, optically

homogeneous wafer. The external surface of the resin must be carefully washed with distilled water to remove any remaining release agent since the latter remains on the resin and not on the glass (use only a very soft rag).

5. The slide with its resin wafer can be observed by light microscopy, and the material selected for sectioning can be photographed at this stage (Fig. 5). The selected material is then marked by a circle inscribed in the plastic by a diamond scriber (Figs. 3 and 6) .

When oil-immersion observations are needed, the cells in the resin wafer can be first covered by a drop of water and then by a coverslip. Direct contact of immersion oil and resin can some- times damage the preparation.

6. With a sharp scalpel blade, a square of about l o x 10 mm is cut from the resin wafer around the inscribed circle marking the site of the cell to be sectioned (Fig. 3).

Smaller squares are not recommended because the mounting glue (see para. 9) may flow over the edges, making them more difficult to observe.

7. The square is removed from the slide using fine tweezers and then carefully washed with distilled water to remove the release agent remaining on the bottom side.

8. The square piece of wafer, placed on a slide, is now examined with the light microscope, and the distance between the selected cell and the two faces of the resin is measured employing the micrometric focusing screw. This operation, although dispensable, can save much time during sectioning, since it allows semi-thick sections to be taken prior to attaining the level of the cell in the resin.

82 Olivier L. Reymond andJeremy D. Pickett-Heaps

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Fig. 3. The wafer of resin containing the cells IS examined wlth light and selected materlal IS clrcled wlth an adjustable diamond scriber (DS). A square (about 10 x 10 mm) I S cut with a sharp scalpel or a knlfe (K) around each of the circles (C).

Fig. 4. View of a section through objective (0), pyramid (P), resin stub (S), block-holder (BH) for ultramicrotome, microscope slide (MS), stage (St) and condenser (Co). The block-holder's central hole permits illumination from below and observation of the biological material. This examination is part'cu- lady useful when the selected cell is very small, virtually invisible with stereo microscope and causes certain problems during pyramid trimming and sectioning.

Flat embedding 83

9. The square piece of wafer is now glued to the smooth side of the prepared stub using translucent glue (Fig. 1, para. 2). The upper side is that side to which the cell is closer, as determined in para. 8.

10. The stub is transfared to the modified block-holder (Fig. 4). 11. The face of the wafer is trimmed to a pyramid, which is then sectioned as usual. During

these operations, if the embedded cell is poorly visible under the stereo microscope, accurate control over the trimming can be exercised by frequent examination of the preparation in the modified block-holder, with a light microscope at high magnification (Figs. 4 and 7).

Further control of specimen orientation may be achieved by modifying the mounting procedure. For example, long cells will settle during polymerization of the resin so that they are predominantly parallel to the surface of the slide. If one needs to section such a cell from one end, a selected cell is then mounted on a stub whose smooth face is slanted or vertical. In this way, accurate control over the plane of sectioning can be achieved ( e g Pickett-Heaps et al., 1978: dia. 1).

Fig. 5. Bacillus polymixa with endogenous spore, flat embedded in a resin wafer. Dark-field microscopy. Fig. 6. The position of the same bacterium (see Fig. 5) (arrow) is marked by a circle (C) made with a dia- mond scriber. Phase-contrast microscopy. Fig. 7. The same bacterium (see Fig. 5) (arrow) still visible at the top of the trimmed pyramid. Bright-field microscopy. Fig. 8. One of the sections of the same bacterium (see Fig. 5) observed with transmission electron micro- scope, showing the endogenous spore.

D I S C U S S I O N This basic method has been successfully applied to diverse material such as bacteria (Figs.

5-8), unicellular or filamentous microalgae, spores or mycelia or fungi, protozoa, moss proto-

84 Olivier L. Reymond andJeremy D. Pickett-Heaps

nemata and leaves, and we feel confident that it can be directly applied to many other biological materials.

This method has certain advantages over other embedding techniques. The preserved specimen can be examined at, or near, the highest resolution of the light microscope, and only a few cells from each sample are used.

The fixed samples are stored on microscope slides, and easily filed in a slide box for future reference. The resin stubs can also be used repeatedly after resmoothing their surface (Fig. 1).

The use of a translucent, non-granular release agent allows handling of both large specimens (10 pm or more), and also, by using phase-contrast optics, very small material (2-5 pm).

In addition, this release agent is inexpensive, has a very low toxicity and can be completely removed from the resin plate.

Teflon (Chang, 1971; Schibler & Pickett-Heaps, 1980) has also been used as a release agent. Release of the wafer from the slide is sometimes difficult and particles of Teflon which remain between the wafer and glass can cause optical problems when the cell is very small and high magnification is required. Teflon, however, is also a very good release agent for these flat embedding methods if used properly.

The flat embedding method of tiny specimens also raises the question of bringing them to the point of embedment. The reply depends upon the material itself which should generally be fixed and dehydrated so that the internal and external morphology are not modified. For example, phytoplankton could be maintained in centrifugation tubes while mycelium needs to be placed.carefully in a shallow dish.

ACKNOWLEDGMENTS We would like to thank Dr J. Fahrni, Dr R. Peck, Dr S. Kantengwa and Dr J. Naef of the

University of Geneva, Dr A. Gautier of the Centre de Microscopie tlectronique de 1’Universite de Lausanne, Dr U.-P. Roos of the University of Zurich, Dr H. R. Preisig of the Culture Centre for Algae and Protozoa at Cambridge, and Dr B. Hickel of the Max-Planck-Institut fur Lim- nologie at Plon, for reading the manuscript, their advice and providing different material to experiment with.

J.D.P.-H. gratefully acknowledges the support of the Systematics Biology Section of the National Science Foundation (grant No. DEB 79-22200).

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Chang, S.-T. & Tanaka, K. (1970) Culturing and embedding of filamentous fungi for electron micrography

Duchateau, A., Zeitoun, P., Escaig, F., Leclerc, A. & Guillerm, J.-F. (1980) Separation of epoxy-embedded

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