a negative staining—carbon film technique for studying viruses in the electron microscope

printed in Sweden CAOvvright © 1974 by Academic Press, Inc.

ii rights of reproduction in any form reserved

j , LILTRASTRUCTURE RESEARCH 47, 361-383 (1974) 361

A Negative Staining-Carbon Film Technique for ~tudying Viruses in the Electron Microscope

I. Preparative Procedures for Examining Iccsahedral and Filamentous Viruses

R. W. HORNE and IVONNE PASQUALI RONCHETTI 1

John Innes Institute, Colney Lane, Norwich NOR 7OF, England

Received November 14, 1973

In several recent publications reference has been made to the possible causes for the limited amount of information seen in electron micrographs of biological material when compared to the potential resolution of the electron microscope. It has been concluded that a large number of biological objects rarely show detail below about 2.5 nm unless they have been prepared or examined by special processes (9, 17, 20). The reasons for the relatively poor level of structural information observed can be attributed to irradiation damage by the electron beam, total specimen thickness, specimen preservation and contrast. In addition, phase contrast effects in the image coupled with a large level of random background noise are known to influence the visible detail recorded from any electron microscope image (5~ , 14, 16, 17).

During a series of studies into certain aspects of the above problems we developed a technique that has allowed viruses and other small biological particulate materials to be prepared from very highly concentrated or crystalline suspensions for examination in the electron microscope and subsequent analysis by optical diffraction (3, 12). The negative stain-carbon technique described below has also provided a means for obtaining a very thin total specimen thickness with some gain in resolution. It has also pointed to additional possible causes of background granulation associated with certain viruses and other biological preparations which are considered to affect seriously the direct visualisation of the small detail in biological particles. It should be stressed here that the viral structures and dimensions together with their relation- ship to the crystalline patterns resulting from these experiments and subsequently analysed by optical diffraction methods will be the subject of a series of separate publications.

z Permanent address: Institute of General Pathology, Via Campi, Modena, Italy.

362 HORNE AND PASQUALI RONCHETTI

V / ] / Z / / / / [ / / / / / / / / ] / / / ] ] / ] / / / / ~ I

< 1 FIG. 1. Diagram showing the shape of mica piece cut before cleaving with a razor blade.

METHODS

Virus material. Highly concentrated and purified suspensions of brome mosaic virus (BMV) containing 7-18 mg/ml virus, pH 5.6. Cowpea chlorotic mosaic virus (CCMV) containing 4-8 mg/ml virus (1). Potato virus X and apple chlorotic leafspot virus (CLSV) were also used in the experiments and prepared according to the methods of Bar-Joseph et al. (2). Preparations of human adenovirus type 5 were kindly prepared and supplied by Dr W. C. Russell of the National Institute for Medical Research, Mill Hill, London. The final concentration of adenovirus was 0.6 mg/ml at pH 7.0.

Negative stains, Various negative stains were used in our studies to establish the optimum conditions for obtaining good contrast and preservation. The stains included 3-10% ana- monium molybdate and were used in a series of concentrations and pH values within the range of 5.2 7.5, 2% sodium tungstate pH 5.6, 2% phosphotungstic acid pH 4 and pH 6.5 by KOH, 2 % potassium silicotungstate pH 6.5, 1% uranyl acetate pH 4.0, 1% uranyl formate pH 3.7, and 1% aluminum uranyl acetate pH 6.5.

Mica substrates. Freshly cleft mica sheets were cut to the approximate shape and size illustrated in Fig. 1 for the purposes of initially spreading the negative stain and virus suspension. The pointed end of the mica sheet was found to assist in the initial release of the final specimens at the liquid/air interface.

Carbon evaporation. Very thin films were produced from a standard carbon arc source of the type originally described by Bradley (4). The carbon arc intensity and duration was controlled by a variable autotransformer inserted into the primary circuit of the arc trans- former supply. Carbon was deposited in a vacuum of about 2 × l0 -6 Torr using a liquid nitrogen-trapped mercury high vacuum pump which was backed by a liquid nitrogen- trapped mechanical pumping line.

Control carbon .films. Carbon films for determining background granulation levels were made directly on to the mica pieces as described above. In addition, samples of mica surfaces containing dried negative stains only were coated with carbon and released from their substrates in the same way as for the mixtures of virus and stain preparations described below.

Specimen support films. The final virus and carbon film specimens were supported on carbon-coated plastic holey films mounted on standard 490-mesh electron microscope copper grids (10, 15).

Electron microscopy. Electron microscopy was carried out on a JEM 100B instrument operating at 80 kV fitted with a standard specimen stage. The contamination rate at the specimen was measured to be 0.1 nm/minute. The microscope magnification was accurately

NEGATIVE STAINING--CARBON FILM TECHNIQUE

~/f/J/f///fJ/Jf/f/f J J ~

363

F1G. 2. The mixture of the first negative stain and virus suspension is spread on the freshly cleft mica surfaces with the aid of a fine pipette.

calibrated using catalase crystals according to the method of Wrigley (21). The micrographs were recorded at × 33 000 and x 66 000.

Optical diffraction. Analysis of the electron micrographs was carried out on an experimental optical diffractometer of the type described by Beeston et al. (3). The diffractometer camera length was calibrated using micrographs of catalase crystals and optical diffraction gratings. Selected area optical diffraction was achieved by carefully marking the appropriate areas on the micrographs and illuminating these areas through a simple adjustable diaphragm stop.

Air-dried preparation of negatively stained virus and carbon films. Suspensions containing virus material (BMV, CCMV, adenovirus, PVX, and CLSV were mixed with an equal volume of one of the negative stains at a pH value adjusted according to the type of virus suspension and mixed mechanically with the aid of a Whirlimixer (Fison Scientific Apparatus Ltd.) for about 10 seconds. The final concentration of the virus in the negative stain was about 0.4-8 mg/ml depending on the concentration of the virus in the original suspensions. A small volume of the negative stain and virus mixture (0.2 ml),was spread carefully on the surface of a precut and freshly cleaved mica sheet as shown in Fig. 2. The excess liquid was then carefully removed with a pointed filter paper leaving a thin liquid film at the mica surface and then allowed to dry at room temperature. After drying, the mica sheets were placed in the evaporator and coated with a very thin layer of carbon (Figs. 3 and 4). It was found necessary to mark gently the carbon films by placing four black spots from a soft felt pen at suitable positions in order to locate the films at a later stage, because the films deposited at the correct thickness were normally invisible to the naked eye (Fig. 3). If the films were easily visible after being released from the mica, they were considered to be too thick for high resolution studies.

The technique of floating the films onto a liquid surface used to separate the films and virus material from the mica surface was essentially the same as for standard carbon films (4), but the normal distilled water trough was replaced by a small petri dish containing a second negative stain (Fig. 5). The second negative stains used included ammonium molybdate, uranyl acetate, uranyl formate, and sodium tungstate. Grids covered with holey films

24-- 741839 Y. Ultrastructure Research


~1111111f/If111 )'I 11111111,1

Fic. 3. Carbon is evaporated on to the air-dried stain and virus. Following removal from the vacuum chamber the carbon surface is carefully marked with four black spots from a soft marking pen.

were placed underneath the floating specimens and raised with a pair of forceps to collect the virus and carbon films.

Freeze-drying procedure. Specimens were frozen-dried under carefully controlled conditions in a freeze-fracture apparatus designed by Mr N. King of the Food Research Institute, Norwich (to be published). The wet specimens were spread according to the methods described above and placed in the apparatus, immediately frozen at -180°C and rapidly pumped to a vacuum of 10-" Torr. Once the vacuum had been established, the temperature of the specimen was raised in controlled stages to -32°C during continuous pumping. The specimens were then coated in the freeze-fracture apparatus with a thin carbon layer as described above and removed from the vacuum chamber. Separation of the frozen-dried specimens from the mica was achieved by the same procedure applied to the air-dried samples.

RESULTS

Brome mosaic virus and cowpea chlorotic mosaic virus

The virus suspensions when mixed with 3 % a m m o n i u m molybdate (pH 5.2) as

the initial negative stain and floated on to the surface of 1% uranyl acetate acting

\D \c F~. 4. Schematic drawing of the mica piece (D), negative stained virus (B and C), and thin carbon layer (A), prior to being floated on to the surface of the second negative stain.

NEGATIVE STAINING-CARBON FILM TECHNIQUE 365

FIG. 5. The mica piece is slowly immersed into the dish containing the second negative stain and the specimen film areas are identified by the four black spots.

as the second stain, gave the best and most consistent results for BMV and CCMV under the experimental conditions we have used and mentioned above. When examined in the electron microscope at relatively low magnification, the preparations of BMV particles had the appearance shown in Figs. 6 and 7. Large areas of two- dimensional hexagonal crystalline assays of virus were seen extending across the holey film supports (Fig. 6). Some areas showed isolated particles in the presence of the negative stains, and occasionally crystalline arrays consisting of two and possibly three layers were observed in the micrographs, but these were relatively rare events.

At higher magnifications the individual virus particles or crystalline arrays gave the appearance of being essentially similar to conventional negatively stained preparations consisting of an electron dense material surrounding the virus capsids and in some areas penetrating the core regions of the particles (Fig. 7). The packing of the BMV virus in hexagonal arrays together with the structural details at the periphery of particles is well resolved in the electron micrographs and optical diffraction patterns shown in Fig. 8. Much of the surface detail relating to the individual capsomeres is less well defined because of the pronounced interference or Moir6 effects resulting from the superimposition of the upper and lower surfaces of the capsid. The complex patterns formed from this type of superimposition have been discussed in detail by Klug and Finch (11). However, some particles have been seen as one- sided structures and their capsomeres resolved on the virus surface, but these were only occasionally observed in these particular BMV preparations.

The preparation of cowpea chlorotic mosaic virus (CCMV) for examination in the electron microscope was carried out in essentially the same way as for BMV,


FIG. 6. Negative stain-carbon film containing an area of crystalline virus shown extending across the holey film support. The difference in visible detail observed on the thin film when compared with overlapping films is striking.

bu t some studies were made on the effects of changing the p H of the initial negative

stain and drying the specimens at low temperatures. When mixtures of C C M V

and 3 % a m m o n i u m molybda te negative stain at p H 5.2 were dried down at r o o m

temperature , the crystal arrays were general ly the same as B M V in a hexagonal fo rm

FIG. 7. (a) An area of BMV in hexagonal crystalline array prepared by the negative stain-carbon film method (3 % ammonium molybdate as first negative stain and 1% uranyl acetate as second stain). The particles are essentially similar to those seen in conventional negatively stained preparations. Some virus capsids have been penetrated by the negative stain, but these structures are small in number when compared to the total virus population forming the crystals. (b) High magnification of a region from CCMV crystalline preparation in hexagonal array. The small angular electron dense material can be seen associated with the virus surface as indicated (arrows) in the electron micrograph.


as shown in Fig. 7, or in some areas as isolated particles. In a few areas on the films examined it was possible to find occasional square packing of the particles. By reduc- ing the negative stain pH to 3.5 and mixing with the virus suspension, it was noted that the packing arrangement was entirely hexagonal and from the large number of specimens examined at the lower pH value, there was no evidence of square arrays. At a higher pH value of 9.0 the CCMV particles formed some small regular or ordered patterns, but many were seen as scattered virus structures. The effect of drying the CCMV preparations at about 20°C and 4°C was also investigated. It was noted that drying down the virus and negative stain mixtures (pH 5.2) at room temperature the majority of the crystal forms were in hexagonal array, but drying at 4°C produced a majority of crystals with square packing as illustrated in Fig. 8. The formation of CCMV in square arrays at 4°C was found to be a consistent arrangement under the conditions we have described.

Examination by optical diffraction of the electron micrographs obtained from BMV and CCMV prepared by the negative stain-carbon film method, provided evidence for the precise way in which the virus particles packed together. The diffraction spectra also indicated repeating features in the micrographs extending out to a resolution of better than 2.0 nm (Fig. 8).

Adenovirus. The suspensions of highly concentrated adenovirus when mixed with an equal volume of ammonium molybdate at pH 7.2 as the first negative stain and air dried at room temperature, produced large sheets of hexagonally packed particles in the positions shown in Fig. 9. The particles shown in Fig. 9 are interpreted as being in positions with a 3-fold rotational symmetry axis perpendicular to the paper, and those illustrated in Fig. 10 appear to be viewed along an axis of 2-fold rotational symmetry. In some areas unpacked adenovirus particles were seen as shown in Fig. 11. Several interesting features were observed in both the crystalline arrays and in separated particles. The individual capsomeres on the intact virus were clearly seen as hollow elongated cylinders where the negative stain had penetrated between capsomeres and into their hollow centres (Fig. 11). These were particularly well resolved at the periphery of a large number of virus particles we have analysed. The detailed structure and dimensions of the intact adenovirus and disrupted components as determined by optical diffraction will be described in a separate publication, but our present findings support the earlier observations of Wilcox et al. (19), who reported that the capsomeres of adenovirus type 5 were polygonal hollow rods (Figs. 10 and l 1). The electron micrographs of intact capsids also showed many of the capsomeres viewed along their long axis when seen in more favourable orientations, but a large number of the images were obscured by the superimposition of the upper and lower surfaces of the virus capsids (Fig. 11) (11).

Many of the capsomeres on both intact and disrupte d adenovirus capsids showed

)


Fro. 8. (a) The area on the left shows a small region f rom a CCMV cubic crystalline array selected for analysis by optical diffraction. The very small visible detail at the surface of individual virus particles results f rom the superimposition of the upper and lower surface structures.

The optical diffraction pat tern shown on the right was recorded f rom the area A. Some indication of the resolution from repeating features within the crystalline virus array can be seen f rom the diffraction spectra (arrows) spacings extending to a value of 1.7 nm. (b) The area on the left contains a small region of BMV particles in hexagonal array ~elected for optical diffraction. A n optical diffraction pat tern f rom B is shown on the right. Note how the particles ~re precisely related to each other. The spots ringed are at a spacing of 2.8 nm.


the presence of small rodlike structures geometrically arranged between the capsomeres as illustrated in Figs. 11 and 12.

In the virus suspensions of adenovirus examined by us under the above preparative conditions, it was not possible to resolve the spikes associated with the B and C antigen complex located at the axes of 5-fold symmetry on the intact packed particles when packed in crystalline arrays, but large numbers of the individual spikes were observed separately or in aggregates as illustrated in Fig. 12 (18). In a limited number of areas it was possible to observe adenovirus cores which appeared to be released f rom disrupted virions. These core components were much more in evidence when the specimens were prepared under the conditions described below (13).

Disruption of adenovirus in the pre~ence of negative stain

In addition to the examination of intact and crystalline arrays of adenovirus by the negative stain-carbon film technique, experiments were carried out on the disruption of particles after preparation of the virus according to the above preparative procedures. Adenovirus was mixed with 3 % ammonium molybdate at p H 7.2 and carbon coated on the mica surfaces as described earlier. Samples were removed f rom the liquid/air interface of the second negative stain which was 3 % ammonium molybdate used in place of the normal solution of 1% uranyl acetate. Crystalline arrays were observed covering large areas and were the same patterns as those shown in Fig. 9. When the films were allowed to remain floating at the surface of the second 3 % ammonium molybdate solution for a period of 48 hours, the adenovirus crystals dispersed and a large proport ion of the virus material present was seen to be in various stages of disruption. Many of the capsids released their capsomeres which recrystallised on the carbon films (Fig. 12). In many of the specimens examined, the crystalline arrays of capsomeres could be seen arranged over large areas and were analysed by optical diffraction methods. As mentioned in the introduction a more detailed account of our current findings relating to the capsomere structure will be reported separately.

Potato virus" X and apple chlorotic leaf~Tot virus

Highly purified suspensions of potato virus X (PVX) and apple chlorotic leaf spot virus (CLSV) were prepared and dried on the mica surfaces using the same conditions for BMV, CCMV, and adenovirus. At high concentrations of virus it was possible

F~G. 9. Crystalline array of human adenovirus type 5 prepared in the presence of 3 % ammonium molybdate as the first negative stain and separated from the mica surface by floating on to 1% uranyL acetate as the second negative stain. The positions of the capsids on the film indicate that they are viewed along an axis of 3-fold rotational symmetry, but are randomly orientated with respect of each other (see Fig. 10).

HORNE AND PASQUALI RONCHETTI 372

FIG. 10. (A) Selected area from a crystalline array of adenovirus. Note the shape of the particle profile compared to those in Fig. 9. (B) Optical diffraction pattern from A indicating the precise orientation of the particles. (C) Optical diffraction from the same areas as B, but underexposed to show spectra closer to the central beam. Two diffuse rings of diffraction spectra can be seen extending beyond the inner groups of spots which indicate hexagonal detail associated with the distribution of small structural features in the capsomeres, which are less ordered between particles than the structures seen at lower resolution [see Beeston et al. (3), pp. 363-365]. The outer diffuse ring with an indication of hexagonal detail (arrows) extends to a value of 2.8 nm.

to find relatively large areas containing filamentous PVX nucleocapsids arranged in semiparallel arrays or as isolated virus. The electron micrographs and optical diffraction patterns are shown in Fig. 13.

From preparations of CLSV it was possible to observe the packing of the virus filaments in large circular arrays as shown in Fig. 14. It is of interest that this type of packing and other patterns associated with CLSV was quite different compared to PVX and other flexuous viruses that we have studied with the aid of the above techniques. A more detailed comparison of these packing arrays as seen in negatively stained-carbon film preparations and thin sections will be published separately.

Examination of reconstituted virus material

The techniques we have employed in the present studies were applied to virus material assembled in vivo. The particles were capable of forming large areas of crystalline arrays in the presence of the negative stains after the suspensions were dried at room temperature or under freeze drying conditions as described earl ier . The optical diffraction patterns indicated that the wild-type virus specimens formed arrays in which the particles were precisely related to each other (Figs. 8 and 10). Since it was demonstrated by Bancroft (1) that it was possible to reconstitute plant viruses of the BMV and CCMV type in vitro, we attempted to compare the packing


FIG. 11. High magnification area of adenovirus showing pronounced interference patterns as a result of underfocusing and the superimposition of the upper and lower surfaces being imaged at the same time. The hollow central regions of the columnar capsomeres are clearly seen (white arrow) at the periphery of the capsids. In suitable orientations small structural components linking capsomeres can be resolved (black arrow).


patterns of reconstituted virus with those from normal virus preparations. Our current experiments have indicated that the particular suspensions of reconstituted virus we examined did not pack into regular arrays, but tended to form sheets of somewhat irregular packing patterns (Fig. 15).

Several preliminary studies were made in an attempt to follow the stages of assembly relating to reconstituted BMV at very high concentrations of BMV protein prepared according to the procedures of Bancroft (1). The electron micrographs shown in Fig. 16 illustrate stages of assembly, as prepared by the above methods for electron microscopy. Many of the structures shown in Fig. 16 A and B are seen as capsomeres distributed over the film background, and in some areas they are associated with recognizable capsids. A large amount of unassembled protein is spread over the film surface, but at a later stage of assembly the particles have the appearance of the structures shown in Fig. 16C, with very few identifiable individual capsomeres in the background and when final reconstitution of the viral products occurs, large sheets of aggregated virus were observed (Fig. 15).

From the specimen samples used in our studies of reconstituted virus it was noted that the suspensions of reassembled capsids contained a high proportion of irregular- shaped particles and the small variations in shape, size, and symmetry prevented their precise packing of the type shown in Figs. 7 and 8.

High resolution electron microscopy of virus preparations

Electron microscopy of the specimens prepared by the negative stain-carbon film procedure and examined at high magnifications showed several features which suggested that the negative stains tended to form very small crystallites. These were particularly noticeable in association with the virus material, but some areas in the background contained similar electron dense structures. The crystallite material is shown in the high resolution electron micrograph illustrated in Fig. 7B, where it is possible to observe the angular shapes and sizes of the electron dense stain. These dense structural features were observed when ammonium molybdate was used both as the first and second negative staining material and also when uranyl acetate or other stains were employed to enhance contrast in the viral preparations. In the absence of virus the level of crystalline particles was considerably reduced, and it was concluded that there was some possible interaction between the negative stains, virus material or residual buffer used in the preparations. It is also possible that the negative stain crystallites are structurally correlated to the viral components. The

F~. 12. Large areas of reaggregated hexon (A antigen) capsomeres resulting from the dissociation of adenovirus in 3 % ammonium molybdate pH 5.2 as the second negative stain for a period of up to 48 hr. Many areas show the capsomeres arranged in hexagonal arrays. The isolated BC antigen spike complex (insert white arrow) separate from the hexon capsomere aggregates. Structural detail in the form of small units can be resolved at their bases.


level of the crystallites and their size range did not appear to vary either with the concentration and pH values of the negative staining solutions, but they did tend to increase in the presence of higher concentrations of buffer material.

The studies carried out using the above techniques on BMV, CCMV, PVX, CLSV and adenovirus also revealed the presence of a large amount of relatively low molecular weight material randomly distributed in the background of the thin support films and in many places extending over the virus particles (Fig. 13). This material suggests that the virus in suspension is probably being disrupted at some stage of the purification preparation procedure or in the presence of the negative stains. Some samples were fixed with buffered glutaraldehyde prior to negative staining, but this did not appear to reduce the amount of background material. Measurements made from highly magnified images of the virus preparations suggested that the components forming the background aggregates were essentially of the same physical dimensions as those seen on certain virus particles.

DISCUSSION

The simple technique we have described here has certain advantages over conventional negative staining procedures. It is possible to produce very thin carbon support films in relation to the physical dimensions of the virus material examined coupled with the high concentrations of particles used in our experiments. With the aid of normal methods for mounting highly concentrated virus there is considerable diffi- culty associated with mechanical damage to thin support films and their subsequent disruption in the electron beam. In addition, virus samples at high concentration frequently tend to form dense random aggregates when pipetted directly on to carbon or carbon/plastic films.

When highly purified suspensions of BMV, CCMV, and adenovirus were dried in the presence of ammonium molybdate they formed regular crystalline arrays which could be reproduced repeatedly provided the physiological conditions of stain and virus suspensions were so arranged that disruption of the virus was avoided. The large areas of crystalline patterns has allowed detailed analysis of the structures to be performed with the aid of optical diffraction techniques. It can be seen from the optical diffraction spectra illustrated in Figs. 8, 10, and 13 that the resolution extends to values which indicate an improvement over normal negative staining methods which may, in part, result from the very thin carbon films. Whether the technique

FIG. 13. (a) Selected area from a sheet of aggregated PVX. Note the large regions surrounding the virus filaments which contain a high proportion of low molecular weight material (3 % ammonium molybdate pH 5.2 as first negative stain and 1% uranyl acetate as second stain). (b) Optical diffraction pattern from A indicating the interparticle distances and periodicity of the protein helix along the PVX filament axis. The layer lines (arrows) are at a distance of 3.6 nm.


i .... ,~, ~i ~il,ii~ii:i~Si~i!

. . . . . . : : : N :


provides some protection against irradiation damage remains to be established, but prolonged exposure of the specimens to the electron beam of up to periods of 20 minutes did not reduce the detail as determined from the optical diffraction spectra. A comparison of the electron low-angle diffraction patterns with those obtained

from the optical diffractometer are in progress and will be the subject of a separate communication, as the former offers a better direct approach to specimen changes

resulting from beam effects (see 6, 17). The aggregates and packing patterns seen in the case of PVX and other flexuous

virus filaments were of particular interest, as it was possible to arrange the highly

concentrated particles in arrays which could be studied by selected-area optical

diffraction methods. In the case of nonrepeating structural detail from disrupted viral components, we

were able to obtain accurate measurements from separated and well resolved struc-

tures distributed over the specimen supports. These structures were of special interest when studying partially assembled reconstituted virus material. The low molecular weight material, on the other hand, may well cover or obscure structural features in

the larger virus capsids. It may also prevent the formation of virus crystalline arrays if present at high concentrations in a given virus preparation.

There is the question of whether the images we have obtained are from a type of

replica? It is clear from some of the experiments on chemically removing the virus from the carbon that the virus and negative stain is present and associated with the carbon. In some areas we did see images that were of a replica form, but the resolution was considered to be very poor and no structural features apart from the gross morphology of the particles and crystal boundaries could be detected. Further evio

dence for the presence of viral material was provided from the experiments on the dissociation of adenovirus in the presence of a second negative stain where not only were the crystalline arrays lost, but clear images of capsomeres and their recrystallisa- tion were recorded. Our analysis of virus material indicates that the increased resolution adds to the problems of the very small details causing pronounced interference

effects resulting from the superimposition of the upper and lower surfaces of capsids which has been previously discussed by Klug and Finch (11). These high resolution

F~G. 14. Packing pattern resulting from CLSV filaments prepared in the presence of ammonium molybdate at pH 8.0 as the first negative stain and 1.0 % uranyl acetate as the second negative stain. FIG. 15. Highly concentrated preparation of reconstituted BMV prepared by the negative stain-carbon method (3 % ammonium molybdate pH 5.2 as first negative stain and 2 % uranyl acetate as second stain). FIG. 16. Partially reconstituted BMV showing isolated capsomeres (a) and incomplete capsids. The capsomeres shown in (b) possess spokelike structures (arrow). The BMV-reconstituted capsids (c) show the variation in shape and size of the capsids together with the penetration of stain within the central region of the virus (3 % ammonium molybdate pH 5.2 as first negative stain and 1% uranyl acetate as second stain).


2 5 - 741839 J. Ultrastructure Research


images are difficult to interpret from direct examination by the eye and must be subjected to analysis by optical diffraction or other methods.

It should be emphasized that the resolution as indicated in the optical diffraction patterns shown in Figs. 8A, B and 10B, C, is derived from the presence of periodic or semiperiodic structural features recorded in the electron micrographs (3). However, the measurements we have made from individual small structural components relating to nonperiodic features and where superimposition effects are not present, have shown that the negative stain-carbon method is capable of giving a resolution approaching 1.5 to 1.0 nm, provided that the specimen thickness, stability, and opti- mal electron optical focusing requirements are met.

From our current studies the three-dimensional preservation of the virus material we have examined suggests that it is better than specimens prepared by conventional negative staining methods. The samples examined following freeze-drying under carefully controlled conditions gave essentially the same results when compared to the air dried specimens for the viruses mentioned above. It must be stressed, however, that some attention should be given to the concentration and pH of the ammonium molybdate or other material when used as the first negative stain in relation to the physiological conditions employed to prepare a given type of virus suspension. The application of the second negative stain acts as an additional form of contrast enhance- ment and in the case of uranyl acetate some partial fixation of certain viral components appears to result. When some specimens were floated on to the surface of a 1% uranyl acetate solution for prolonged periods (10-15 minutes), the viral material became stabilized and in several instances acted as a positive stain for the viral core material. The use of other negative stains as second stains, on the other hand, when adjusted to suitable pH values, can be used to some advantage in slowly disrupting the highly concentrated virus as demonstrated in the case of adenovirus (Fig. 12) and certain filamentous particles.

The examination of the virus suspensions at high resolution showed the presence of large amounts of low molecular weight material forming the background. It is our view that this material and other organic components present in distilled water introduced at some stage of the virus preparation, together with negative stain solutions and film production, are also likely to cause serious limitations in resolving detail below 20 nm from bfological objects of small physical dimensions.

SUMMARY

A simple technique has been developed for the preparation of highly concentrated or crystalline suspensions of viruses in the presence of negative stains. Several ico- sahedral and filamentous viruses have been studied in concentrations from 1.0-20


mg/ml. The technique has several advantages over conventional negative staining procedures by forming two-dimensional and three-dimensional crystalline arrays of highly concentrated viruses. The packing arrangements of the virus in the crystalline arrays can be varied according to the type of negative stain and pH used during the preparation. High resolution electron micrographs of material prepared by the method have been analysed by optical diffraction procedures.

The relative thickness of the carbon supporting film in relation to the physical dimensions of viruses is considerably improved.

The authors wish to thank Professor Roy Markham and Dr J. Bancroft for many valuable discussions and Mrs J. M. Hobart for technical assistance. Acknowledgement is also due to Mr S. Frey and Mr L. S. Clarke for their help with the photography.

REFERENCES

1. BANCROFT, J. B., aT. Gen Virol. 14, 223 (1972). 2. BAR-JOSEPH, M., LOEBENSTEIN, G. and COHEN, J., Virology 50, 821 (1972). 3. BEESTON, B., HORNE, R. W. and MARKHAM, R., in GLAUERT, A. M. (Ed.), Electron

Diffraction and Optical Diffraction Techniques. Practical Methods in Electron Micros- copy. North-Holland/American Elsevier, Amsterdam, 1973.

4. BRADLEY, D. E., in KAY, D. (Ed.), Techniques for Electron Microscopy, p. 96. Black- well, Oxford, 1965.

5. COSSLETT, V. E., in OSTER, A. W. and POLLISTER, G. (Eds.), Physical Techniques in Biological Research, Vol. 1, p. 46. Academic Press, New York, 1956.

6. GLAESER, R. M., J. Ultrastruct. Res. 36, 466 (1971). 7. GLAESER, R. M., BUD1NGER, T. F., AEBERSOLD, P. M. and THOMAS, G., in FAVARD, P.

(Ed.), Proc. Seventh Congr. Electron Microsc. Vol. 1, p. 463, 1970. Grenoble. 8. HARRIS, W. W., in PARSONS, D. (Ed.), Some Biological Techniques in Electron Micros-

copy, p. 147. Academic Press, New York, 1970. 9. HORNE, R. W. , J. Microsc. 98, 286 (1973).

10. JAFFE, M., J. Appl. Phys. 19, 1191 (1948). 11. KLU6, A. and FINCH, J. T., J. Mol. Biol. 11, 403 (1965). 12. KLUO, A. and DE ROSIER, D. J., Nature (London) 212, 29 (1966). 13. NERMUT, M. V., J. Microsc. 96, 351 (1972). 14. RUSKA, E., J. Roy. Microsc. Soc. 84, 77 (1965). 15. SJOSTRAND, F. S., in SJOSTRAND, F. S. and RHODIN, J. (Eds.), Proc. Eur. Conf. Electron

Microsc. 1st, p. 120. Stockholm, 1956. 16. STENN, K. and BAHR, G. F., J. Ultrastruct. Res. 31, 526 (1970). 17. UNWIN, P. N. T., Proc. Eur. Congr. Electron Microsc. 5th, p. 232. Institute of Physics,

London, 1972. 18. VALENTINE, R. C. and PEREIRA, H. E., J. Mol. Biol. 13, 13 (1965). 19. WILCOX, W. C., GINSBERG, H. S. and ANDERSON, T. F., J. Exp. Med. 118, 307 (1963). 20. WILLIAMS, R. C. and FISHER, H. W., J. Mol. Biol. 52, 121 (1970). 21. WRIGLEY, N. G., J. Ultrastruct. Res. 24, 454 (1968).

a negative staining—carbon film technique for studying viruses in the electron microscope

Documents