Conditions Critical for Optimal Visualization of Bacteriophage

Transcription

1 JOURNAL OF VIROLOGY, June 1975, p Copyright American Society for Microbiology Vol. 15, No. 6 Printed in U.S.A. Conditions Critical for Optimal Visualization of Bacteriophage Adsorbed to Bacterial Surfaces by Scanning Electron Microscopy GWEN WENDELSCHAFER-CRABB,1 S. L. ERLANDSEN,2 AND D. H. WALKER, JR.* Department of Microbiology, University of Iowa, Iowa City, Iowa Received for publication 21 January 1975 The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of' infected cells which were fixed with glutaraldehyde followed by postfixation in Os04 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of' infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of' resolving viral particles on cell surf'aces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used. The use of the scanning electron microscope (SEM) to identify surface features of virusinfected cells as virus particles has been reported by several investigators. De Harven et al. (3) described "knobs" visualized on murine Friend erythroleukemia cells as RNA tumor viruses which spontaneously bud from the cell line. This identification, based only on morphological criterias, was not wholly convincing since the cell surfaces in the specimens were not well preserved due to the air drying preparation of the specimens for SEM. Virus antigens have been localized by using nonspecific virus markers distinguishable by SEM. Nemanic et al. (6) developed a technique for the localization of antigens on budding mammary tumor virus by using the characteristic morphology of tobacco mosaic virus as a recognizable marker for SEM. A hapten sandwich technique in which tobacco mosaic virus was immunologically linked by an antibody bridge to mammary tumor virus antigens was used and resulted in several tobacco I Present address: Infectious Disease Laboratory, Veterans Administration Hospital, Iowa City, Iowa Present address: Department of Anatomy, University of Minnesota, Minneapolis, Minn mosaic viruses being bound to budding mammary tumor viruses. Fonte and Porter (5) emphasized the importance of the correlation of transmission electron microscopy (TEM) to SEM. They determined the stereo relationship of viruses within cells with high voltage electron microscopy and then studied the same cells by SEM to localize herpes simplex virus type I. Although they demonstrated that viruses can be visualized on cell surfaces by SEM and TEM, the effect of both fixation and drying techniques used in the preparation of specimens was not discussed or compared. The purpose of this paper is to report the effects of cell preparation, i.e. fixation and drying technique, on the visualization of viruses by SEM using a model system employing the adsorption of bacteriophage P1 to Shigella dysenteriae. P1 was chosen as the virus in these studies because of its distinctive morphology characterized by a large isometric head, 80 nm in diameter, and tail, 200 nm in length (10). S. dysenteriae was used as a host cell since it is free of flagella and other surface features which could interfere with the visualization and identification of adsorbed phage.

2 VOL. 15, 1975 SEM VISUALIZATION OF PHAGE ADSORPTION 1499 (A preliminary report of this work was published in Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, p. 244.) MATERIALS AND METHODS Bacteria and viruses. Cultures of S. dysenteriae strain 16 (Sh-16) (2) were infected at multiplicities of 100 with bacteriophage Plkc (2). Escherichia coli K-12 strains DW101 and DW103 were used for the propagation of high titer stocks of Plkc using the method of Swanstrom and Adams (8) as modified by Walker and Anderson (10). All strains were obtained from D. H. Walker, Jr., University of Iowa. Media. Media was prepared as described by Walker and Anderson (10). Fixation and electron microscopy. Sh-16 was grown in L broth at 37 C with slow agitation to a concentration of 3 x 108 cells per ml. The culture was assayed by viable count. A high titer stock culture of P1 phage was diluted and mixed with the appropriate concentration of cells to obtain a multiplicity of infection (MOI) of 100. PFU were assayed by plate count. Plkc was adsorbed to Sh-16 at 37 C for 10 min in 0.05 M cacodylate buffer (ph 6.1). The infected bacteria and uninfected control culture were centrifuged, and the pellets were resuspended in fixative. The fixative consisted of 2% glutaraldehyde (wt/vol)-0.05 M cacodylate buffer (ph 6.1) with 0.2% MgSO4 added. The fixation was carried out at 25 C for 30 min. Samples for SEM were collected on 0.1-,gm pore size nucleopore filters which were then refixed in glutaraldehyde-cacodylate for 30 min. One-half of each filter was postfixed in a 2% solution of osmium tetroxide in 0.05 M cacodylate buffer for 1 h, whereas the other half was held in buffer. Samples postfixed with OSO4 were washed, and portions were removed for air drying from water. The remaining specimens were dehydrated through a graded series of ethyl alcohol, and samples were also removed from alcohol for air drying. The remaining specimens were changed from alcohol to amyl acetate and dried by the critical point method of Anderson (1). All samples were mounted on aluminum specimen stubs with conductive copper tape, coated once with gold palladium, and viewed in a Kent Cambridge 4S stereoscan microscope at an accelerating voltage of 20 kv. After glutaraldehyde fixation, samples for TEM were centrifuged and resuspended in two to three drops of molten 1.2% noble agar. The agar was allowed to solidify on a flat surface, sliced with a razor blade, and then placed in a vial with buffer. Portions of each sample were postfixed in 2% OSO4 for 1 h and then fixed and stained en bloc with uranyl acetate by the method of Terzakis (9). All samples were dehydrated through a series of ethyl alcohol, placed in propylene oxide, embedded in Newcomb araldite-epon, and polymerized at 60 C for 48 h. Thin sections (40 to 60 nm) were cut on a Huxley ultramicrotome, stained with uranyl acetate (4), where necessary, and lead citrate (7), and viewed in either a JEOL 100B or a Philips 300 transmission electron microscope at an accelerating voltage of 60 kv. RESULTS Illustrated in Fig. 1 is a comparison of control and infected cells with SEM and TEM. Samples were fixed in glutaraldehyde and osmium tetroxide. Samples for SEM were dried by the critical point method. Numerous phage profiles are clearly visible in the transmission micrograph of the infected cells (Fig. ld). Note, in the inset, the large head, contracted sheath, and tail tube of the virus which is attached to the cell. No phage were seen by TEM on uninfected Sh-16 cells (Fig. lc). This difference between infected and uninfected Sh-16 is also very apparent by SEM. Numerous phage particles attached to the surface of Sh-16 cells are visible (Fig. lb). The general appearance and distribution of these particles is comparable to that seen by TEM. Gold-palladium coating tends to obscure the distinct structure of heads and tails; however, in some areas (see arrows Fig. lb) there is an indication of heads attached by tail-like structures to Sh-16 cells. The uninfected control Sh-16 cells are free of such particles (Fig. la). Using SEM, distinct comparisons between infected and uninfected cells could not be made without the use of proper fixation (Fig. 2). Different fixatives have different effects on the appearance of infected and uninfected cells; cells fixed only in glutaraldehyde (Fig. 2a, c, and e) are compared with cells fixed with both glutaraldehyde and OsO (Fig. 2b, d, and f). The micrographs of uninfected cells illustrates an artifact of glutaraldehyde fixation: cells are not smooth and rounded but are wrinkled and appear to have collapsed slightly. After postfixation with OsO there is marked contrast between the infected (Fig. 2d) and control cells (Fig. 2b). Viruses are clearly discernible on the surface of the infected cells, whereas the uninfected cells are smooth, cylindrical and well rounded. Another common artifact seen by SEM after glutaraldehyde fixation is a surface film covering the cells (Fig. 2c). This film apparently masks the structure of the viral particles; however, TEM of glutaraldehyde-fixed infected cells reveals several phage attached to the cell wall (Fig. 2e). The ultrastructure is not preserved as well as with OsO fixation (Fig. 2f), but, nevertheless, the viruses are clearly distinguishable on the cell surface. Although viruses remain attached to glutaraldehyde-fixed cells, they are not resolvable by SEM under these conditions. The drying technique used also has a critical effect on the visualization of phage by SEM, as

3 1500 WENDELSCHAFER-CRABB, ERLANDSEN, AND WALKER J. VIROL. c FIG. 1. Comparison of infected and uninfected cells by SEM and TEM. All specimens were fixed in glutaraldehyde and postfixed in OsO4. SEM specimens were prepared by critical point drying. TEM specimens were stained en bloc with uranyl acetate and embedded in araldite epon; thin sections were stained with lead citrate. (a) Uninfected Sh-16 by SEM; (b) SEM of P1 adsorbed to Sh-16 (MOI = 100), head and tail-like structures are visible (arrow); (c) TEM of uninfected Sh-16; (d) TEM of P1 adsorbed to Sh-16 (MOI = 100), inset higher magnification of Plkc. Magnification bar represents 0.5 Am (0.1 Am in inset). illustrated in Fig. 3, which shows cells which have been air dried from water (Fig. 3a, b), air dried from absolute ethanol (Fig. 3c, d), and dried by the critical point method (Fig. 3e and f). These are the three most common drying techniques used in the preparation of specimens for SEM. Cells with multiplicities of 100 (Fig. 3b, d, and f) are compared with uninfected control cells (Fig. 3a, c, and e). All samples received a double fixation with glutaraldehyde and osmium. Samples air dried from water after fixation appear to be collapsed on the nucleopore filter (Fig. 3a and b). No appreciable difference can be seen between control (Fig. 3a) and infected cells (Fig. 3b). The viruses are not detectable except for a few protrusions, which may be viruses, on the cell surfaces. Similar results were obtained with specimens which were air dried from absolute ethanol * Differences in infected (Fig. 3d) and control (Fig. 3c) cells are very minimal. The cells are less collapsed with this method than with drying from water; however, the surfaces are not smooth and well rounded in appearance. Particles approximating the size of viruses (unadsorbed) can be seen on the surface of the nuclepore filter of the infected sample and occasionally on the surface of infected Sh-16 cells (Fig. 3d). Bacteriophage on the surface of infected cells prepared by the critical point method are clearly discernible (Fig. 3f0. Uninfected cells prepared by this method also show good ultrastructural preservation (Fig. 3e). DISCUSSION Our experiments describe parameters concerning the visualization of bacteriophage by.*

4 VOL. 15, 1975 SEM VISUALIZATION OF PHAGE ADSORPTION 1501 Downloaded from FIG. 2. Comparison of glutaraldehyde fixation to glutaraldehyde, post-osmium fixation by SEM and TEM. Samples for SEM were prepared by critical point drying. Samples for TEM were treated as indicated in (e) and (f). (a) SEM of uninfected Sh-16 fixed with glutaraldehyde alone; (b) SEM of uninfected Sh-16 fixed with glutaraldehyde-oso4; (c) SEM of P1 adsorbed to Sh-16 (MOI = 100) fixed with glutaraldehyde alone; (d) SEM of P1 adsorbed to Sh-16 (MOI = 100) fixed with glutaraldehyde-oso4; (e) TEM of P1 infected Sh-16 (MOI = 100) fixed with glutaraldehyde and stained, after thin sectioning, with uranyl acetate and lead citrate; (f) TEM of P1-infected Sh-16 (MOI = 100) fixed with glutaraldehyde-oso4, stained en bloc with uranyl acetate, and stained, after thin sectioning, with lead citrate. Magnification bars represent 0.5,m. f 0 K, on December 31, 2018 by guest

5 1502 WENDELSCHAFER-CRABB, ERLANDSEN, AND WALKER J. VIROL..1im W.17! AN, Lb I -F A. ft sommom -h Jo, Downloaded from FIG. 3. Comparison of drying techniques. All specimens were fixed in glutaraldehyde followed by OsO4. (a) Uninfected Sh-16 air dried from water; (b) P1-infected Sh-16 air dried from water; (c) uninfected Sh-16 air dried from absolute ethanol; (d) P1-infected Sh-16 air dried from absolute ethanol; (e) uninfected Sh-16 prepared by the critical point drying method; (f) P1-infected Sh-16 critical point dried. Magnification bar represents 0.5,um. on December 31, 2018 by guest SEM. The results clearly demonstrate that phage particles can be visualized by SEM, but that this visualization is completely dependent on the techniques used in the preparation of the specimens. Detection of viral particles by SEM is entirely dependent upon both the fixation and drying methods used. Glutaraldehyde fixation alone proved to be inadequate for SEM visualization of P1 attached to shigella cells even though

6 VOL. 15, 1975 SEM VISUALIZATION OF PHAGE ADSORPTION 1503 examination of this same preparation by TEM revealed large numbers of P1 attached to the cell surfaces. This inability to detect attached P1 on shigella surfaces after glutaraldehyde fixation seemed to be related to the presence of a surface film which covered the cell surfaces and thus obscured the attached viral particles. The nature of this surface film is unknown since after OsO, postfixation of glutaraldehyde-fixed cells this film was absent and P1 particles were easily distinguishable on infected cells. After fixation in glutaraldehyde followed by OsO, the viruses can be visualized only if infected cells are prepared by the critical point method. With this method alteration of the cell by surface tension forces is minimized, if not entirely avoided, such that the P1 phage particles remain attached to the cells and may be visualized by SEM. Air drying from water after fixation, or from absolute ethanol after dehydration, results in poorly preserved surface structure which does not even permit discrimination of phage-infected cells from uninfected control cells. Numerous particles approximating the size of P1 can be visualized on the surface of the nucleopore filter containing the sample which was air dried from ethanol. Perhaps the particles are phage which were removed from the cell surface by the changes in surface tension that occurred during the air drying procedure. By correlating SEM and TEM it is possible to identify particles as viruses distinct from surface protrusions which could easily be interpreted as viral particles. By comparison of infected and uninfected preparations, particles tentatively identified as viruses by SEM can be confirmed as viruses by TEM. In summary, the SEM is capable of resolving bacteriophage P1 adsorbed to Shigella dysenteriae. Positive identification of particles on cells as viruses can be confirmed with TEM. The method of fixation and drying are of utmost importance for the detection of viral particles. The fixation must include OsO since glutaraldehyde alone is not sufficient in preserving the structure of phage-infected cells. The infected cells must be dried by the critical point method to prevent alteration of the phage adsorbed to the cells. The SEM reveals the surface features of virus-infected cells under these conditions. When correlated with TEM, the SEM may be of value in studying adsorption, penetration, and budding of viruses in animal virus systems. ACKNOWLEDGMENTS We thank Ching-Yuan Shih and John Puffer for excellent technical assistance. LITERATURE CITED 1. Anderson, T. F Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci. 13: Bertani, G Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62: De Harven, E., N. Lampen, T. Sato, and C. Friend Scanning electron microscopy of cells infected with a murine leukemia virus. Virology 51: Erlandsen, S. L., and D. G. Chase Paneth cell function: phagocytosis and intracellular digestion of intestinal microorganisms. I. Hexamita muris. J. Ultrastruct. Res. 41: Fonte, V. G., and K. R. Porter Visualization in whole cells of herpes simplex virus using SEM and TEM, p In 0. Johari (ed.), Scanning electron microscopy/1974. HIT Research Institute, Chicago, Ill. 6. Nemanic, M. K., J. M. Shannon, D. P. Carter, and L. Wofsy Immunospecific labeling of cell surface antigen with a marker visible in the scanning electron microscope (SEM). J. Histochem. Cytochem. 22: Reynolds, E. S The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17: Swanstrom, M., and M. H. Adams Agar layer method for production of high titer phage stocks. Proc. Soc. Exp. Biol. Med. 78: Terzakis, J. A Uranyl acetate, a stain and a fixative. J. Ultrastruct. Res. 22: Walker, D. H., Jr., and T. F. Anderson Morphological variants of coliphage P1. J. Virol. 5: