Experimental Cytomegalovirus Ophthalmitis - NCBI

Experimental Cytomegalovirus Ophthalmitis Jared N. Schwartz, MD, Charles A. Daniels, MD, PhD, Jeffrey C. Shivers, BS and Gordon K. Klintworth, MD, PhD

Cytomegalovirus can produce a severe necrotizing chorioretinitis in patients on immunosuppressive therapy and infants born with congenital cytomegaloviral inclusion disease. To study the effect of cytomegalovirus on the eye, murine cytomegalovirus was injected into the eyes of nonimmunosuppressed Swiss CD-1 weanling mice. The eyes were then prepared for virus titer, as well as light and electron microscopy at variable periods after inoculation (1 to 28 days). From days 2 to 6, the hallmarks of cytomegalovirus infection, intranuclear and intracytoplasmic viral inclusions, were evident within cytomegalic cells. The major site of reaction was in the uveal tract, where necrosis and inflammation were prominent. Viral particles budding through the nuclear membranes into the perinuclear cisternae and vacuoles with viral particles could be seen in the cytoplasm of infected cells. In lesions older than 2 weeks, only a mild mixed inflammatory infiltrate and fibrosis were observed. Morphologic alterations unaccompanied by inflammation occurred in the outer sensory retina overlying infected retinal pigment epithelial cells. Multiple necrotic foci with inclusion-bearing cells in the liver indicated the systemic spread of virus from the eye. The titer of virus recovered from the eye peaked at day 4 and then declined to low levels, but infectious virus could still be isolated at day 28, even though viral particles were not seen morphologically at or after day 14. Many of the alterations seen in the model resemble those found in the human cytomegaloviral ophthalmitis (Am J Pathol 77:477-492, 1974).

IN MAN, cYToMEGALovIRus (CMV) can cause a disseminated infection of newborns and debilitated adults. If the virus involves the eye, blindness can result from a severe necrotizing chorioretinitis. In 1947, Kalfayan described, for the first time, ocular lesions in a 2-monthold patient with a fatal CMV infection.' Following tis report, ocular involvement has become recognized as an important component of congenital cytomegaloviral infection.' In 1964, Smith described CMV chorioretinitis in an adult who died with Hodglin's disease.9 Recently, an increasing number of adult patients who developed CMV ophthalmitis while on immunosuppressive or antineoplastic therapy have been

reported.913 From the Department of Pathology, Duke University Medical Center, Durham, NC. Supported in part by Research Grants EY00146-03 and EYCA 00881-02, Training Grant 2T01GN-0726 and Contract NIDR 72-2402 from the US Public Health Service; Dr. Klintworth is the recipient of Research Career Development Award 1 K04 EY 44795-04 from the National Eye Institute, National Institutes of Health and an R. P. B. Louis B. Mayer Scholarship. Accepted for publication August 7, 1974. Address repnnt requests to Dr. J. N. Schwartz, Department of Pathology, Duke University Medical Center, Durham, NC 27710.

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Tissue studies of human cases of CMV chorioretinitis have been observed at a late stage in the disease. Therefore, it was felt desirable to develop an experimental model in which the pathogenesis of the ocular lesions could be studied under control conditions. Due to the inability of human CMV to infect and produce disease in experimental animals, the morphologically identical murine cytomegalovirus (MCNIV) was used to study the alterations in tissues due to CMV.15-7 This report describes the sequential changes that occur in the eye of nonimmunosuppressed mice infected with murine CMV. Materials and Methods Virus and Assay MCMV was prepared by injecting 3- to 4-week-old male weanling Swiss CD-1

mice (Charles River Farms, Wilmington, Mass) intraperitoneally with 105 plaqueforming units (PFU) of a salivary gland homogenate of the Smith strain MCICMV.18 Salivary glands of these animals were harvested 3 weeks postinoculation and homogenized in Eagles' minimal essential medium (MEM) containing 10% fetal calf serum, penicillin (100 units/ml) and streptomycin (50 m/l). Monolayers of fibroblasts were prepared by trypsinization of 14 to 16 day mouse embryos.'9 Virus was assaved on the second passage of these cells using a methyl cellulose

overlay.20 XThere indicated, a 1-ml sample of the above prepared MCMV was inactivated by exposing it for 10 minutes 1 cm from an ultraviolet light source (Lamp No. 782L30, Atlantic UV Corporation, Long Island, NY). After ultraviolet irradiation, the titer of virus was reduced from 1063 to less than 1001 PFU 'ml.

Animal Studies

Twenty-four animals were injected intraocularly behind the lens with 5.0 IL of a salivary gland suspension containing 104 PFU of MICMIVI. For control purposes, 4 animals were injected with an identical volume of a sterile salivary gland suspension, and 4 animals were injected with ultraviolet-inactivated MCMV. A Hamilton syringe (Whittier, Calif) equipped with a 30-gauge needle was used for all intraocular injections. On days 1, 2, 4, 6, 14 and 28 postinjection, groups of 4 mice which had received infectious virus were anesthetized with ether and sacrificed. The two groups of controls were killed on day 6. Preparation of rTssue Immediately after death, 2 mice from each group of 4 were infused intravenouslv with 20 ml of 0.05 M cacodylate-buffered 4% glutaraldehyde (pH 7.4), and the eves were removed for light and electron microscopy. One eye from each remaining pair of animals not infused was removed for virus titer, and the other eve 'was fixed for light microscopy by immersion in 0.02 M phosphate-buffered 4i formaldehyde (pH 7.0). A 10% XV/V homogenate in MENM was made of the eyes used for viral assav, and this was stored at -70 C until titered. Since the liver is a primarv target for an acute 'MCMIV systemic infection, hepatic tissue was also prepared for light microscopic examination. ne eves and liver were embedded

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in paraffin, sectioned at 6 u, and stained with hematoxylin and eosin. For electron microscopy, perfused eyes were immersed in 0.05 M cacodvlate-buffered 4% glutaraldehyde (pH 7.4) for 4 hours, postfixed in 0.2 MI S-collidine-buffered 1% osmium tetroxide (pH 7.4). After being dehydrated in graded alcohols and embedded in Epon 812, 0.5-u-thick sections were cut, stained with toluidine blue, and examined by light microscopy. Thin sections (700 A) cut from selected areas were stained with 0.4% lead citrate and 7.5% uranyl magnesium acetate and examined at 50 kAV in a Hitachi HS-8 electron microscope.

Results

In the 8 control animals examined, the fixation bv intravenous infusion of glutaraldehyde was found to be superior to that of fixation bv immersion. Although MCMV is known to be an endemic infection in certain strains of mice, no evidence of this virus or morphologic abnormalities could be found in any of the control eyes examined by light and electron microscopy, as well as virus assay. Ught Microscopic Findings

A neutrophilic infiltrate was present only in the ciliary bod,y, iris and cornea on day 1. This inflammatory response progressively increased in degree, reached its maximum intensity on day 4, and was then composed of mononuclear cells as well as polymorphonuclear leukocytes (Figures 1-3). At this time, the sclera, optic nerve and anterior chamber were also involved by the inflammatory infiltrate, and vascularization of the corneal stroma was evident. The intensity of the inflammatory response was more prominent in the normallv vascular uvea than in the cornea. By day 14, the major change in the anterior uveal tract was fibrosis with both anterior and posterior synechiae. Enlarged cells with eosinophilic intranuclear and/or intracytoplasmic inclusions were first seen on day 2. These findings were prominent in the epithelial cells of the ciliary body and iris, endothelial cells of the cornea, the retinal pigment epithelium (Figures 5 and 7) and in large mononuclear cells in the optic nerve, but were not observed in the sensory retina. With time, the inclusions progressively became more numerous and, like the inflammatory infiltrate, reached maximum intensity at day 4; however, the number of inclusions found varied in different regions of the eye. The inclusions gradually decreased and were conspicuously absent 14 and 28 days after intraocular inoculation (Text-figure 1). The greatest number of inclusions and the most marked inflammatory cell infiltrate was consistently found in the angles of the anterior chamber. Major pathologic alterations occurred in the outer nuclear layer and visual receptors of the sensory retina by day 4. Overlying the focal areas

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of infected retinal pigment epithelium, the visual receptors were absent. Their cell nuclei in the outer nuclear layer of the retina were decreased in number and replaced by bizarre, intensely hematoxylinophilic structures of variable shape and size (Figures 4 and 6). These marked morphologic abnormalities were accompanied neither by an inflammatory infiltrate nor by pathologic change in the inner portion of the retina. Examination of the liver for evidence of systemic spread of the virus showed a focal neutrophilic infiltrate, cytomegaly and intranuclear inclusions in the region of the central veins only on day 6. Elen

scopic Findings

Examination of random sections of the ocular tissues by electron microscopy.disclosed no viral particles at day 1; however, by day 2, evidence of vims replication was conspicuous. Cells of the ciliary body, iridial epithelium, corneal endothelium and the retinal pigment epithelium were enlarged and contained both intranuclear and intracytoplasmic viruses and inclusions (Figure 8). Although many viral particles were present in the nuclei of these cells, few had dense nuclear



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cores. Viral particles were seen budding through the nuclear membrane into a dilated perinuclear cisternae and receiving their membranous envelope (Figure 9). Once in the cytoplasm, the virus was observed in one of four forms: a) enveloped virus free in the cytoplasm (Figure 10), b) virus and membranous debris inside vacuoles (Figure 8), c) virus in large clumps associated with granular electron-dense material that was not membrane bound (Figure 8) and d) viral particles in small numbers inside membrane-bound homogeneous electron-dense bodies (Figure 10). The latter form was often detected outside the cells (Figure 10) and inside phagocytic vacuoles of leukocytes (Figures 13 and 14). Virus and viral inclusion bodies were observed from days 2 to 6, but neither were noted 14 or 28 days after the intraocular inoculation. The cellular changes in infected cells were first apparent on day 2. Migration of nucleoli to the periphery of the nucleus, peripheral clumping of nuclear chromatin, dilatation of the perinuclear cisternae and its interconnected endoplasmic reticulum, and slightly swollen mitochondria were evident (Figure 8). In addition, the Golgi apparatus was prominent, and the homogeneous electron-dense bodies were often seen closely associated with it (Figure 11). At days 4 and 6, the above ultrastructural alterations were accentuated, revealing many mitochondria was disrupted cristae, reduplication of the nuclear membrane, and many vacuoles containing cellular debris and viral particles. Morphologic evidence of neutrophils and macrophages phagocytizing viral parficles and attaching to the surface of injured cells accompanied these virusinduced cellular alterations (Figures 12-14). On days 14 and 28, the acute reaction had subsided and no evidence of an active infection was observed. However, a few necrotic cells were present, as were macrophages containing cellular debris. By day 4, alterations markedly different than those seen in the cells infected with virus were apparent in the cells of the sensory retina. At this time, the visual receptors overlying infected retinal pigment epithelial cells had undergone degeneration. Although not evident by light microscopy, marked fragmentation of the visual receptor apparatus with membranous fragments still attached to the degenerating retinal pigment epithelium was demonstrated by electron microscopy. A slight accumulation of this membranous debris was evident between the retinal pigment epithelium and the outer nuclear layer. In addition to the ultrastructural changes which characterized all infected cells, the retinal pigment epithelium contained myelin-like membranous accumulations within phagosomes. The latter structures appeared to be

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about as numerous in infected cells as in the normal retinal pigment epithelium. Focal areas of degenerative change in the outer nuclear layer of the retina consisted of extremely electron-dense, finely granular, clumped chromatin. In comparison to controls, the nuclei of the infected cells had indistinct nuclear membranes and varied from one another in size and shape (Figures 15 and 16). These focal alterations in the sensory retina were stll evident at days 14 and 28. Despite the marked degenerative changes, inflammation was not a significant component of this reaction. Even though the virus was initially inoculated immediately adjacent to the inner surface of the retina, no viral particles or cellular changes were noted in the ganglion cell, inner nuclear or inner plexiform layers of the retina. Virologic Findings

After an initial drop in infectious virus recovered at day 1, the titer of virus increased, reaching a peak at day 4. Recoverable infectious virus rapidly declined and remained at low levels thereafter (Textfigure 1). Discussion

This study demonstrates that MCMV can replicate and cause extensive alterations in a variety of cell types in the eyes of nonimmunosuppressed Swiss CD-1 mice. In the murine eye, many of the hallmarks of cytomegaloviral infection which are also seen in other cells of the mouse were observed. However, due to the unique anatomy of the eye, certain alterations occurred which have not been described in other organs. Cytomegalovirus assembles in the nucleus of infected cells and passes through the nuclear membrane, where it acquires a membranous envelope."6 Once in the cytoplasm, the enveloped virus associates itself with electron-dense homogeneous material. In studies with human fibroblasts,2, this cytoplasmic material was thought to be synthesized by the Golgi apparatus and microtubular system. This also appears to be true in the eye, since in this study the Golgi apparatus was consistently one of the first organelles to undergo morphologic alterations secondary to the viral injury. Once in the cytoplasm, the virus undergoes maturation and is released. The infected cells die, and lysis occurs. The recovery of infectious virus at days 14 and 28 may indicate persistence and/or latency of the virus, since morphologic evidence of MCMV was not present. A similar finding in mouse salivary glands was



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made by Brodsky et al.22 Thus, this experimental ocular disease may also be of use in the study of latent and/or persistent viral infection. The inflammatory cells and necrotic debris were most intense at the angles of the anterior chamber and around the optic nerve. Since intraocular fluid drains from the eye of the mouse at these locations, the virus and cellular debris probably accumulated in high concentrationsin these areas. In this regard, it is of interest that herpes simplex inoculated into the anterior chamber of rabbits also produces a marked inflammatory reaction in the chamber angles.23 The presence of inflammatory cells correlated directly with the cell necrosis and the cellular release of virus, as reflected by the rising titer of the recoverable virus. This early inflammatory reaction in the experimental ocular infection could have been initiated by one or more mechanisms. First, humoral antibody might have been elaborated against the invading viral agent and led to antigen-antibody complexes which resulted in chemotactic factors being elaborated through the complement system.24 Secondly, lymphocytes may have become sensifized to viral antigens and liberated chemotactic lymphokines.25 Also, cellular necrosis is known to lead to the infiltration of inflammatory cells.24,26 There is evidence, however, that MCMV suppresses the huune response in mice during acute infecmoral 2728 and cellular 29 tion. The alterations in the outer sensory retina, which were neither associated with virus nor accompanied by an inflammatory reaction, deserve comment. The occurrence of the above changes in the sensory retina probably does not reflect a specific reaction to the virus, for no evidence of viral replication was found in these cells. The outer retina is known to be especially sensitive to a wide variety of noxious stimuli including intense visible light (as in xenon and laser photocoagulation), x-ray irradiation, chloroquine and intraocular iron.- An intimate structural and functional relationship normally exists between the retinal pigment epithelium and the visual receptors.31-- An injury induced by the infection of retinal pigment-epithelial cells hence might be expected to cause significant alterations in the visual receptors and in the outer nuclear layer which contains the nuclei of these cells. The cellular trophism of MCMV for certain cells of the eye is in keeping with the known predilection of pathogenic viruses for specific cell types. In general, cytomegalovirus readily infects cells that constantly go through a cell cycle in vivo." In the present study, the neurons, including those in the ganglion cell and inner and and outer nuclear layers disclosed no evidence of viral replication. Since these cells do

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not undergo cell division in the mature retina, the cytomegalovirus may not be able to replicate in the neurons of the sensory retina, and this may be the reason why virus was not seen in these cells. The lesions described in this study are similar in several respects to the human ocular infection. Necrosis, cytomegaly and viral inclusions have all been reported in the human disease, and so has major involvement of the uvea and retinal pigment epithelium.1- As might have been anticipated, differences exist between the murine model and the human disease. The main differences relate to the degree of destruction produced by the chorioretinitis which occurs in man. In human eyes with CMV infection, which have been examined histologically, the sensory retina has been so necrotic and disrupted that the exact cells infected with the virus could not, as a rule, be positively identified. The reasons for the differences between the human disease and the murine model remain to be determined. References 1. Kalfayan B: Inclusion disease of infancy. Arch Pathol 44:467-476, 1947 2. Christensen L, Beeman H1V: Cytomegalic inclusion disease. Arch Ophthalmol 57:90-99, 1957 3. Burns RP: Cytomegalic inclusion disease uveitis. Arch Ophthalmol 61: 376-387, 1958 4. Dvorak-Theobald G: Cvtomegalic inclusion disease. Am J Ophthalmol 47: 52-56, 1959 5. Maschot WA, Daamen CPT: A case of cytomegalic inclusion disease with ocular involvement. Ophthalmologica 143:137-140, 1962 6. Miklos G, Orban T: Ophthalmic lesions due to cytomegalic inclusion disease. Ophthalmologica 148:98-106, 1964 7. Smith ME, Zimmerman LE, Harley RD: Ocular involvement in congenital cytomegalic inclusion disease. Arch Ophthalmol 76:696-699, 1966 8. Tsukamari T, Uneo I, Kawanishi H: Retinal changes in human cytomegalovirus infection. Am J Ophthalmol 62:1153-1160, 1966 9. Smith ME: Retinal involvement in adult cytomegalic inclusion disease. Arch Ophthalmol 72:44-49, 1964 10. Aaberg TM, Cesarz TJ, Rytel MV: Correlation of virology and clinical course of cytomegalic retinitis. Am J Ophthalmol 74:407-415, 1972 11. Wykinny CJ, Apple DJ, Guastella FR, Urgantas CM: Adult cytomegalic inclusion retinitis. Am J Ophthalmol 76:773-781, 1972 12. DeVenicia G, ZuRhein GM, Pratt M, Kisken W: Cytomegalic inclusion retinitis in an adult. Arch Ophthahmol 86:44-57, 1971 13. Editorial: Eyes after renal transplantation. Br Med J 3:127-128, 1972 14. Smith MG: Propagation in tissue culture of a cytopathogenic virus from human salivarv gland virus (SGV) disease. Proc Soc Exp Biol Med 92:424430, 1956 15. McCordock HA, Smith MG: The viscersal lesions produced in mice by the salivary gland virus of mice. J Exp Med 63:303-313, 1936



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16. Henson D, Strano AJ: Mouse cytomegalovirus: necrosis of infected and morphologically normal submaxillary gland acinar cells during termination of chronic infection. Am J Pathol 68:183-201, 1972 17. Reubner BH, Miyai K, Slusser RJ, Wedemeyer P, Medearis DN: Mouse cytomegalovirus infection: an electron microscopic study of hepatic parenchymal cells. Am J Pathol 44:799-821, 1964 18. Smith MIG: The salivary gland virus of man and animals. Prog Med Virol 2:171-202, 1969 19. Younger JS: Monolayer tissue cultures. I. Preparation and standardization of suspensions of trypsin-dispersed monkey kidney cells. Proc Soc Exp Biol Med 85:202-205, 1954 20. Plummer G, Benyesh-Melnick M: A plaque reduction neutralization test for human cytomegalovirus. Proc Soc Exp Biol Med 117:145-150, 1964 21. Craighead JE, Kanich RE, Ahmeida J: Nonviral microbodies with viral antigenicity produced in cytomegalovirs infected cells. J Virol 10:766-775, 1972 22. Brodsky I, Rowe WP: Chronic subclinical infection with mouse salivary gland virus. Proc Soc Exp Biol Med 99:654-655, 1958 23. Mlartenet A: Herpes simplex uveitis. Arch Ophthalmol 76:848-865, 1966 24. Snyderman R, Wholenberg C, Notkins AL: Inflammation and viral infection: chemotactic activity resulting from the interaction of antiviral antibody and complement with cells infected with herpes simplex virus. J Infect Dis 126:207-209, 1972 25. Glasgow LA: Cellular immunity in host resistance to viral infections. Arch Intern Med 126:125-134, 1970 26. Hill J, Ward P: C3 leukotactic factors produced by a tissue protease. J Exp Med 130:505-518, 1969 27. Osborn JE, Blazkovec AA, Walker DL: Immunosuppression during acute murine cytomegalovirus infection. J Immunol 100:835-844, 1968 28. Howard RJ, Kunerth V, Najarian JS: Interactions of cytomegalovirus (CMV) and the immune system. Fed Proc 32:3554, 1973 29. Miller J, Howard RJ, Hattler BG, Najarian JS: Correlation of MLC reactivity after experimental and clinical transplantation: virus and cytophilic antibody-sources of false negatives. Transplant Proc 5:1771-1774, 1973 30. Burger PC, Klintworth GK: Experimental retinal degeneration in the rabbit produced by intraocular iron. Lab Invest 30:9-19,1 974 31. Kroll AJ: Secondary retinal detachment: electron microscopy of retina and pigment epithelium. Am J Ophthalmol 68:223-237, 1969 32. Dowling JE, Sidman RL: Inherited retinal dystrophy in the rat. J Cell Biol 14:73-110, 1962 33. Young, RW: The renewal of rod and cone outer segments in the rhesus monkey. J Cell Biol 49:303-318, 1971 34. Haflick L, Moorhead PS: The serial cultivation of human diploid cell strains. Exp Cell Res 25:585-621, 1961

Acknowledgments We would like to thank Brenda Montgomery, Carol Carmthers, Linda Tomlinson, Gloria Dean and Bernard Lloyd for preparation of the tissue for light and electron microscopy, Jessie Calder and Carl Bishop for preparation of the photographs, and Donna Shumaker for typing the manuscript.

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Key to Figures CB = ciliary body, I = iris, R = retina, C = comea, GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer, VR = visual receptors, RPE = retinal pigment epithelium, Nu = nucleolus, IN = intranuclear inclusion, ICI, ICs = intracytoplasmic inclusions, VP = viral particle, PNC = perinuclear cisternae, CV = cytoplasmic vacuoles, E = envelope, GA = Golgi apparatus, HB = homogeneous body, NP = neutrophil, CE = comeal epithelium, P = pseudopod, NM = nuclear membrane, CC = clumped chromatin

Legends for Figures Fig 1-The normal-appearing anterior segment of a mouse eye inoculated with sterile salivary gland suspension and sacrificed at day 4 (H&E, x 230).

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Fig 2-The anterior segment of a mouse inoculated with infectious MCMV and sacrificed at day 4. Disruption of the normal architecture has occurred with an infittration of inflammatory cells (H&E, x 230).

Fig 3-A higher magnification of the ciliary body and adjacent structures (Figure 2) demonstrating the intense inflammatory reaction (H&E, x 400).

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Fig 4-The normal multilayered retina of a mouse inoculated with ultraviolet-irradiated virus and sacrificed at day 4 (H&E, x 300). Fig 5-The retinal pigment epithelium of an animal inoculated with a sterile salivary gland suspension and sacrificed at day 4. Note the normal size of the cells, as compared to those in Figure 7 (H&E, x 920). Fig 6-The retina of an infected animal at day 4. The visual receptors are absent, and the outer nuclear layer contains varying sized and shaped bodies replacing the normal cells. On the right is a more normal appearing outer nuclear layer. The retinal pigment epithelium is not shown (H&E, x 300). Fig 7-The retinal pigmented epithelium in an infected animal at day 4. Cytomegalic retinal pigmented epithelial cells containing intranuclear inclusions are present (H&E, x 920). Fig 8-A cytomegalic cell in the ciliary body seen at day 2. Note the peripherally marginated nucleoli, intranuclear and intracytoplasmic inclusions, viral particles, dilated perinuclear cistemae and cytoplasmic vacuoles. Inset-The viral particles are shown at a higher magnification (Uranyl magnesium acetate and lead citrate, x 10,000, inset, x 37,500).

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Fig 9-Virus particle budding from the nuclear membrane into the perinuclear cistemae and receiving its outer membranous envelope (Uranyl magnesium acetate and lead citrate, x 120,000). Fig 10-The membrane-bound, electron-dense homogeneous cytoplasmic bodies containing viral particles seen in the ciliary body at day 4 (Uranyl magnesium acetate and lead citrate, x 34,000). Fig 11-Dilated and prominent Golgi apparatus with virus and the electron-dense bodies closely related to it (Uranyl magnesium acetate and lead citrate, x 17,480).

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Fig 12-Neutrophil attaching to an altered comeal endothelial cell at day 6. Note the pseudopod protruding into the endothelial cytoplasm (Uranyl magnesium acetate and lead citrate, x 9850). Figs 13 and 14-Two neutrophils which have ingested cellular debris and virus at day 4 in the anterior chamber (Uranyl magnesium acetate and lead citrate, 13, x 9500; 14, x 12,000).

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Fig 15-A normal outer nuclear layer on day 4, seen in a mouse inoculated with a sterile salivary gland suspension. Note the distinct nuclear membrane and large nucleus with finely granular chromatin (Uranyl magnesium acetate and lead citrate, x 19,000). Fig 16-The outer nuclear layer at day 4 in an animal infected with MCMV. The nuclear membranes are indistinct, and the nucleus contains extremely electron-dense clumps of chromatin (Uranyl magnesium acetate and lead citrate, x 19,000).