DEMONSTRATION OF VIRAL IN UNSTAINED TISSUE SECTIONS WITH THE AID OF THE PHASE MICROSCOPE I. THE INCLUSION BODIES OF YELLOW , HERPES SIMPLEX, FOWL POX, AND DISTEMPER JUAN J. ANGULO, OSCAR W. RICHARDS, AND AGUST!N L. ROQUE Department of Experimental , School of Medicine, University of Havana, Havana, Cuba, and the Research Department, Scientific Instrument Division, American Optical Company, Buffalo, New York Received for publication December 3, 1948 The phase microscope provides means for observing structures too transparent to be seen with the ordinary bright-field microscope, such as details of the struc- ture of living, or unstained, fixed microorganisms and tissues. Thus, chromo- somes (Michel, 1941; Bosshard, 1944), the Golgi apparatus (Brice, Jones, and Smith, 1946; Jones, 1947a), and mitochondria (Albertini, 1946c; Jones, 1947b; Zollinger, 1948) have been observed without any staining, even in living cells. The process of mitosis (Michel, 1941), a serologic reaction with a protozoon (Harrison, Richards, Maurer, and Fowler, 1946), and other biological phenomena have been recorded in motion pictures. Extensive use of the phase microscope was made by Albertini (1945, 1946a,b, 1947), who applied it to pathology, espe- cially to the diagnosis of tumors and exudates by examining fresh as well as unstained fixed tissues and cells. Reviews of the applications of the phase microscope in biology are available elsewhere (K6hler and Loos, 1941; Loos, 1941; Bosshard, 1944; Richards, 1947a). These results suggested to us the feasibility of phase microscopy for the identi- fication of inclusion bodies. In a study of mitochondria and other cellular constituents, Zollinger (1948) noticed inclusions in cells from several tumors, some of which happened to be virus tumors, although no mention of this etiology was made in his paper. In this paper the results of a phase microscope examination of the inclusion bodies of , herpes simplex, fowl pox, and distemper as they appear in unstained tissue sections are presented. MATERIAL AND METHODS The yellow fever inclusion bodies were examined in the from a rhesus monkey inoculated with yellow fever virus. The herpes simplex material con- sisted of a liver specimen from a 9-day-old child that died from a generalized herpes . A strain of herpes simplex virus was isolated from the skin lesions of this case and identified (Quilligan, 1948). Besides this, the pathological findings and the clinical and epidemiological data contributed to the diagnosis. The fowl pox specimen was obtained from the skin of a chicken that had been inoculated 6 days before with fowl pox virus. Distemper material was obtained 297 1298 J. J. ANGULO, 0. W. RICHARDS, AND A. L. ROQUE IvoL.57 from the bladder of a mink with clinical and pathological pictures characteristic of distemper. All the specimens' were fixed and embedded by standard procedures. Sections were cut 4 microns thick. One section was stained with hematoxylin and eosin, and an adjacent one from the same specimen was mounted unstained in paraffin oil. The cover slip of the latter preparation was sealed with "gold size" to facilitate the use of an immersion objective. These stained preparations served as a control for checking the identification of the inclusion bodies made with the phase microscope. A field of the section the position or cell arrangement of which was such that it could be identified easily in the stained section as well as in the unstained one was selected for study. For comparison also, the unstained sec- tions were examined with an ordinary bright-field objective. The exposure time was double for the unstained preparations owing to the smaller amount of light transmitted by the phase system (Richards, 1947b). A Spencer phase micro- scope was used. The immersion objective employed contained a medium bright contrast, 0.14 A + 0.25 X diffraction plate. OBSERVAIONS The yellow fever intranuclear inclusion bodies (Torres bodies) were seen as somewhat refractile, coarsely granular structures. The inclusion bodies were readily detected in the sections studied, because normal nuclei showed a trans- parent, dark content except for the refractile nucleoli and occasional fibers or very small masses of opaque material. Councilman bodies were also easily noticed in the cytoplasm of some cells, as well as free among the masses of de- generating parenchyma. Although Councilman bodies can scarcely be regarded as true inclusion bodies, their occasional intracytoplasmic occurrence together with their diagnostic value leads us to mention their appearance with the phase microscope. Councilman bodies were more refractile than the liver cell cyto- plasm. The presence of vacuoles of different size and shape was apparent inside the bodies. Intracellular Councilman bodies were often surrounded by a narrow, dark, transparent zone. The distinctness of contour, shape, and size were also characteristic of Councilman bodies. The herpes simplex inclusion bodies (Lipschutz bodies) were also readily de- tected. Nuclei harboring those inclusion bodies showed an opaque granular mass that sometimes filled the entire nucleus. Structures that looked like the Councilman bodies of the yellow fever specimen were accidentally observed with the phase microscope. The identification was confirmed by an examination of the control stained preparation. In this section, the eosinophilic property of the bodies was also evidenced. Fowl pox inclusion bodies (Bollinger bodies) were seen without difficulty as 1 The authors are greatly indebted to Dr. Madureira Para from the Oswaldo Cruz Insti- tute, Rio de Janeiro, Brazil, for the yellow fever material; to Dr. A. James French, Depart- ment of Pathology, University of Michigan, Ann Arbor, Michigan, for the herpes specimen; and to Dr. John R. Gorham, Fur Animal Disease Research Laboratory, Pullman, Washing- ton, for the distemper sections. 1949] DEMONSTRATION OF VIRAL INCLUSION BODIES 299 refractile masses of irregularly granular structure contrasting with the almost transparent, dark background of the remaining cytoplasm. Some inclusion bodies resembled refractile rings owing to the presence of a central vacuole. It

Figure 1. Liver section from a monkey with yellow fever; unstained, bright-contrast, phase microscope. Intranuclear inclusion bodies in several cells. On the right, an intra- cellular Councilman body. 800 X. Figure 2. Magnified portion of figure 1 showing a cell with a small intranuclear inclusion body and sharp nucleolus. 2,500 X. Figure S. Magnified portion of figure 1 showing another cell with an intranuclear in- clusion body. 2,500 X. Figures 4, 5. Herpes inclusion body. Unstained, bright-contrast, phase microscope. 2,100 X. was interesting to note the sharpness of the structural details as well as the marked contrast of the inclusion bodies. The latter was favored by the balloon- ing degeneration of the cells harboring inclusion bodies. 300 J. J. ANGULO, 0. W. RICHARDS, AND A. L. ROQUE [VOL. 57

Figure 6. Three Councilman bodies in human liver with a generalized herpes infection. Unstained, bright-contrast, phase microscope. 800 X. Figure 7. Skin section of a chicken with fowl pox. Inclusion bodies in most cells. Un- stained, bright-contrast, phase microscope. 800 X. Figure 8. Magnified portion of figure 7 showing cell with an inclusion body. The nu- cleus, on the left, is degenerated. 2,500 X. Figure 9. Magnified portion of figure 7; another cell with an inclusion body. 2,500 X. The cytoplasmic inclusion bodies of distemper (Lentz bodies) were conspicuous because of the marked contrast of the inclusion body with the remaining cellular content. The clear, transparent zone around cytoplasmic inclusion bodies that 19491 DEMONSTRATION OF VIRAL INCLUSION BODIES 301 is seen in stained preparations of distemper showed in the bright-contrast, phase microscope as a dark, transparent zone. No structure could be detected in these unstained inclusion bodies with the phase microscope nor in the stained control preparation with the bright-field microscope. A few intranuclear inclusion bodies, showing a granular structure, were noticed in this specimen when studied unstained with the phase microscope or when examined stained in the ordinary bright-field microscope. ,AffA

Figmue 10. Bladder of mink with distemper. Unstained, bright-contrast, phase micro- scope. Only cytoplasmic inclusion bodies are in focus. 800 X.

DISCUSSION Despite the limited material studied, the results obtained suggest that no great amount of experience wvith phase microscopy is necessary for the identification of inclusion bodies in unstained tissue sections. The inclusion body ,as seen with marked contrast in every case. Besides, intracellular position, shape, size, and internal structure details, which were clearly seen with the phase microscope, helped to identify readily the inclusion bodies studied. There was a distinct difference, for instance, between the image of a normal nucleus and that of an inclusion-bearing one in the fixed tissue sections studied. In the normal nucleus the only structures visible with the bright-contrast, phase microscope were the nuclear membrane and the nucleolus. The remainder of the nucleus was dark and transparent except for some fine fibers or very small masses of opaque ma- terial. This image somewhat resembles that from dark-field microscopy. When a nucleus harbored an inclusion body, this was readily detected as a slightly refractile mass situated in the above-mentioned dark, transparent space of the nucleus, and the refractile nucleolus was in many cases missing. Cytoplasmic inclusion bodies could be identified similarly. The phase microscope seems to be a promising instrument for the study of inclusion bodies in unstained sections because of the structural details revealed and the avoidance of artifacts that might be due to staining. It also saves time, equipment, and reagents. Further use of the phase microscope in the study of 302 J. J. ANGULO, 0. W. RICHARDS, AND A. L. ROQUE [VOL. 57 living material will probably yield other advantages. In addition, the phase microscope appears to be useful in the diagnosis of virus diseases, and further study should be made to determine its usefulness in this field. The finding of Councilman bodies in the liver of a generalized herpes case is interesting. According to Klotz and Belt (1930), Councilman bodies may be taken as pathognomonic of yellow fever. Penna and Figueiredo (1929) and Villela (1941) share this opinion. However, Belt (1939) found Councilman bodies in the liver of each of four humans suffering fatal burns.

SUMMARY By means of bright-contrast, phase microscopy, virus inclusion bodies were easily detected in unstained sections of tissues from yellow fever, herpes simplex, fowl pox, and distemper cases. The marked contrast and structural details shown by the inclusion bodies helped in their identification. The phase micro- scope offers promise for studies on the structure of inclusion bodies as well as in the diagnosis of virus diseases. Councilman bodies were observed in the liver of a child suffering a generalized herpes infection. REFERENCES ALBERTINI, A. VON 1945 Zur Anwendung der Phasenkontrastmikroskopie in der patho- logischen Histologie. Schweiz. Z. allgem. Path. Bakt., 8, 298-310. ALBERTINI. A. VON 1946a Erfahrungen und Ergebnisse mit dem Phasenkontrastverfahren in der normalen und pathologischen Histologie. Praxis, Schweiz. Runds. Med., 35,107- 112. ALBERTINI, A. VON 1946b Cytologische Exudatbefunde mit dem Phasenkontrastverfah- ren. Schweiz. Z. allgem. Path. Bakt., 9, 701-706. ALBERTINI, A. VON 1946c Pflasterepithelzellen in Phasenkontrastbild. Acta Anat., 1, 463-468. ALBERTINI, A. VON 1947 Vergleichende histologische Geschwulstuntersuchungen mit dem Phasenkontrastverfahren. Schweiz. Z. allgem. Path. Bakt., 10, 4-29. BELT, T. H. 1939 Liver necrosis following burns, simulating the lesions of yellow fever. J. Path. Bact., 48, 493-498. BOSSHARD, F. 1944 Phasenkontrast-Mikroskopie. Schweiz. Brau. Rundschau, 55, 99- 131. BRICE, A. T., JONES, R. P., AND SMITH, J. D. 1946 Golgi apparatus by phase contrast microscopy. Nature, 157, 553-554. HARRISON, J. A., RICHARDS, 0. W., MAURER, J. A., AND FOWLER, E. H. 1946 Serologic reactions with two strains of Paramecium bursaria. Anat. Record, 94 (suppl.), 39. JONES, 0. P. 1947a The Golgi element in primitive erythroblasts of the 11-day rat em- bryo. Anat. Record, 97 (3), 77. JONES, 0. P. 1947b Mitochondria and their relation to the so-called hyaloplasm. J. Lab. Clin. Med., 32, 700-719. KLOTZ, O., AND BELT, T. H. 1930 The pathology of the liver in yellow fever. Am. J. Path., 6, 663-687. KOHLER, A., AND Loos, W. 1941 Das Phasenkontrastverfahren und seine Answendung in der Mikroskopie. Naturwissenschaften, 29, 49-61. (A translation is available in the Textile Research J., 1947, 17, 82-95.) Loos, W. 1941 Das Phasenkontrastverfahren nach Zernicke als biologisches Forschung- smittel. Klin. Wochschr., 20, 849-853. MICHEL, K. 1941 Die Darstellung von Chromosomen mittels des Phasenkontrastverfah- ren. Naturwissenschaften, 29, 61-62. 1949] DEMONSTRATION OF VIRAL INCLUSION BODIES 303

PENNA, 0., AND FIGUEIREDO, B. 1929 Contribucao ao estudo da histo-pathologia do figado na febre amarella. Folha med., 10, 229-233. QUILLIGAN, J. J. 1948 To be published. RICHARDS, 0. W. 1947a Biological phase microscopy. Cold Spring Harbor Symposia Quant. Biol., 11, 208-214. UICHARDS, 0. W. 1947b Phase photomicrography. J. Biol. Phot. Assoc., 16, 29-38. VILLELA, E. 1941 Histology of human yellow fever when death is delayed. Arch. Path., 31, 655-669. ZOLLINGER, H. U. 1948 Cytologic studies with the phase microscope. II. The mito- chondria and other cytoplasmic constituents under various experimental conditions. Am. J. Path., 24, 569-589.