DEMONSTRATION OF VIRAL INCLUSION BODIES IN UNSTAINEDTISSUE SECTIONS WITH THE AID OF THE

PHASE MICROSCOPEI. THE INCLUSION BODIES OF YELLOW FEVER, HERPES SIMPLEX, FOWL POX,

AND DISTEMPER

JUAN J. ANGULO, OSCAR W. RICHARDS, AND AGUST!N L. ROQUEDepartment of Experimental Pathology, School of Medicine, University of Havana, Havana,

Cuba, and the Research Department, Scientific Instrument Division, AmericanOptical Company, Buffalo, New York

Received for publication December 3, 1948

The phase microscope provides means for observing structures too transparentto 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, andSmith, 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 phenomenahave been recorded in motion pictures. Extensive use of the phase microscopewas 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 asunstained fixed tissues and cells. Reviews of the applications of the phasemicroscope 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 virus inclusion bodies. In a study of mitochondria and other cellularconstituents, Zollinger (1948) noticed inclusions in cells from several tumors,some of which happened to be virus tumors, although no mention of this etiologywas made in his paper.

In this paper the results of a phase microscope examination of the inclusionbodies of yellow fever, herpes simplex, fowl pox, and distemper as they appear inunstained tissue sections are presented.

MATERIAL AND METHODS

The yellow fever inclusion bodies were examined in the liver from a rhesusmonkey 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 generalizedherpes infection. A strain of herpes simplex virus was isolated from the skinlesions of this case and identified (Quilligan, 1948). Besides this, the pathologicalfindings and the clinical and epidemiological data contributed to the diagnosis.The fowl pox specimen was obtained from the skin of a chicken that had beeninoculated 6 days before with fowl pox virus. Distemper material was obtained

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from the bladder of a mink with clinical and pathological pictures characteristicof distemper.

All the specimens' were fixed and embedded by standard procedures. Sectionswere cut 4 microns thick. One section was stained with hematoxylin and eosin,and an adjacent one from the same specimen was mounted unstained in paraffinoil. The cover slip of the latter preparation was sealed with "gold size" tofacilitate the use of an immersion objective. These stained preparations servedas a control for checking the identification of the inclusion bodies made with thephase microscope. A field of the section the position or cell arrangement of whichwas such that it could be identified easily in the stained section as well as in theunstained one was selected for study. For comparison also, the unstained sec-tions were examined with an ordinary bright-field objective. The exposure timewas double for the unstained preparations owing to the smaller amount of lighttransmitted by the phase system (Richards, 1947b). A Spencer phase micro-scope was used. The immersion objective employed contained a medium brightcontrast, 0.14 A + 0.25 X diffraction plate.

OBSERVAIONS

The yellow fever intranuclear inclusion bodies (Torres bodies) were seen assomewhat refractile, coarsely granular structures. The inclusion bodies werereadily detected in the sections studied, because normal nuclei showed a trans-parent, dark content except for the refractile nucleoli and occasional fibers orvery small masses of opaque material. Councilman bodies were also easilynoticed in the cytoplasm of some cells, as well as free among the masses of de-generating parenchyma. Although Councilman bodies can scarcely be regardedas true inclusion bodies, their occasional intracytoplasmic occurrence togetherwith their diagnostic value leads us to mention their appearance with the phasemicroscope. Councilman bodies were more refractile than the liver cell cyto-plasm. The presence of vacuoles of different size and shape was apparent insidethe bodies. Intracellular Councilman bodies were often surrounded by a narrow,dark, transparent zone. The distinctness of contour, shape, and size were alsocharacteristic of Councilman bodies.The herpes simplex inclusion bodies (Lipschutz bodies) were also readily de-

tected. Nuclei harboring those inclusion bodies showed an opaque granularmass that sometimes filled the entire nucleus. Structures that looked like theCouncilman bodies of the yellow fever specimen were accidentally observed withthe phase microscope. The identification was confirmed by an examination ofthe control stained preparation. In this section, the eosinophilic property of thebodies was also evidenced.Fowl pox inclusion bodies (Bollinger bodies) were seen without difficulty as1 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.

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refractile masses of irregularly granular structure contrasting with the almosttransparent, dark background of the remaining cytoplasm. Some inclusionbodies 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 inclusionbody 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 themarked contrast of the inclusion bodies. The latter was favored by the balloon-ing degeneration of the cells harboring inclusion bodies.

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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 conspicuousbecause of the marked contrast of the inclusion body with the remaining cellularcontent. The clear, transparent zone around cytoplasmic inclusion bodies that

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is seen in stained preparations of distemper showed in the bright-contrast, phasemicroscope as a dark, transparent zone. No structure could be detected in theseunstained inclusion bodies with the phase microscope nor in the stained controlpreparation with the bright-field microscope. A few intranuclear inclusionbodies, showing a granular structure, were noticed in this specimen when studiedunstained with the phase microscope or when examined stained in the ordinarybright-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 greatamount of experience wvith phase microscopy is necessary for the identificationof inclusion bodies in unstained tissue sections. The inclusion body ,as seenwith 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 distinctdifference, for instance, between the image of a normal nucleus and that of aninclusion-bearing one in the fixed tissue sections studied. In the normal nucleusthe only structures visible with the bright-contrast, phase microscope were thenuclear membrane and the nucleolus. The remainder of the nucleus was darkand transparent except for some fine fibers or very small masses of opaque ma-terial. This image somewhat resembles that from dark-field microscopy. Whena nucleus harbored an inclusion body, this was readily detected as a slightlyrefractile mass situated in the above-mentioned dark, transparent space of thenucleus, and the refractile nucleolus was in many cases missing. Cytoplasmicinclusion 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 revealedand 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

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living material will probably yield other advantages. In addition, the phasemicroscope appears to be useful in the diagnosis of virus diseases, and furtherstudy 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 betaken as pathognomonic of yellow fever. Penna and Figueiredo (1929) andVillela (1941) share this opinion. However, Belt (1939) found Councilmanbodies in the liver of each of four humans suffering fatal burns.

SUMMARY

By means of bright-contrast, phase microscopy, virus inclusion bodies wereeasily detected in unstained sections of tissues from yellow fever, herpes simplex,fowl pox, and distemper cases. The marked contrast and structural detailsshown by the inclusion bodies helped in their identification. The phase micro-scope offers promise for studies on the structure of inclusion bodies as well asin the diagnosis of virus diseases. Councilman bodies were observed in the liverof a child suffering a generalized herpes infection.

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