APPLIED MICROBIOLOGY, Jan. 1972, p. 141-144Copyright © 1972 American Society for Microbiology

Vol. 23, No. 1Printed in USA.

Electron Microscope Methods for theIdentification of Adenoviruses Isolated in Micro

Tissue Cultures1M. J. ROSENBAUM, R. C. KORY, K. A. SIEGESMUND, H. J. PEDERSEN, E. J. SULLIVAN, AND R.

0. PECKINPAUGHVirology Division, Naval Medical Research Unit No. 4, Great Lakes, Illinois 60088; Veterans AdministrationHospital, Wood, Wisconsin 53193; and Department of Anatomy, Medical College of Wisconsin, Milwaukee,

Wisconsin 53232

Received for publication 4 August 1971

A tissue culture micromethod is described for adenovirus isolation and prep-

aration for presumptive identification by electron microscopy. These proce-dures are easier, more economical, and faster than conventional methods. Themicro techniques make it more feasible to utilize direct visualization of virus ininfected cells as an adjunctive diagnostic and research tool.

Micromethods for cell cultivation in dispos-able plastic plates have been shown to be relia-ble, rapid, and economical techniques for thesero-diagnosis of viral infections (3, 4). Recentinvestigations have demonstrated that thesemethods also can be used for the isolation ofvarious respiratory disease viruses, particu-larly, the adenoviruses (E. J. Sullivan, manu-script in preparation). In addition to the tech-nical advantages mentioned above, the recog-nition of viral cytopathic effect (CPE) is facili-tated by the micro technique. It was hypothe-sized that if electron microscopy could be ap-plied to the specimen, at the time CPE wasfirst evident, then direct visualization of viralmorphology might be obtained. This procedurewould further decrease the time required foridentification of a viral isolate.

In 1968 Rosenbaum et al. reported a simpli-fied method for electron microscopy of tissueculture cells by employing the micromethod(2). Modifications of these techniques wereused in the present investigation of virus-in-fected cells. The results obtained verified theusefulness of the method as a practical andeconomical tool for improving the identificationof viral agents.

MATERIALS AND METHODSThe procedures for the cultivation, inoculation,

and preparation of infected tissue cultures for elec-tron microscopy are diagrammed in Fig. 1.

I This investigation was done in connection with researchproject MF12.524.009-4019AF61 of the Bureau of Medicineand Surgery, Navy Department, Washington, D.C.

The technique for cultivation of cells in micro-plates has been previously described (4). For virusisolation, HeLa cells were added to each well of amicroplate at a concentration of 7,500 cells per 0.025ml of growth medium (Eagle's minimum essentialmedium plus 10% fetal calf serum). Then one drop(0.025 ml) of throat-swab specimen obtained from apatient with acute respiratory disease was placed ineach of eight wells containing the seed cells. Cellsand inoculum were mixed by shaking and incubatedin a humidified CO2 incubator (2% CO2 in air) at 34C. When cultures showed CPE involving approxi-mately 25% of the cell monolayer, the media in wells

2 31

SEED CELLS MONOLAYERIUAE 72 I FORMS

(361)

W 5

RX ANDIENYDRATE

CFE

EMIED AND TRIM PELLET MOUNTINCUBATE 36 SECTION

(6rc) IFIG. 1. Preparation of microplate cell cultures for

embedding and ultrasectioning.141

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ROSENBAUM ET AL.

FIG. 2. Appearance of uninoculated control (A) and infected HeLa cells exhibiting viral cytopathology (B).x 300.were removed and the residual cells were gentlywashed three times with 0.1 M phosphate-bufferedsaline (PBS). The cells were fixed for 30 min with a2.7% solution of osmium tetroxide in PBS. The fixedcells were then quickly washed with PBS to removeexcess fixative. This step was followed by cell dehy-dration through graded concentrations of ethyl al-cohol (50, 75, and 95%), allowing 3 to 5 min in each

bath. Final dehydration was accomplished by two10-min changes in absolute ethyl alcohol.

Neither propylene oxide nor acetone could be usedeither as a transitional solvent or for infiltration,since these chemicals would have dissolved theplastic wells. Therefore, after the absolute alcoholwas removed, the cells were immediately embeddedin Epon 812 by the method of Luft (1) using three

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IDENTIFICATION OF ADENOVIRUSES

parts of Epon A to seven parts of Epon B. The resinwas polymerized at 60 C for 36 hr.

After polymerization, a plastic well containing theembedded cells was selected and cut away from themicroplate with a razor blade. An LKB Pyramitomewas used to section through the plastic until the in-terface between the plastic and the resin wasreached. The block was then transferred to an LKBUltrotome III where thin sections for electron mi-croscopy were prepared with a DuPont diamondknife.The sections were mounted unsupported on a 300-

mesh grid and counterstained with uranyl acetateand lead citrate. The tissue was examined and pho-tographed with an RCA EMU-3G electron micro-scope at 50 kv.

RESULTS AND DISCUSSIONThe appearance of uninoculated cell control

and cultures with evidence of CPE are shownin Fig. 2. Although specific viral cytopathologycan be recognized, the causative agent usuallycannot be definitely ascertained until virustyping tests are performed.

Figure 3 is an electron micrograph of a thinsection prepared from a replicate culture inoc-ulated with the same specimen. Intranuclearinclusions with crystalline packing typical ofadenovirus can be seen. Also the characteristicmorphology of the adenovirus particle isreadily apparent. This will suffice to identifythe viral group of the infecting agent, but sero-logical tests still must be done to establish itstype specificity. Nevertheless, the time forpresumptive diagnosis of the etiological agentmay be decreased from several weeks to a fewdays by eliminating the need to apply physico-chemical procedures where tissue cytopathol-ogy is uncertain or misleading.

It is recognized that not all groups of virusesproduce distinctive cellular inclusions, buttentative diagnoses may be deduced from themorphological appearance of the virus particleand its site of cellular localization. Applicationof ferritin antibody techniques in conjunctionwith the method described may aid in obscurecases.

FIG. 3. Electron micrograph of a HeLa cell showing intranuclear adenovirus particles in crystalline array.(NM, nuclear membrane; x 39,500).

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144 ROSENBAUM ET AL.

These micro techniques would be particu-larly useful for the identification of coronavi-ruses or other viruses which produce little orno discrete cytopathology. Organ cultures inmicroplates could be inoculated with such sus-pected viruses and later thin sections could beprepared in situ and examined for the pres-ence of the coronavirus particles. It is conceiv-able that viral agents, heretofore unrecognized,may be detected by employing these tech-niques.

APPL. MICROBIOL.

LITERATURE CITED

1. Luft, J. H. 1961. Improvements in epoxy resin embeddingmethods. J. Biophys. Biochem. Cytol. 9:409-414.

2. Rosenbaum, M. J., A. M. Earle, A. L. Chapman, and E.J. Sullivan. 1968. Simplified procedure for in situembedding cell monolayers for electron microscopy.Lab Pract. 17:713-714.

3. Rosenbaum, M. J., E. J. Sullivan, and E. A. Edwards.1970. Micromethods for respiratory virus seroepide-miology. Health Lab. Sci. 7:43-52.

4. Sullivan, E. J., and M. J. Rosenbaum. 1967. Methods for.preparing tissue culture in disposable plastic plates andtheir use in virology. Amer. J. Epidemiol. 85:424-437.

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