In a previous paper we have described a method for the segregation of viruslike bodies from cancerous animal tissues by the use of nonpolar organic solvents that form emulsions with aqueous liquids containing highly dispersed tissues. These solvents were selected in order to have greater control over the specific gravity and viscosity of the isolating medium and, at the same time, to avoid the difficulties usually encountered in aqueous systems.We had first selected monochlorbenzene for the organic solvent since its low viscosity and specific gravity of 1.1 produced the effect that long periods of high centrifugation were avoided. In a search for solvents similar but possessing an absolute minimum of toxicity, we found the fluorocarbons to be highly suitable. Particularly, the toxicities of Freon 112 t (CCl aF-CClaF) or Genetron 226t (CFaCl-CClaF), are of the order of that of carbon dioxide. Their specific gravities of 1.6 allowed easy adjustment with an admixture of n-heptane to resemble the specific gravity of proteins. N-heptane is a saturated hydrocarbon possessing a density of Ca. 0.7, and the qualities and low toxicity of petrol ether (n-h ex an e). The latter hydrocarbon has been frequently mentioned in the literature as a useful agent to use in extracting fat without affecting the viability of viruses contained in the tissue. The surface tensions of both Freon 112 and Genetron 226 are very low, producing the effect that their emulsions with water break easily and cleanly upon standing or at low-speed centrifugation.For a suitable test virus we selected vaccinia virus grown on the chorioallantoic membranes of Ll-day-old fertile chicken eggs. The homogenization of the infected membranes and the segregation of the viruses contained in them required a total time of less than 2 hours. The viability of the obtained vaccinia viruses compared very favorably with our controls. In reality, dilutions could be made after our Freon-heptane treatment that showed a titer of 10-9 , while our control showed a titer of 10. 7 • In view of the ease of operation of this method and the freedom of the isolated viruses from nonviral proteins and lipids, we believe that the
Two forms of a high speed type of microtome are described which are able to cut cross sections to the thinness required for electron microscopic study (0.1 to 0.8 micron, depending on the material). Techniques of microtome operation are suggested together with a few methods of sample preparation and subsequent section treatment, namely, collecting, selecting, and mounting. Embedding materials which sublime readily are described; these have been used successfully with this high speed microtome to support many types of materials. These volatile embedding materials have the advantage that they eliminate the difficulties that on some occasions arise with the use of solvents in the process of solvent extraction of embedding materials, such as Carbowax and paraffin, from the sections. A number of photographs are shown of the high speed microtome as well as electron micrographs of a few sections produced by the instrument. These pictures of rubber, rayon, Lucite, block Nylon, and animal tissue indicate some of the fields of application of the microtome and demonstrate the effectiveness of the necessary auxiliary techniques for it, particularly sample hardening and embedding, and section collecting and mounting.
In 2 accompanying papers we have described a method for the isolation of animal viruses by fluorocarbon emulsification. Vaccinia virus and Rous sarcoma fowl virus had been selected for test materials. They were chosen because they can be grown easily and because their dimensions represent 2 extremes of virus sizes. Vaccinia virus is one of the largest whose bricklike shape has the average dimensions of 250 x 350 m/-L. Rous virus is of globular form, and its size, as isolated by our method, is near the threshold of visibility of the electron microscope, which is approximately 5 m/-L. Notwithstanding this 1:50 difference in linear-size levels, the Freon-heptane emulsification technique worked equally well in either case, producing what we believe to be satisfactory results as to the ease of the method and the purity of the segregated viruses. Some unreported forms of the Rous virus were observed, and a discussion of the experimental procedure and the results seems necessary.With the availability of an efficient method for the separation of nonviral proteins from the nucleoproteins of virus particles, a very thorough mincing and homogenization of virus-bearing tissue becomes practicable.
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