Acrolein - a Fine Structure Fixative for Viral Cytochemistry

The simplest architectural arrangement favored for infectious virus particles involves the encapsulation of a core of nucleic acid (either DNA or RNA) in a coat or capsid of antigenic protein. For a fine structure fixative to be a suitable candidate for viral cytochemistry, it must allow at least the identification and localization of these two fundamental moities. Such a fixative should preserve the antigenicity of viral proteins and allow their specific demonstration with labeled antibody conjugates (3, 24). The fixative should also preserve viral nucleic acids so that they may be identified and located by means of simple fluorochrome procedures (1, 15). A satisfactory performance includes the ability to carry out the relevant enzyme digestion tests necessary to establish specifically the nucleic acids identified. In addition, a degree of fine structural preservation of both virus particles and cell organelles should be attained so that examination of ultrathin sections of virus-infected cells in the electron microscope becomes a meaningful endeavor. Most viral antigens are well fixed by acetone (4), but this agent is a totally inadequate fixative for viral or cellular nucleic acids or for fine structure. Alcoholic fixatives such as Carnoy's fluid (60 per cent ethanol, 30 per cent chloroform, 10 per cent acetic acid), ethanol, ether-alcohol, and acetic alcohol allow a brilliant demonstration of both cellular and viral nucleic acids with acridine orange staining techniques (1, 16, 14). Both proteases and nucleases are fully active on such fixed material, enabling a confident identification of nucleic acid type. However, these fixatives do not preserve viral antigens and, although useful for demonstrating nucleic acids in purified virus preparations (30), are totally inadequate preservatives of cellular fine structure. Osmium tetroxide and potassium permanganate are excellent fine structural fixatives for electron microscopy, and enzyme digestion tests can be carried out on permanganate-fixed viruses (7, 8). However, although a few bacterial antigens appear to survive osmium tetroxide fixation (27), both the aforementioned fixatives denature viral antigens and are also quite inadequate for routine fluorescence microscopy. Formol (29, 10) and formol-calcium (24, 20, 10) have been the most successful fixatives, to date, for microcytochemistry and, in combination with water-soluble embedding materials, lead to identifiable electron images (2). However, cell fine structure is not well preserved routinely, and the results in the fluorescence microscope are not impressive. Use of these fixatives has led to an elegant demonstration of viral antigens in the electron microscope with ferritin-labeled antibodies (26, 24, 20), but postfixation with osmium tetroxide is necessary for a full revelation of cellular fine structure, and staining with specific antibody conjugates cannot, therefore, be carried out directly on ultrathin sections. In Table I the performances of a number of the so called "conventional" fixatives, as assayed from observations in both the fluorescence and electron microscopes, are summarized. Interest in acrolein as a fine structure fixative for electron microscopy was stressed by Luft (12) who was particularly impressed by its rapid penetration of tissue components in contrast to osmium tetroxide. Feder (9) pointed out the superiority of highly polar organic solvents,