Principles of biological microtechnique; a study of fixation and dyeing

Abstract

The Reactions of Fixatives with Proteins, i. The Visible Effects 31 3 The Reactions of Fixatives with Proteins. 2. The Chemical Changes 4 The Reactions of Fixatives with Tissues and Cells: Methods of Research 5 Primary Fixatives Considered Separately, i . Coagulants 6 Primary Fixatives Considered Separately. 2. Noncoagulants III 7 Fixative Mixtures PART II: DYEING 8 Introduction to the Chemical Composition of Dyes 9 The Classification of Dyes 10 The Direct Attachment of Dyes to Tissues 1 1 The Indirect Attachment of Dyes to Tissues 12 The Differential Action of Dyes 13 Metachromasy 14 The Blood Dyes 15 '^e specially the latter's lactophenol. Amann called his fluids Beohachtiingsmedien, to indicate that they not only prevented the decay of tissues, but were media in which cells might remain during microscopical examination. It is desirable that research FIXATIONLiving cytoplasm commonly has a refractive index (r.i.) in the neighbourhood of 1-353;*^^'*^^t hat is to say, not very much higher than that of the saline solutions in which cells are commonly immersed for vital study. An aqueous solution of sodium chloride at 0*9% has an r.i. of i*335.^^-"-When cells are examined alive in such media, a w^ater-immersion objective w^ill give almost as good resolution as a first-rate oil-immersion objective, for the high numerical aperture of the latter will be partly wasted. One may surround living cells with innocuous media of the same r.i. as the cytoplasm ** and thus obtain slightly higher resolution (as well as gaining other advantages), but the difference will not be great. As soon, however, as a fixative acts, a profound change occurs. The evidence suggests ** that the protoplasm is now represented by interlacing sub-microscopic fibres having the r.i. of dry protein (about 1*54). These fibres lie in water, if the fixative is aqueous; this can be replaced by media of any desired refractive index. If a medium of r.i. close to that of dry protein is used (Canada balsam, for instance), two results ensue: almost perfect transparency is obtained (which may be modified as desired by the use of dyes), and oil-immersion objectives can be used at their full aperture. This fact should not, however, be too strongly stressed, for the making of a permanent microscopical preparation involves considerable shrinkage of the tissues of organisms (p. 76), and this reduces or nullifies the advantage of higher microscopical resolution. The advantage can be fully secured only if the object to be examined happens to be unshrinkable by the processes involved in making a permanent preparation. The valves of diatoms provide an example. Some of the constituent parts of organisms do not require fixation, because they are not subject to autolysis and are resistant to bacteria and moulds and to most of the reagents ordinarily used in microtechnique. Examples are chitin, cellulose, scleroproteins, certain inorganic crystals, and amorphous silica. Most such substances not only do not need fixation but are not acted upon by fixatives; or, if acted upo...

Ethane-Freezing/Methanol-Fixation of Cell Monolayers: A Procedure for Improved Preservation of Structure and Antigenicity for Light and Electron Microscopies

“…Light microscopic observations of structural changes in living cells under aldehyde treatment have shown that many organelles, such as mitochondria (Bereiter-Hahn and Voeth, 1979), chromosomes (Skaer and Whytock, 1976), or cytoskeletal elements (Luther and Bloch, 1989) undergo significant changes in shape or position in the early seconds of fixation, before gelation of cytoplasm (Millonig and Marzzoni, 1968). Furthermore, as aldehydes cannot effectively cross-link membrane-lipids, membrane artefacts are often observed (Baker, 1968;Bereiter-Hahn and Voeth, 1979;Hasty and Hay, 1978). Fixation-induced fusion of vesicles has been observed (Bretscher and Whytock, 1977;Doggenweiler and Heuser, 1967;Hausmann, 1977).…”

Coagulant and Non-Coagulant Fixation of Plant Cells

“…Though the subject has not yet been analysed by a competent scientific historian, it seems likely that coagulant fixatives have been retained in plant microtechnique because they give better results with the paraffin procedure than either osmium tetroxide or formaldehyde (see Baker 1958). The paraffin wax procedure does have some advantages over any plastic-embedding procedure so far devised since it yields ribbons of sections.…”

“…It is clear that Baker's (1958) term "coagulant" is ideally suited to describe the effects of the traditional botanical fixatives (FAA, CRAF), since these fixatives convert the fine structure of the cell into a stringy precipitate that is somewhat coarser than that produced by boiling. The action of the coagulants upon nuclear chromatin in the interphase nuclei illustrated here produces massive alteration in structure.…”

Electron Microscopy of Retinal Photoreceptors

“…All these techniques have in common the prolonged treatment of the tissue by dichromate solutions. The staining of lipids is a result of the binding capacity of lipids for chromium (3,16) which then acts as a mordant for hematoxylin. Furthermore, cbromation stabilizes some lipid fractions by rendering them insoluble in organic solvents (3,5).…”

“…The staining of lipids is a result of the binding capacity of lipids for chromium (3,16) which then acts as a mordant for hematoxylin. Furthermore, cbromation stabilizes some lipid fractions by rendering them insoluble in organic solvents (3,5). It was particularly this last property that seemed advantageous in considering the maintenance of the structure in lipoprotein systems for their study at the level of the electron microscope.…”