This paper describes an investigation by ultra-violet spectrometry of the physical and chemical changes caused by treating interphase chick heart fibroblasts, grown in tissue culture, with a variety of fixatives. The same cells, before and after fixation, were photographed either at 265 and 313 mµ, or at a series of wavelengths required for an absorption spectrum.
2. The nucleus of the living interphase cell, apart from nucleoli, is nearly homogeneous, and non-specific light loss measured by the optical extinction at 313 mµ (E313) is not detectable. After fixation the nuclear material is redistributed into filamentous and rounded structures. These nuclear structures are least apparent after osmium tetroxide, methanol freezing-substitution, and freezing-drying; may be more marked after 10 per cent, neutral formalin; and are very marked after Carnoy fixation. E313 is measurable in a fixed nucleus and the ratio of optical extinctions E313/E265 is always less than about 15 per cent. E265increases as a result of the shrinkage of the nuclear area which ranges from about 8 to 16 percent, for osmium tetroxide, about 5 to 35 percent, for neutral formalin, about 19 to 47 per cent, for Carnoy, and about 20 to 40 per cent, for freezing-substitution. Apart from occasional cracks in cells, freezing-substitution and freezing-drying preserved nuclear and cytoplasmic structure as well as osmium tetroxide.
3. (i) Total nuclear extinction is defined as average nuclear extinction times nuclear projected area. Decreases in total nuclear extinction at 265 m µ, after fixation in Carnoy and neutral formalin, range from 10 to 35 per cent, and may be attributed to losses of absorbing material. After formalin fixation there is a decrease of approximately 40 per cent, in the total cytoplasmic extinction at 265 mµ The method of correcting for nonspecific light loss and other possible sources of error are discussed.
(ii) Osmium tetroxide itself absorbs in the ultra-violet and ‘stains’ the cell, thereby increasing the optical extinction. The staining reaction was studied by fixing films of protein, fat, and nucleic acid.
(iii) After methanol freezing-substitution the total extinction of the nucleus increases slightly.
4. The ratio of optical extinctions E265/E280for the living nucleus is about 2.0 and by applying the methods of curve analysis due to Caspersson it is concluded that the amount of ‘standard protein’ in the nucleus is small compared with the amount of nucleic acid. After all methods of fixation E265/E280 decreases to about 1.3 and similarly it is concluded that the fixed nucleus contains about ten to twenty times as much ‘standard protein’ as nucleic acid. This curious result is discussed and several possible explanations suggested. It is concluded that the derivation of relative amounts of protein and nucleic acid by analysis of the absorption spectra of either living or fixed cells should be treated with caution.