s-Collidine as a Basis for Buffering Fixatives

Abstract

In 1952 Palade introduced the use of Michaelis' veronal-acetate buffer for regulating the pH of osmium tetroxide-fixing solutions for electron microscopy (1, 2). This buffer has enjoyed wide popularity and is regarded as satisfactory by many cytologists. The veronal-acetate buffer suffers from several limitations, however. I t is not particularly stable, since it supports the growth of mould or bacteria. Hence it is not practical to make up large amounts in bulk nor to store it over a considerable period of time. I t is not possible to make up a solution much more concentrated than that recommended by Palade, which approaches saturation for veronal (diethyl barbituric acid). The veronal-acetate mixture was developed by Michaelis (3) to meet the need for a relatively non-toxic buffer system capable of broad pH range and constant ionic strength. For this it works very well. I t is actually two buffer systems in one; i.e., sodium acetate-HCl and sodium veronal-HC1. The former is most efficient between pH 4.2 and 5.2 (pK for acetic acid is 4.76), but is nearly inoperative at pH 7 to 8. The veronalHCI system is most efficient between pH 7.5-8.5 (pK for veronal is 7.96 (4)). As recommended by Palade, osmium tetroxide is most commonly used at pH 7.2-7.5. At these pH's the veronal-HC1 system is operating at the acid edge of its range, where it is not very efficient in suppressing changes toward further acidity (2). Thus the combined veronal-acetate buffer system is not particularly effective in the pH region between its two buffer systems, namely between pH 5.2 to 7.5. This has been confirmed by titration curves. Even though our experience with Palade's buffered osmic fixative has been satisfactory, we undertook a search for a medium which might offer improvements from the point of view of stability and buffering capacity. Our attention was focused on s-collidine ('y-collidine or 2,4,6-tr imethylpyridine), which has a pK of about 7.4 (7.37 in