Koelle's copper thiocholine method performed with a low-pH phosphate buffer, followed by osmification of the precipitate: revival of two abandoned procedures.

Using a glutaraldehyde-fixed mouse neuromuscular junction, a fine precipitate of copper thiocholine was obtained with Koelle's medium prepared by a mixture of phosphate buffer (pH 5.6-5.9) and copper glycine solution (strongly acidic). The final pHs of these incubation media were very low, being situated between 3.8 and 4.2, respectively. It is well known that phosphate buffer, at such a low pH value, has no buffering effect on the acetic acid of enzymatic hydrolysis. This probably caused a sharp drop of the pH value in the vicinity of the enzymatic site and allowed a fine localization of copper thiocholine, the precipitation of which is pH dependent. Furthermore, the osmification of copper thiocholine in the same phosphate buffer provided a finely localized electron dense product. The chemical nature of the osmified copper thiocholine is discussed.