The stainability of neural elements is determined during the various stages of tissue preparation which precede impregnation.
The hypothesis (Romanes, 1950) that variations in the physiological state of the neurones at the time of fixation might influence the stain was examined. Feeding and anaesthesia did not influence the staining of gastric nerve fibres under the conditions of the experiments.
A study was made of fixing fluids for Bodian staining. A picric-acetic-alcohol mixture and alcoholic Bouin were convenient and effective, each being superior to formaldehyde-acetic-alcohol. For overnight fixation formaldehyde solution neutralized with calcium carbonate was satisfactory, but where prolonged fixation is permissible formaldehyde neutralized with excess of magnesium carbonate gave superior results.
Attention was drawn to the probability that the final stain could be improved by ‘pretreatment’ of mounted sections before silvering. Dewaxed sections were treated individually with 27 substances from a variety of chemical groups to determine the effects on subsequent Bodian staining. Nitric and sulphuric acids, formaldehyde solution neutralized with magnesium carbonate, methyl alcohol, and perhaps sodium carbonate contribute in different ways towards better preparations. In particular pretreatment with nitric acid suppresses the background stain, especially in smooth muscle; sulphuric acid increases the number of fine nerve fibres displayed, and methyl alcohol facilitates the staining (in formaldehyde-fixed tissues) of nerve fibres that are otherwise difficult to demonstrate, such as those in the renal cortex, which are almost certainly post-ganglionic sympathetic fibres.