Alcoholic Bouin fixation of insect nervous systems for Bodian silver staining. II. Modified solutions

A previously devised synthetic equivalent of 'aged alcoholic Bouin (Duboscq-Brasil) fixative was modified in various ways to discover which of the chemical changes brought about by aging were important in improving fixation and staining. Effects were tested with ventral nerve cord ganglia of the cockroach Periplaneta americana, locust Schistocerca gregaria, and honey bee Apis mellifera. Formation of reaction products, chiefly ethyl acetate and diethoxymethane, seemed to play only a subsidiary role: neither individually appeared essential as long as a sufficient quantity of one or the other was present. In place of diethoxymethane, ethyl acetate concentration could be increased to 25% with little effect on results. Reduction in concentration of two of the original constituents, formaldehyde and ethanol, appeared to be the principal factor in improving fixation. Varying the concentration of each original constituent individually revealed that formaldehyde mainly increased glial staining, ethanol increased tissue shrinkage and reduced overall staining intensity, acetic acid improved preservation, and picric acid decreased glial staining but produced few other effects within a wide range of concentrations, though its omission seriously impaired overall preservation and staining. Varying the ethanol and acetic acid concentrations simultaneously confirmed that they acted in opposite ways. A decrease in ethanol and an increase in acetic acid both improved results. The optimum mixture, 'improved synthetic alcoholic Bouin' (40% formaldehyde 0-15: ethanol 25: acetic acid 5: ethyl acetate 5: diethoxymethane 15: picric acid 0.5: water to 100), gives better preservation and more intense staining, and formaldehyde content can be varied to give the degree of glial staining and more intense staining, and formaldehyde content can be varied to give the degree of glial staining required. Without formaldehyde glial staining is virtually eliminated, while preservation and staining of the neurons appears unaffected. This modification seems to offer a valuable advance in technique.