The effect of chemical fixatives on cell walls of Bacillus subtilis

Cell walls of Bacillus subtilis were treated with several chemical fixatives which are commonly used preparatory to electron microscopy; i.e., osmium tetroxide, formaldehyde, acrolein, crotonaldehyde, and glutaraldehyde. Dimensional analysis was performed on thin sections of fixed walls from plastic embeddings and, by means of the statistical technique of multiple comparisons, significant differences were found between wall thicknesses from the various fixations. These differences varied with the fixation time and the type of fixative used in the reaction. When compared to embedded walls which had been stained before fixation, the overall effect was a reduction in wall thickness which was attributed to fixative action and not to the embedding or staining processes. The reduction of wall thickness was even more apparent when dimensions of fixed walls were compared to published dimensions of both frozen sections and freeze-etch profiles. Since these fixatives bind to reactive sites within the wall fabric, a change in electrochemical charge density is effected which can be monitored in terms of heavy-metal-binding capacity. Most monoaldehyde fixatives and osmium tetroxide render the wall as reactive, or less reactive, to uranyl acetate as unfixed walls, whereas glutaraldehyde can significantly increase the binding capacity.