We investigated the use of safranin O and astra blue dissolved in ethyl alcohol as differential stains to distinguish between lignified and unlignified tissues in microtome sections of tension and normal wood of sugar and red maple. Normal wood was used as a control for the histochemical analysis. Lignified and unlignified tissues were found in the same section for both tension and normal wood of each species. These results were confirmed in unstained samples using ultraviolet light. Unlignified libriform fibers were detected using both techniques. Libriform fibers did not fluoresce in UV light, although fluorescence was observed in some of the cell corners. The astra blue in ethyl alcohol and the UV wavelength we used differentiated syringyl from guaiacyl lignins. Ethyl alcohol solutions of these dyes provide an effective and reliable method to distinguish lignified and unlignified tissues.

Our earlier studies on Acer have shown morphological and chemical differences between two fiber types. Our new research applies the soft-rot cavity method using the soft-rot fungus Chaetomium globosum to measure the microfibril angle (MFA) in these two fiber types in fifteen species of Acer (maple). Microfibril angles in fiber type 1 were significantly larger than those in fiber type 2. The greatest difference (11.6°) was noted in radial sections of Acer floridanum, in which the average MFA (n =100) obtained from soft-rot cavities for fiber type 1 was 23.3 degrees (95 % C.I. ±1.4°) and for fiber type 2 11.7 degrees (95 % C.I. ±1.2°). The second greatest difference (10.0°) was in Acer nigrum, where the average MFA for type 1 was 16.4 degrees (95 % C.I. ±1.07°) and for type 2 6.4 degrees (95 % C.I. ± 0.64°). When transverse sections were examined with polarized light, areas of fiber type 1 were darker (less birefringent) than those of type 2, which indicates that the MFA of the two cell types differs. After four weeks of exposure to C. globosum the fibers of type 1 were more intensely attacked by the fungus than were the fibers of type 2, suggesting a difference in cell wall chemistry. Fluorescence spectra of the two types of fibers support that observation. Differences in MFA supplement the differences in morphology and chemistry and demonstrate that for Acer species the two fiber types are fundamentally different. These two types of fiber differ in the distribution of their pits. In type 1 the pits are concentrated mainly toward the center of the fiber while in type 2 the pits are distributed along the fiber length.

Fifteen Acer species were examined to study distribution and percentage by area of their libriform fibers. Safranin-O and astra blue dissolved in alcohol solution is an effective differential staining method to identify and localize libriform fibers. They have larger lumens than fiber tracheids, intercellular spaces, and occur in various patterns, ranging from large groups to wavy bands. In some cases they occur in radial files. The percentage by area of libriform fibers ranges from 14 to 40%. Inconspicuous to moderately visible spiral thickenings are part of the Acer libriform fibers and fiber tracheids. Differences in stain reactions and fluorescence indicate that libriform fibers differ in lignin concentration or composition from fiber tracheids – the concentration of syringyl lignin is greater in libriform fibers.