Comparative effects of fixation methods using tannic acid on contrast of stained and unstained sections from Spurr-embedded plant leaves

Abstract

The comparative effects of different fixation methods using tannic acid (TA) on contrast of stained and unstained sections from Spurr-embedded apple leaves were examined. A tetrafixation method, glutaraldehyde (GA) – osmium tetroxide (OsO4) – TA – OsO4, gave the best contrast in the unstained sections while double-fixed (GA – OsO4 and GA∙TA – OsO4) specimens showed low contrast. The unstained sections from triple-fixed (GA – OsO4 – TA and GA – TA – OsO4) leaves resulted in medium contrast. Another tetrafixation method (GA – TA – OsO4 – TA) gave fairly good contrast in unstained sections. The enhancing effect of TA on contrast was still more increased by brief double stain (uranyl acetate – lead citrate) or lead citrate alone. The results showed that the former tetrafixation method was very useful for observing the fine ultrastructure of stained and unstained sections from Spurr-embedded plant tissues. In the tetrafixed leaf cells, electron-dense cellular structures were found at the peripheral area of cells. Some rough endoplasmic reticulum (ER), some Golgi bodies, vesicles beneath plasma membranes, cell walls, plasma membranes, and amorphous materials between cell walls and plasma membranes showed increased contrast in the stained and unstained tissues. The vesicles were often found to be continuous with plasma membranes. Taking into account the distribution and function of the electron-dense organelles, the ER, Golgi bodies, and vesicles may be associated with cell wall synthesis.