EFFECT OF DIFFERENT PREPARATION SCHEDULES FOR ELECTRON-MICROSCOPY ON LOSS OF LABELED SUBSTANCES IN ROOTS OF PISUM AFTER FEEDING WITH GLUCOSE-C-14

The losses of marked metabolites of 14C-glucose in Pisum roots were determined by means of liquid scintillation counting during different electron microscopic preparation schedules. 14C-glucose was applied to the roots. Metabolites were identified in thinlayer chromatographs and radioautographs. Fixation with glutaraldehyde (glut.) followed by OsO4 rendered the radioactivity in the tissue less susceptible to ethanolic extraction than treatment with either glut. or OsO4. The ethanolic extracts showed 76 per cent of their radioactivity in glucose, the remainder in amino acids and nitrogen-free acids. Continuous dehydration by acetone instead of stepwise dehydration by acetone, ethanol, and propylene oxide further reduces extraction. Even better results are achieved by adding sucrose to the fixation and dehydration media. Finally, after fixation with OsO4 fumes followed by continuous dehydration, most of the incorporated radioactivity is retained in the tissue. The passage through high acetone and ethanol concentrations as well as through propylene oxide and epon does not cause significant losses. Glut. and acroleine do not differ significantly with respect to the retention of radioactivity. In all preparations the larger part of the retained radioactivity is found in glucose. It is suggested that glucose is predominantly retained because of membrane changes during fixation, whereas the amino acid retention is enhanced by reactions with the fixing agents. © 1978 Springer-Verlag.