CHILLING OF TISSUE BEFORE GLUTARALDEHYDE FIXATION PRESERVES FRAGILE INCLUSIONS OF SEVERAL PLANT-VIRUSES

Glutaraldehyde-osmium fixation showed the presence of crystalline inclusions of brome mosaic virus (BMV), turnip yellow mosaic virus (TYMV) [and its protein], and potato virus X (PVX) in host tissues provided the tissue was vacuum-infiltrated with buffer and chilled a minimum of 2 hr before chilled glutaraldehyde was allowed to diffuse in. Tobacco mosaic virus (TMV) inclusions could only be preserved if osmium tetroxide postfixation was omitted. Cowpea mosaic virus (CPMV) crystalline inclusions were not observed with this method. Glutaraldehyde did not fix gelatin at concentrations below 2.5% unless the protein was first gelled at 5°C. Bovine serum albumin was not crosslinked by glutaraldehyde at concentrations of less than 3% at 5°C or of 4% at 25°C. Attempted fixation of low concentrations of gelatin and bovine serum albumin by glutaraldehyde at 5 or 25°C paralleled preservation or nonpreservation, respectively, of viral crystalline inclusions of TYMV, BMV, PVX, and TMV in expanded leaf tissue. The inability of glutaraldehyde to crosslink low concentrations of protein has consequences for the interpretation of the intracellular ultrastructural location of virus or virus-related products in expanded leaf tissues. It is proposed that a minimum protein concentration is required for glutaraldehyde to fix cells and that this minimum protein concentration decreases with decreases in temperature. © 1979 Academic Press, Inc.