Using Microwave Irradiation to Improve Preservation of Female Nematodes and Gall Tissues for TEM Observations

Abstract

Microwave irradiation of glutaraldehyde-immersed samples was evaluated for the chemical fixation of 3-week-old galls that resulted from the infection of tomato roots (Lycopersicon esculentum) by a root-knot nematode, Meloidogyne incognita. Observation by transmission electron microscopy indicated that the best results were obtained when vials containing the intact galls were immersed in buffered glutaraldehyde and irradiated for 10 seconds then allowed to cool for 30 seconds; this procedure was repeated two additional times. Galls that were fixed by this method and subsequently embedded in resin provided thin sections that remained stable in the electron beam so that fine structural details could be evaluated and photographed. Root cortical cells displayed no indication of osmotic stress, which usually results in plasmolysis or displacement of the cytoplasm toward the interior of the cell. All organelles in the giant cells appeared normal and well fixed. Cross sections near the center of the gall showed that the hypodermis of the female was not separated from the cuticle, which in turn was appressed to the outer cell walls of the giant cells. No obvious evidence of shrinkage, distortion, or failure of resin infiltration into the female nematode was apparent. High magnifications of the female nematode indicated that fine structural features of the tissues were also well preserved. Immersion fixation combined with microwave irradiation not only improved fixation of older tissues but enabled preservation of stages and feeding sites that could not be easily obtained by conventional methods.