| Abstract: | Hyphae of the fungus Basidiobolus magnus were embedded in extractable polyethylene glycol (PEG) or diethylene glycol distearate (DGD) to prepare embedment-free sections in order to seek components of the cytoskeleton that may be obscured in epoxy-embedded sections. All methods showed that hyphae possess an intricate filamentous matrix, which is apparently unoriented. However, the appearance of the cytoskeleton depended on the embedding medium, the solvent used during embedding, and whether cells were fixed by conventional fixation or freeze-substitution. PEG proved to be the best embedding medium for fungal cells because DGD caused cell shrinkage and produced a more lamellar than filamentous matrix. Also, the cytoskeletal filaments were clearer in freeze-substituted cells than in conventionally-fixed cells. Since we failed to find microtubules in the embedment-free sections, we re-embedded cells in Epon to discern whether microtubules or other cytoplasmic components had changed. Neither PEG nor DGD adequately preserved microtubules as compared to regular Epon-embedded sections, and other cellular structures were significantly altered. Therefore, alternative methods need to be employed to further characterize fungal cytoskeletons. |
