Chemical Drying of Nematodes for Scanning Electron Microscopy Observations

Use of the Scanning Electron Microscopy (SEM) to study nematode morphology and taxonomy is extremely important tool and it has become an essential component of new species description. However, the general preparation procedures for SEM can frequently result in specimen distortion during fixation and other different drying procedures such as critical point dry. Here, a chemical preparation method using hexamethyldisilazane (HMDS) was employed to dry nematodes for SEM. The plant parasitic nematode Globodera sp., and the soil free-living nematode Caenorhabditis elegans were dried without any deformities. Adult females and Cyst stages of Globodera, and the synchronized adults of C. elegans hermaphrodites were used in this test. Globodera adults and cysts were obtained in FAA, and the synchronized C. elegans hermaphrodites were killed, and fixed in FAA for 48 hours. All nematode specimens were dehydrated through an acetone series to 100% acetone. Nematodes then were transferred gradually to 100% hexamethyldisilazane (HMDS). All nematode specimens in 100% HMDS were left overnight under the hood to dry. Dried nematodes were mounted on SEM aluminum stubs, sputter coated with gold and viewed with SEM using accelerated voltage of 10, 15, or 20 KV. SEM Images of C. elegans and Globodera sp., proved that drying of nematodes using HMDS were as good/or better than those regularly dried by critical-point dryer. Using HMDS to prepare nematodes for SEM is efficient, safe and easy to use.

Keywords: C. elegans; Chemical drying; Globodera; Hexamethyldisilazane; HMDS; Method; Nematodes; SEM; Technique