A method of processing third-stage larvae of the nematode Trichostrongylus colubriformis for electron microscopy

A method for processing third-stage larvae for electron microscopy is described. Using simple apparatus larvae are separated from faecal material by migration to a warmed area; they are then immobilized and chopped up in agar prior to fixation. No cellular distortion is observed in ultra-thin sections. Certain difficulties may be encountered when processing nematodes for electron microscopy. The most notable of these is fixation. Penetration of the usual cytological fixatives is poor, probably due to the relative impermeability of the cuticle. Lee (1966) found that whole larvae of Nippostrongylus brasiliensis were completely unfixed after 3 days in 6.5% glutaraldehyde, and in the present investigation, larvae of Trichostrongylus colubriformis were found to be still living after 1 h in 1% osmium tetroxide. One is, therefore, obliged to cut up the larvae so that the fixative will penetrate. Third-stage larvae usually have to be harvested from frecal material and this also may present certain problems. Material is usually centrifuged to concentrate the larvae, and during this process faecal debris may also be deposited. The amount of debris can be minimized by washing the nematodes and discarding the supernatants. However, sufficient hard debris may be retained to make subsequent ultra-thin sectioning troublesome. In order to overcome these difficulties a technique was devised and is described here.