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Zoology Publications from Victoria University of Wellington—Nos. 58 to 61

Material and Methods

Material and Methods

The collection of animals and their maintenance in the laboratory has been described (Wineera, 1969). Worms were fixed in buffered 4% formaldehyde (Pease, 1964, p.52) for 18 hr. Some of these worms were then postfixed in 1% osmium tetroxide in distilled water for 4 hrs. at room temperature. Specimens fixed in these two ways were embedded in araldite according to the method of Richardson, Jarett and Finke, (1960). Propylene oxide (two changes of 15 mins. each) was used between dehydration and resin infiltration stages. Polymerisation was carried out at 60°C for 8-12 hrs. Blocks were cut on a LKB ultramicrotome using a glass knife. Section thickness of approximately 0.25μ. and 0.5μ was obtained by advancing the block manually after each cut by means of the "microfeed" control. The sections produced were placed on microslides. Unstained sections were examined by phase contrast microscopy, and sections were subjected to the following staining procedures: (i) Methylene blue-borax/Azure II (Richardson et al, 1960)*; (ii) The Falg technique (Gurr, 1965).

Remarks: Most conventional staining methods give negative results with tissues fixed by osmium, and tissue constituents which are usually distinguished by a characteristic staining reaction may prove difficult to identify. However, the alkaline methylene blue/Azure II staining method is designed for use with osmium fixed material, with which it gives excellent results, even in extremely thin (0.25μ.) araldite sections. Results with thin formaldehyde fixed material are poor, but by comparing (i) formalin fixed sections stained with methylene blue/Azure II with those stained by more conventional methods such as the Falg technique and (ii) osmium and formaldehyde fixed sections stained with methylene blue/Azure II it is possible to identify in osmium fixed material the "eosinophil" and "basiphil" substances of conventional staining methods.

Some worms fixed in buffered formaldehyde were embedded in 20% gelatin and sectioned longitudinally at 10μ. on a freezing microtome. The sections produced were placed on slides, mounted in glycerogel and were examined by phase contrast microscopy.

For observation of lving cells, a modification of the disassociation procedure of Pedersen (1961a) was used. Worms were placed on cavity slides in a few drops of 0.5% trypsin in sea water. The worms were then cut into small pieces using fine needles and the preparations were covered page 3 by a petri dish and left at room temperature for 3-4 hrs. After this time the small pieces of worm were placed on microslides in a drop of the trypsin/sea water solution and were gently squashed beneath a coverslip. The preparations were ringed with petroleum jelly and were examined by phase contrast and interference contrast microscopy. This technique resulted in fairly good cellular dissociation and the cells remained alive (as judged by contraction of muscle cells and the beating of cilia on epithelial cells and in flame cells) for 4-5 hrs.