methods1

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Table of contents   -    Introduction   -    Methods   -    Results   -    Discussion   -    References

METHODS

(a) Culture
(b) Observation of living specimens
(c) Feulgen staining

(a) Culture

The culture of C. elegans has been described by Brenner (1974).

(b) Observation of living specimens

A drop of 5% agar, de-aerated by heating at 100°C, was placed on a clean microscope slide, and flattened into a layer about 0.5 mm thick by means of a second, siliconized, slide supported on spacers. Care was taken to avoid imperfections due to bubbles or dust, which might subsequently trap the nematode during a series of observations. After 2 min the siliconized slide was removed; 2 Ál of 10 % polyvinylpyrrolidone in S medium (Sulston & Brenner 1974) was placed on the agar, and a selected nematode was transferred to this drop. The centre of a 12 mm x 12 mm cover slip was very thinly coated with bacteria (E. coli, strain OP50) scraped from a plate culture, and the cover slip was lowered gently onto the agar. Excess agar was trimmed away, and the edges of the assembly were sealed with silicone grease. Most of the air bubbles were soon absorbed by the de-aerated agar, and after a short period of quiescence (due probably to anoxia) the nematode generally moved into the bacterial lawn and started to feed. Non-optimum temperature and shortage of food were found to be conducive to escape.

A Zeiss Universal microscope was equipped with a Plan x 100 objective and Nomarski differential interference contrast optics, and was kept at an air temperature of 20-22°C. Photographs were taken on Ilford Pan F 35 mm film, with a Zeiss microflash illuminator as the light source, and developed in M. & B. Promicrol.

During periods of cell division and migration, the ventral cord was observed continuously and sketched at 15 min intervals; during the much longer periods of quiescence, observations were made at 1 or 2 h intervals. When a single individual was to be followed for more than one day, it was refrigerated overnight at 6-8°C during a suitable quiescent period. This cooling was found practically to arrest development, and yet on returning to 20°C the animal continued to grow normally.

(c) Feulgen staining

Nematodes were transferred from plates by means of a paper strip or pointed stick to a small drop of 10% ovalbumin on a microscope slide; the drop was smeared out, and the slide was plunged into Carnoy's fixative. For multiple samples, the slide was first divided into zones by Indian ink lines, and was kept cool during transfers in order to avoid drying of the drops. After hydration, Feulgen staining, and dehydration, the nematodes were mounted in methyl salicylate or Depex.

Web adaptation, Chris Crocker, for Wormatlas, 2008