Worm Breeder's Gazette 15(3): 14 (June 1, 1998)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Improved visibility of the defaecation motor programme steps at low magnification for routine analysis of the defaecation rhythm in <I>C. elegans</I>

Sonia Kowal, Fred Kippert

Biological Timing Lab, Institute of Cell, Animal and Population Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JT, Scotland, UK. E-mail: fkippert@srv0.bio.ed.ac.uk

The ultradian rhythm of defaecation in C. elegans has attracted considerable interest. Analysis of mutants with altered periods may provide important clues as to the molecular mechanisms underlying such short period oscillators. We aim to test genes of C. elegans homologous to those identified in our screen for ultradian clock mutants in yeast for a potential role in the defaecation oscillator. Since the number of genes be required for normal clock function in yeast, and thus to be tested in C. elegansis high, we were interested in setting up large scale routine experiments on the ultradian defaecation rhythm in C. elegans.

A major problem with the existing protocol is that the animals have to be observed at rather high magnification and therefore must be followed under the microscope manually. This requires permanent attendance which makes large scale screening impractical. A fully automated system is not feasible with this rhythm. We therefore settled for observations at a much lower magnification to reduce the attendance requirement considerably. Animals are recorded on time lapse video for later analysis.

In order to achieve reliable visibility of the defaecation motor programme steps, we developed the following method. Three different parameters were found to improve visibility independently of each other; in combination their effects are additive. First, a rich medium (NGM with peptone at 5% and additional yeast extract at 5%), with a resulting thick layer of E. coli, improves the optics (and also reduces variability in period length due to differences in the availability of food). Second, adding neutral red to a concentration of 0.0002 % (w/v) specifically stains the intestine cells. We achieved best results when animals were maintained on neutral red medium. Third, a ~ 2 mm thick layer of silicon oil (dimethyl polysiloxane, viscosity 5-50 centistokes, Sigma) has a twofold effect. It improves the optics in a way that otherwise could only be achieved by covering the sample with a coverslip, i.e. the animals become transparent instead of the more three-dimensional im! age typical with a dissecting microscope. In addition it prevents desiccation and thus keeps the osmolarity of the medium at a constant level. None of the components of this new protocol affect the period or other aspects of the defaecation behaviour to any noticeable degree.

With the described method we are routinely able to observe animals at a magnification of 10-15 times, which reduces the attendance required considerably. We have the whole system mounted in an incubator (to assure constant temperatures, crucial for chronobiological experiments) and intermittent inspection (to bring the animal to the centre of the image) is required. The occasional temporary loss of an animal out of the observational field is more than compensated for by the time saved. Additionally, the focus needs to be readjusted rarely if at all because of the low magnification.

The described method is also suited to study of defaecation in larval stages. In addition, because the movement of the gut contents can be followed under conditions which do not affect the normal behaviour of the worms, this protocol may also serve in future analysis of the ingestion and digestion processes. Several photographs and movie sequences of adult worms and larvae monitored according to the new protocol can be seen on the web page http://helios.bto.ed.ac.uk/icapb/fyc/nroil.html.