Basic Caenorhabditis elegans Methods: Synchronization and Observation

Summary

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Abstract

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism ¹. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years ². Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved ³.

C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage ^4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology ⁶, aging ^7,8, stem cell biology ⁹ and germ line biology ¹⁰.

An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others.

Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Protocol

1. Protocol A: Culturing Worms for Bleaching ¹¹

Large populations of C. elegans can be obtained by culturing them either in liquid media or on solid media in plates. They are usually grown on solid NGM (Nematode Growth Media) and fed with E. coli bacteria, which is added to the plates either alive or dead (killed by UV¹², by heat¹³ or by cold¹⁴). The most common procedure uses live OP50 E. coli, which is defective in the synthesis of uracil and cannot overgrow into a thick layer that would obscure the worms.

1. Mix 3 g of NaCl, 17 g of agar and 2.5 g of pepton....

Discussion

Nematode Synchronization

Several bleaching solutions have been described. We tried five different recipes (Table I) and, in our hands, they did not show significant differences in the synchronization of worm populations (Fig. 1). However, our experiments did show that parameters such as temperature (Fig. 2), the ratio bleaching solution:volume of worms (Fig. 3) and the volume of M9 with which the embryos are incubated for hatchin.......