Sexing and Staging Worms | Growing and Manipulating Worms | Performing a Standard Genetic Cross
With practice, the sex and developmental stage of a nematode worm can be readily determined by microscopic examination. For our purposes, it is only necessary to distinguish hermaphrodites and males in adulthood and the immediately preceding (fourth) larval stage.
To ensure the desired genetic cross, hermaphrodites in the fourth larval (L4) stage, which have not yet reached reproductive age and are therefore still virgin, are typically mated. L4 hermaphrodites have a smoothly tapered tail and are distinguished by a white crescent with a small black dot in the middle of the animal. This corresponds to the developing vulva, the structure used to lay eggs. In contrast, adult hermaphrodites lack this crescent and eventually fill with eggs.
Males are distinguished by a fan-shaped tail, their slightly thinner and smaller body, and their distinctive behavior (they move rapidly and tend to stroke other worms and, by doubling back, themselves). Identification of L4 males may require extended practice. The distinguishing characteristic is a swollen tail, not yet hook-like as in adult males, which is often accompanied by a lightly colored streak down the posterior half. Vigorous males in the L4 stage or early adulthood are used to insure a successful cross.
C. elegans is normally a free-living soil nematode that feeds on bacteria. In the laboratory, C. elegans is grown on agar plates spread with E. coli bacteria. These "seeded" plates will be available in the lab. The worms are typically incubated at 20°C, although they grow well at temperatures between 15°C and 25°C.
With the aid of a binocular dissecting microscope and a "worm pick", the following procedure is used to transfer worms:
Two notes of advice:
Two notes of caution:
Genetic crosses are typically performed by placing two or three L4 hermaphrodites together with five or more males on a small seeded plate. After transferring, check your plate carefully to be sure you did not transfer larvae or eggs, which could invalidate your experiment.
To observe self-progeny, a single virgin hermaphrodite (L4 or earlier) is placed on a small, seeded plate. The individual is transferred daily to a fresh plate until no more eggs are produced.
IMPORTANT NOTE: Daily transfer of the crossed adults is essential. Otherwise you may not be able to score all progeny, they may run out of food and starve, and you may confuse generations.
To complete the genetic crosses in a reasonable period of time, the worms will be incubated at 20°C, where C. elegans has a generation time of about 4 days. Consequently, you will be required to perform most of these procedures outside the regularly scheduled laboratory period. 108 Mateer will be available for use whenever the building is open and another laboratory is not scheduled. If additional assistance is required outside the normally scheduled laboratory period, feel free to consult me in 307 Mateer. Plan ahead; in some cases, a mutually convenient appointment may need to be scheduled.
William R. Morgan wmorgan@acs.wooster.edu