Liquid culture of worms

3) Swirl the flask to resuspend the pellet. Add more 0.1 M NaCl to make up to a total volume of 50 mls. Distribute 25 mls into each of two 50 ml centrifuge tubes, and place on ice for several minutes to chill. Add 25 mls ice cold 60% sucrose to each tube, mix, and spin 3.5 K for 5 minutes in a table top centrifuge. The sucrose solution damages and eventually kills the worms; work fast here.

4) After the spin you should see a large dark brown pellet at the bottom of the tube (bacteria, debris, dead worms), and a large light brown layer of worms floating at the top. Use a broken off Pasteur pipette to carefully remove the worms, to a new centrifuge tube (it's ok to take about the top 20 mls of liquid here).

5) Quickly dilute the worm suspension 4 fold with ice cold 0.1 M NaCl, and spin 3.1 K 3 min. Remove the supernatant. The pellet should contain reasonably clean worms.

6) Resuspend the worms in 100 mls (i.e. fill up two 50 ml tubes) of ice cold 0.1 M NaCl, and spin 3.1 K for 3 min. to remove more sucrose. Pour the supernatant off the very soft pellet with great care. At this point the pellet should contain almost entirely healthy worms, with only a very small amount of debris and bacteria. The pellet can now be flash frozen in liquid nitrogen to later prepare RNA, or eggs can be prepped to start synchronized liquid cultures. Note that this last pellet is extremely soft, especially when it contains older worms, so it is impossible to pour off all the supernatant without losing the worms. Just do the best you can do, and freeze or proceed to prep the resulting suspension, which will be about 50% worms, 50% 0.1 M NaCl. If the worms will be used for an RNA prep, it is best to have less than 5 mls of worms in a 50 ml tube, to allow room to add the necessary guanidinium solution.

7) The above prep takes two hours.

PREPARING EGGS TO START SYNCHRONIZED LIQUID CULTURES

1) Start with 20 mls of packed worms prepared as above. Make sure that this contains lots of gravid adults.

2) Resuspend the worms in 1.5 N NaOH, 12% NaOCl, to a total volume of 100 ml (in two 50 ml centrifuge tubes). Mix gently at room temperature for 5 minutes. This will kill all the adults and larvae, but not the eggs, which are protected by their shells.

3) Spin in a table top centrifuge 3.1 K for 5 min. Pour off the sup. and wash in 100 mls 0.1 M NaCl. Spin again, and wash again twice more in 50 mls 0.1 M NaCl each time, consolidating into one tube.

4) Resuspend in 45 mls of S basal. Examining in the microscope at this point, you will see lots of flaccid dead worms, and very few free eggs. Most of the (viable) eggs are inside the dead adults.

5) Our regimen has been to set up four 500 ml cultures from the eggs, and to harvest these synchronized cultures over the next 4 days to get variously staged worms. In order to get an equal mass of worms from the various stages we seed the cultures with 30, 9, 3, and 3 mls of the 45 ml egg suspension. These cultures are havested after 1, 2, 3, and 4 days respectively. Each 500 ml culture is initially fed with 25 mls of packed bacteria. Harvesting the first flask after 21 hours we got ~2 mls of packed L1/L2s (mostly L2s). Harvesting the second flask after 48 hours we got ~4 mls of packed L3s and early to mid L4s. Harvesting the 3rd flask after 72 hours we got ~ 4 mls packed adults, a mixture of ~2/3 young gravid adults and ~1/3 pregravid young adults.