Start a liquid culture of OP50 bacteria each week to serve as a food source for your worms
1. To a sterile snap-cap tube transfer 2 ml of LB broth using a 10-ml sterile pipet. (Use good sterile technique!)
2. Using a sterile wooden stick, gently scrape some OP50 bacteria from the OP50 culture plate. Inoculate the snap-cap tube containing 2ml of LB broth by dipping the stick into the liquid and shaking it a bit.
3. Let the liquid culture grow at room temperature for 2 days. Just place the tube in a test tube rack and let it sit on the bench at room temperature (it does not need to be shaken). After two days tap or gently vortex this OP50 liquid culture to resuspend any cells that may have settled. The culture should be very cloudy with bacterial growth. You will need this liquid culture to seed EZ worm plates. You may store this liquid culture in the refrigerator for weekly use. After about one month it is best to throw out this culture and start a fresh liquid culture.
Seed two EZ worm plates with the liquid culture of OP50
1. Take two EZ Worm plates out of the refrigerator and put them on the bench for at least 1 hr. to allow it to warm to room temperature.
2. Using a sterile 10-ml pipet, take up approx. 0.5 ml of your OP50 liquid culture. Gently touch the pipet tip to the surface of an EZ worm plate and allow some of the culture to flow out into the surface of the plate. Make a sweeping S-shaped motion with the pipet to create a large S-shaped puddle of liquid to span the plate. Using the same pipet, do the same for the other EZ worm plate. Leave both plates on the bench for several hours to overnight to allow the liquid on them to dry. Store your OP50 liquid culture in the refrigerator for future use.
1. Using a burner, flame the end of the worm pick to sterilize it. Wave the pick in the air for a few seconds to allow it to cool down. (Alternately, sterilize the end of the worm pick by inserting it into a small container of 95% ethanol, then wave it for a few seconds to allow the ethanol to evaporate.) Place you starter plate of lin-2 worms under the microscope. Make sure that the ocular (viewing) lenses of the microscope are facing the same side as the stage platform onto which you put your worm plate. The top portion of the microscope, which contains the two ocular lenses, can be rotated to face toward or away from the stage platform.
2. Transfer approximately 10 medium sized worms to each seeded EZ worm plate. After every few worms it is best to re-sterilize your worm pick.
3. Store your worm-containing plates upside-down on the bench at room temperature. It is best to have two plates of worms rather than one just in case you encounter a contamination problem on one plate. Also, you may need two plates of worms to provide you with enough worms for testing the human cDNA library.
Since C. elegans life-cycle is very brief at room temperature (2-3 days), after about one week your plates will be very crowded with two to three generations of worms. Therefore, each week you will need to re-seed two new EZ worm plates with fresh OP50 then add approx. 10 worms to each.