OP50 stock at 20%w/v in S-medium

From Wikionchus

This concentrated OP50 stock is used for feeding worms in liquid cultures. Takes 3 days to prepare, so keep ready in advance.

• Day 1:

1. Take 400 to 800ml of LB-Agar in a 3L flask. Fluted flasks are preferred as they produce more OP50.
2. Pick one colony of OP50 into this LB-Agar.
3. Incubate overnight in a shaking incubator at 37C. Shaker : 120rpm for fluted flask; 180rpm for non-fluted flasks.

• Day 2:

1. Pour the overnight culture into centrifuge bottles for SLA-3000 rotor.
2. Spin at 4C, 5000rpm, 10 minutes (no braking). Discard supernatant.
3. Take an empty falcon tube and note down its weight.
4. Re-suspend the OP50 pellet in ~50ml of S-Medium. (Re-suspension is easy if pellets are left at 37C for 20minutes!)
5. Spin at 4C, 4000rpm, 30minutes. Discard supernatant.
6. Freeze the pellet at -20C overnight to kill all bacteria (otherwise they metabolize the antibiotic in the liquid culture!)

• Day 3:

1. Thaw the OP50 pellet.
2. Note the weight of the tube+pellet. Substract the weight of empty tube to get amount of OP50 (e.g. x grams).
3. Add S-medium to a final concentration of 20% w/v. (e.g. 5x ml of S-Medium)
4. Store at 4C.

Links

• Back to Liquid cultures for large scale dauer cultures page
• Back to Pristionchus pacificus Protocols page