Liquid culture of worms

By Michael Koelle and Tory Herman, adapted from Mir Hengartner 4/6/94

Media

1) superbroth (5 X 2 1/2 liters autoclaved in 6 liter flasks)

5 X 30 g Bactotryptone

5 X 60 g yeast extract

5 X 20 mL 50% glycerol stock solution

5 X 2.25 liters DDW

Hint: use a funnel to pour measured ingredients into the flasks.

Autoclave 30 minutes. Cool to < 60 deg.C.

Then add 250 mL of sterile 0.17M KH2PO4, 0.72M K2HPO4 to each flask.

To prepare 0.17M KH2PO4, 0.72M K2HPO4 :

46.2g KH2PO4

250.8g K2HPO4 per 2 liters; make 250 mL aliquots and autoclave.

2) S- basal (4 X 1 500mls autoclaved in 2 liter flask)

0.5 L 2 L

2.9g 11.6 g NaCl

25 mL 100 mL 1M KHPO4, pH 6.0

136.1 g KH2PO4 per 1000 mL; start with 800 mL and adjust to pH 6.0 with solid KOH (approx. 15g) before bringing up to volume. Make 100 mL aliquots.

475 mL 1.9 L DDW

0.5 mL 2 mL 5 mg/mL cholesterol in 95% EtOH

Warm to 37deg.C O/N to dissolve. Don't worry if it comes out of solution.

Aliquot to four 2L flasks; Swirl to disperse; autoclave.

Sterilely supplement EACH 500 mls with:

1.5 mL 1M MgSO4

3 mL 0.5M CaCl2

5 mL 100X trace metals solution

0.346 g FeSO4.7H20

0.930 g Na2EDTA

0.098 g MnCL2.4H20

0.144 g ZnS04.7H20

0.012 g CuSO4.5H20 per 500 mL

Autoclave. Keep in dark (wrap in aluminum foil).

5 mL 1M KCitrate, pH 6.0

21.01 g citric acid, monohydrate per 100 mL; start with 80 mL and adjust to pH 6.0 with solid KOH (approx. 17 g) before bringing up to volume.

5 mL Gibco 100X Pen/Strp/Neo (, buy from Gibco, keep in freezer)

5 mL 100X Nystatin (buy from Gibco, keep in freezer).

3) worm plates- approximately 5 large plates of N2, more of mutant. Grow until the plates almost starve, or if you want to stage the worms, grow until the plates starve (they'll be mostly L1s).

GROWING THE BACTERIA (WORM FOOD)

1) Prepare 5 x 2.5 L of Superbroth in 5 x 6 L flasks (see above for recipe)

2) Innoculate ~5 mL of HB101 in Superbroth into each of the 5 6-liter flasks; grow O/N @ 37deg. C with shaking.

Warning: Be sure to have enough worm food! It is very bad to run out of food in the middle of a growth.

3) Spin down the bacteria in 1-liter jars in the J6B centifuge (15 min / 4000 rpm / 4deg.C). This will require two spins of 6 bottles each ; just pour off the first sup. and add the rest of the culture on top of the first pellets and spin again. Resuspend the combined pellet in a bit (3 mL per bottle works fine) of S Basal. A convenient method is to put the 1-liter jar back on a platform shaker for ~15 minutes. Transfer to a 50 mL polypropylene tube and store in fridge for up to 2 weeks, or store @ -20 or -80 deg.C for unlimited time. The yield of bacteria per volume of superbroth is disappointing in these large cultures relative to what you would get in a 2 ml tube, presumably because of aeration problems. We have been getting about 9 mls of this bacterial suspension per liter of culture.

GROWING THE WORMS

Start with 5-8 almost starved large plates of N2 (more of mutant) containing a mixture of adults and young larvae. Wash the worms off the plates with S basal, and wash the plates again with more S basal to be sure to get all the worms. Add the worms to 1 liter of S-basal (in two 2 liter flasks containing 500 ml each.) Add 12.5 mls of bacteria to each flask. Shake @ 20deg. C on a platform shaker at ~240 RPM. You may need to start the shaker at slower RPMs and slowly turn it up to 240 in order to get the liquid swirling correctly in the flasks. Take approx. 1/5 mL aliquot every day and dump it on an unseeded worm plate to check that the worms are growing and whether they need more food. After a couple of days you will see tons of oval browinish pellets in the culture, presumbly worm debris and/or waste. If dauers are forming you need more food. Our regimen has been to add 25 mls more of bacteria to each flask after two days, and to harvest the culture 4 days after it was started. If there aren't enough worms, it is possible to wait one extra day to harvest. Waiting 6 days, however, we got lots of dauer larvae, even though the culture wasn't starved; when the culture is dense enough dauer pheromone causes this to happen.

AFTER THE CULTURE HAS GROWN

0) Prepare some ice cold 0.1 M NaCl (500 ml), and ice cold 60% sucrose (100 ml).

1) Spin down worms in 3 500 mL jars 3K for 3 min in a Beckman JA-10 rotor. The speed and timing of this and the subsequent spins are fairly critical; we start the timing when the rotor is up to speed, then turn the machine off to start the deceleration when 3 minutes is up. Pour off the supernatant, being very careful and leaving some liquid in order not to lose any of the soft pellets. Expect a huge pellet with tons of bacteria, containing several different colored layers..

2) Resuspend the pellets in 0.1 M NaCl, combine in one jar, and make up to 500 mls with 0.1 M Nacl. Spin again, this time at 2 K for 3 min in the JA-10, timing after the machine is up to speed as above. Pour off the supernatant, again being careful not to lose any, leaving some liquid.