Liquid culture of worms

互联网2013-09-06

832

<center> By Michael Koelle and Tory Herman, adapted from Mir Hengartner</center>

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 °C.

Then add 250 mL of sterile 0.17M KH2 PO4 , 0.72M K2 HPO4 to each flask.

To prepare 0.17M KH2 PO4 , 0.72M K2 HPO4 :

46.2g KH2 PO4

250.8g K2 HPO4 per 2 liters; make 250 mL aliquots and autoclave.

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

0.5 L           2 L

2.9g           11.6 g         NaCl
25 mL       100 mL      1M potassium phosphate, pH 6.0

136.1 g KH2 PO4 per 1000 mL; start with 800 mL and adjust to pH 6.0 with solid KOH (approx. 15g) before bringing up to volume.

475 mL 1.9 L DDW
0.5 mL 2 mL 5 mg/mL cholesterol in 95% EtOH

Warm to 37°C O/N to dissolve. When this is added to an aqueous solution some cholesterol will precipitate; don't worry about this.

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 .7H2 0
0.930 g Na2 EDTA
0.098 g MnCL2 .4H2 0
0.144 g ZnS04 .7H2 0
0.012 g CuSO4 .5H2 0 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. Autoclave to sterilize.

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 for sick mutants) are needed to start each liter of liquid culture. 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 @ 37° 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 / 4°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 Medium. 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 °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 medium, and wash the plates again with more S medium to be sure to get all the worms. Add the worms to 1 liter of S-medium (in two 2 liter flasks containing 500 ml each.) Add 12.5 mls of bacteria to each flask. Shake @ 20° 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. 200 µl 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 4-4.5 days after it was started. If there aren't enough worms, it is possible to wait 1-3 extra days to harvest. Some mutants grow more slowly than N2 and will definitely require these extra days of growth.

Judging when the culture is ready to harvest takes a bit of experience. In the very best cultures, the bacteria are beginning to clear at the time of harvest (culture turns from milky to a clearer yellowish greenish brownish, and bacteria are less evident under the microscope), the worms are extremely dense, all stages of worms are present, and no dauers visible. If the worms get too dense all the worms will go dauer even if the food hasn't run out due to the accumulation of dauer pheromone. Of couirse, if the food runs out, the worms will also go dauer. Looking at 200 µl of liquid culture dumped onto an unseeded plate, when the culture is ready to harvest you should see about as many worms as you would find on a plate of worms (grown normally, on a seeded plate) that is about 0.5-1 days before starving.

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.

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.

8) Expected yield: about 15-20 mls of purified, packed, mixed stage worm pellet per liter of liquid culture. The contaminating bacteria should be <5% of the mass of the worms.

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 medium. 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.