11/12/1998

We make our E.Coli for liquid culture worm food in large batches that then are frozen at -80° C and thawed when needed. Lately we have used the DH10B strain. The E. Coli growth is done in a 200L fermenter for several hours, spun down in large centrifuges and has yielded 2-3.5 kg pure bacteria (in paste form). This is then resuspended in a combination of M9 and glycerol until it is a "pipettable" liquid. The following ingredients are needed (based on 200L reaction):

1. 4 L of 5X KH₂PO₄/K₂HPO₄ solution. -----> 462 g KH₂PO₄, 2508 g K₂HPO₄, 4 L H₂O
2. 2.4 kg tryptone
3. 4.8 kg yeast extract
4. 1.6L 50% glycerol
5. 6 L of DH10B starter cultures

Items 2-4 are placed in the fermenter with enough water to fill up tp 200L and then sterilized. The phosphate solution is then sterilely added as are the starter cultures. The bacteria is grown at 37° C for ~4.5 hours and then collected. For resuspending you will need to have autoclaved the following:

1. 4-5L of M9
2. 3 large buckets and spoons

You will also want to mix in some glycerol once resuspended (~20%).

Start by growing 5-10 medium (100 mm) plates of your worms containing a mixture of young and adult worms. When they have nearly starved the plates, wash the worms off with S-basal or M9 solution, pipetting the liquid onto each plate and swirling before pipetting off. Wash 2or 3 times to maximize the number of worms you get. You can wash them directly into 500 mL of S-basal in a baffled flask or if you want to quantitate the volume of worms, collect in a 50 mL conical first (or 15 mL conical) and spin down. Then pipette into your flask. Add 1-3mL E. Coli (amount depends on amount of worms and/or quality of batch of E.Coli but generally speaking start with no more than 1 mL). Shake @20° C on a platform shaker at ~240 rpm. Take approximately 100-200 uL aliquot every day and dump it on an unseeded worm plate to check that the worms are growing and whether they need more food. (It is perhaps easiest to judge food necessity by how clear/opaque the liquid appears after a few minutes of letting the worms settle to the bottom of the flask.) In general, we have found that feeding a culture 1-3 mL a day works well. After a few days you will notice the culture become cluttered with brown oval pellets, presumably worm debris/waste. After 4-5 days (longer for some mutants) your worms should be gravid adults and ready to harvest. If dauers are forming you have probably not added enough food and/or the culture is too dense. However, care must be taken to not add too much food at any one time as the worms can suffocate from lack of oxygen. If after 5 days you do not have enough gravid worms, it is possible to let the culture grow another generation but you will have to clean the culture by sucrose floating (see below) and then resuspend only 0.75-1.0 mL worms per flask.

Once you have gravid adults, you will want to first separate the live worms from all the debris/dead worms in the culture. This can be done by sucrose floating as follows:

1. Prepare ice cold 0.1 M NaCl and ice cold 60% sucrose. (We like to keep 1-1.5L stocks in the cold room)
2. Spin down the worms in 50mL conicals in a clinical centrifuge (highest or 2^nd highest setting; this will take you 2-3 rounds of 4 conicals at a time). Spin time is not that critical, approx. 1 minute is sufficient to yield a soft brown pellet at the bottom of your tubes.
3. Pour off supernatant , resuspend the pellet s in the 0.1 M NaCl and spin to wash, ultimately combining everything into 1 or two tubes depending on your pellet volumes. It is not advisable to have more than 15 mL in each tube.
4. Add NaCl to bring the volume of worms/liquid up to 25 mL in each tube. Mix well to disperse pellet and let sit on ice for > 10minutes. Then add 25mL cold 60% sucrose to each tube, mix, and spin on high for 5 minutes in the clinical centrifuge. The sucrose solution damages and eventually kills the worms so work fast here. Don't let them be exposed to the sucrose for much more than the 5 minutes.
5. After you spin, you should see a brown pellet at the bottom of the tube and a light brown layer of worms floating at the top. Use a broken pasteur pipette tip or a 10mL glass pipette to carefully remove the worms into a new 50 ml conical with approx. 35 mL cold 0.1 M NaCl.
6. Spin the diluted worms for ~1 minute, pour off supernatant and repeat the wash 2-3 more times to get rid of all sucrose.
7. At this point you are ready to collect eggs. To your worm pellet add 2.5 times the volume of hypochlorite solution ( 40 mL 5% bleach, 5 mL 10M NaOH, fill to 100 mL volume with H₂O). Mix gently at room temperature for 5 minutes. This will kill the adults and larvae but not the eggs. It should start disintegrating the carcasses as well though usually not completely. Spin 1 minute and decant supernatant. Wash 3-4 times with 0.1 M NaCl, at some point consolidating into 1 tube. Wash the eggs into 100 mL of fresh S. Basal and let shake O/N at 20° C to hatch.

Note: for doing timecourses you will need to grow up 3-5 cultures of worms rather than 1.

3-4 days before you know you will be bleaching your worms and collecting eggs, you will want to pour ~60 large plates (150mm) with the following media:

1. Add together:

4.5 g NaCl
3.75 g Bactopeptone
30g Bactoagar (Difco)
7.5 g Bactotryptone
3.75 g Yeast extract
3.0 mL cholesterol (5 mg/mL in ethanol)
1.5 Liter distilled water

2. autoclave 30 minutes
3. mix in, using sterile technique, in the following order:

1.5 mL CaCl₂ (1M)
1.5 mL MgSO₄ (1M)
37.5 mL Potassium Phosphate (1M, pH 6) mix thoroughly

4. pour into plates using sterile technique (~4.5 L = 60 plates). Once they are cooled, spread with E. Coli (not from the frozen down stocks but from a flask of LB broth that has been innoculated with E.Coli) and let grow 2-3 days.

The day after you bleached your worms you will want to plate the eggs onto these plates. We have found it useful to sucrose float once more to separate the L1's from the carcasses from which they hatched. A manageable mount of worms per plate is approx. 100 uL spun-down worms pre-sucrose floating or a proportional amount of sucrose-floated worms (approximately 10-50 ul). Plates are then put at 20° C.

For L3 staging timecourse, you will want to begin monitoring your worms under the Nomarski scope 32-36 hrs after plating. We usually begin collecting 1/7^th of the plates when the Pnp cells are indicative of early L3 stage, that is they are fairly big and look ready to divide for the first time. Time collection will vary depending on the specific experiment you want to do.

You will want to keep a large stock of M9 at 20° C in preparation for harvesting. Doing almost everything at 20° C, pipette M9 onto the plates you want to collect (several mLs per plate) and let shake or swirl by hand for a few seconds/minutes . Then pipette off the liquid with worms into 50 ml conicals. Repeat step again and wash into conicals. Spin worms down briefly in clinical centrifuge and wash with M9 2-3 times to get rid of as much bacteria as possible. In the past we have had trouble with bacteria washing off in flakes and then spinning down with the worms, thereby inflating the real volume yield of worms. For 7 plates we usually see ~1 mL worms. Transfer to 15 mL conical and add 4x volume of trizol reagent. Vortex briefly, flash freeze in liquid nitrogen, thaw at 37° C and repeat sequence once more. Freeze at -80° C until ready to make RNA.