Maintaining the worm

Requesting strains

See our article on strains.

Preparation of growth media

Preparation of bacteria food source

C. elegans is usually grown in the lab using the E. coli strain OP50 as a food source. A small bacterial lawn is preferred so that the mating of worms can be properly observed. A starter culture of E. coli OP50 can be requested from CGC. Use the starter culture to isolate single colonies on a streak plate of a rich medium, such as LB agar 10 g Bacto-tryptone, 5 g Bacto-yeast, 5 g NaCl, 15 g agar, H2 O to 1 litre, pH 7.5] (Byerly et al., 1976). Aseptically inoculate a rich broth, such as L Broth [10 g Bacto-tryptone, 5 g Bacto-yeast, 5 g NaCl, H2 O to 1 litre, pH to 7.0 using 1 M NaOH. Put 100 ml into 250 ml screw-cap bottles and autoclave. The bottles of media can be stored at room temperature for several months. Allow inoculated culture to grow overnight at 37°C. If not used, the streak plate and liquid culture should be stored at 4°C.

Preparation of NGM petri plates

The Nematode Growth Medium (NGM) agar medium can be poured into petri plates easily and aseptically using a peristaltic pump. Whenever necessary, drugs can be added to the medium just before being poured.

Equipment and Reagents

• NaCl
• Agar
• Peptone
• 5 mg/ml cholesterol in ethanol (Do not autoclave!)
• 1 M KPO₄ buffer at pH 6.0 (108.3 g KH₂PO₄, 35.6 g K₂HPO₄, add H₂O to 1 L)
• 1 M MgSO₄
• Petri plates
• Peristaltic pump

Method

1. Mix 3 g NaCl, 17 g agar, and 2.5 g peptone in a 2 litre Erlenmeyer flask. Add 975 ml H2O. Cover mouth of flask with aluminium foil. Autoclave for 50 min.
2. Cool flask in 55°C water bath for 15 min.
3. Add 1 ml 1 M CaCl2, 1 ml 5 mg/ml cholesterol in ethanol, 1 ml 1 M MgSO4 and 25 ml 1 M KPO4 buffer. Swirl to mix well.
4. Using sterile procedures, dispense the NGM solution into petri plates using a peristaltic pump. Fill plates 2/3 full of agar.
5. Leave plates at room temperature for 2-3 days before use to allow for detection of contaminants, and to allow excess moisture to evaporate. Plates stored in an air-tight container at room temperature will be usable for several weeks.

Seeding NGM plates

Using proper sterile technique, transfer approximately 0.05mL of E. coli OP50 liquid culture to medium NGM plates or 0.1mL to large NGM plates. Spread the drop with pipette tip or glass rod but keep try to keep the lawn in the center and away from the edge of the plate. Then allow the E. coli OP50 lawn to grow overnight at room temperature or at 37° C for 8 hours (cool plates to room temperature before adding worms). Seeded plates stored in an air-tight container will remain usable for 2-3 weeks.

Culturing C. elegans on Petri plates

Transferring worms: C. elegans is transparent, and can be observed using a dissecting stereomicroscope equipped with a transmitted light source. One method of transferring the worm from on dish to another is “chunking”—simply use a sterilized scalpel or a spatula to remove a chunk of agar containing the worms from one plate to another. This method is sufficient for transferring homozygous worm stocks, but is not advisable if the population is heterozygous or if a stock must be maintained by mating.

Long-Term Storage

There are two methods for freezing worms, soft agar freezing and liquid freezing. Soft agar freezing is performed at –80° C; liquid freezing is performed at –196° C and uses liquid nitrogen. From both, stocks over ten years old can be recovered, though soft agar freezing is more suited to shorter-term freezes. Starved L1 and L2 larvae survive the best.

Liquid Freezing

Materials

• Mix up 1 L of S buffer:
  • 129 mL 0.05 M K2HPO8
  • 871 mL 0.05 M KH2PO4
  • 5.85 g NaCl
• Make a solution of 70% S buffer and 30% glycerin (by volume) using 411 mL of the above and 176 mL of glycerin. The protocol requires equal amount of pure S buffer and S/glycerin mix; these instructions prepare about 587 mL of each. This is enough to freeze about a thousand vials of worms. You may want to divide all of the numbers in these first two bullet points by ten. Autoclave both afterwards.
• 1.8 mL cryotube vials (e.g. Nunc #65234)
• 1 large NGM plate with lots of freshly-starved L1 and L2 animals (which have recently been moved off a plate with food). 2-3 medium plates or 5-6 smaller plates will suffice as well.

Freezing

1. Wash the plates with 0.6 mL of S buffer for each vial you intend to freeze. One vial must be thawed the next day to make sure the freeze was successful, so freeze at least two. Ten is probably better, and more may be appropriate if you expect to be recovering worms as general stock.
2. Collect the liquid from this wash in a sterile test tube.
3. Add an equal volume of S/glycerin mix, doubling the amount of liquid in the test tube. Mix well.
4. Aliquot 1.0 mL of mixture into 1.8 cryotube vials.
5. Label and store in a styrofoam microtube holder.
6. The holder should then be placed in a –80° C freezer overnight (at least 12 hours). Recover one of the tubes to ensure that the freezing was done correctly, and then transfer the rest to liquid nitrogen storage.

Recovery

1. Remove vial from freezer. Let thaw until all ice has melted.
2. Pour contents onto large plate seeded with OP50 lawn. Worms should begin wiggling after a few minutes.
3. After two to three days, transfer groups of 10–15 animals to new plates. Take care to do this worm-by-worm. After reproducing for one generation, the worms can be confirmed as expressing the correct phenotype.

Soft Agar Freezing

Materials

• Mix up 1 L of S buffer:
  • 129 mL 0.05 M K2HPO8
  • 871 mL 0.05 M KH2PO4
  • 5.85 g NaCl
  • Autoclave.
• Soft Agar Freezing Solution:
  • 0.58 g NaCl
  • 0.68 KH₂PO₄
  • 30 g glycerol
  • 0.56 mL 1 M NaOH
  • 0.4 g agar
  • Top up to 100 mL with H₂O
  • Autoclave.
• 1.8 mL cryotube vials (e.g. Nunc #65234)
• 1 large NGM plate with lots of freshly-starved L1 and L2 animals (which have recently been moved off a plate with food). 2-3 medium plates or 5-6 smaller plates will suffice as well.

Freezing

1. Melt Soft Agar Freezing Solution in autoclave or microwave and place in 50° C bath for at least 15 minutes.
2. Wash plates with 0.6 mL of S buffer for every vial you intend to freeze. Collect the runoff in a sterilized test tube and put this on ice for 15 minutes.
3. Double the volume of the test tube by adding Soft Agar Freezing Solution. Mix well.
4. Aliquot 1 mL of this into labelled cryotubes.
5. Pack in styrofoam and store in –80° C. After 12 hours, check part of one tube to make sure it froze correctly.

Thawing

Work quickly, to prevent thawing of the whole tube, and keep the vial on ice in styrofoam.

1. Flame a small scoop or spatula and use it to remove 1/4 to 1/3 mL of the frozen solution. Place this on an NGM plate with an OP50 lawn, and return vial to freezer. Worms should begin wiggling after a few minutes.
2. After two to three days, transfer groups of 10–15 animals to new plates. Take care to do this worm-by-worm. After reproducing for one generation, the worms can be confirmed as expressing the correct phenotype.