Caenorhabditis elegans

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The Worm

Common Name : The Worm

Scientific Name : Caenorhabditis elegans

Multicellular eukaryotic nematode

Biosafety Level: BSL1

Benefits

• As a model organism, C. elegans is a species of nematode worm, and the adults are
  approximately 1 mm in length with 959 somatic cells. Its transparent body consists of
  three layers; an epidermal layer, an intestinal layer, and a muscle layer.Other systems
  such as the nervous system and reproductive systems are found between the three
  layers.There are physiological overlaps between humans and C. elegans, such as the
  digestive system, the nervous system, and the reproductive system.

• Size and transparency allows for easy anatomy and developmental examination under a
  microscope.

• C. elegans has a much simpler anatomy than humans (it doesn’t have bones, a heart or
  a circulatory system) but it does share many genes and molecular pathways with
  us.Many of the genes in the C. elegans genome have functional counterparts in humans
  which makes it an extremely useful model for human diseases.
• Easy maintenance, short lifespan and easy breeding makes C.elegans a good model
  organism.
• The lifespan of C. elegans is 2-3 weeks. Developing from eggs, an individual goes
  through four larval stages before reaching maturity. Short lifespan helps study disease
  progression.
• C. elegans display two sexes; male or hermaphrodite. The hermaphrodites are able to
  self-fertilize and can give birth to hundreds of progeny.This ability to self-fertilize is a
  useful tool for its use as a model organism, not only because these hermaphrodites can
  reproduce in high numbers quickly, but also because certain mutated strains of C.
  elegans may be paralyzed but do not need to find a mate to reproduce.
• Healthy cultures of C. elegans can be frozen and then defrosted and revived when
  needed.

Applications

• C. elegans as a model organism has advantages including having all the physiological
  properties of an animal, the ability to replicate human diseases and a fast life cycle. C.
  elegans has been used as a model organism to study human diseases ranging from
  Parkinson’s disease to mitochondrial diseases, as well as studying the immune system.
• C. elegans mutants can be screened with thousands of potential drugs for important
  diseases.
• Studying cell death or ‘apoptosis’ in the C. elegans could hold the key to counteracting
  the effects of aging in humans as well as providing clues about cancer, diabetes and
  other diseases. It is found that human genes responsible for a range of mitochondrial
  diseases have orthologous genes in C. elegans. Hence, C. elegans is a possible model
  organism for the study of mitochondrial diseases.
• C. elegans only has innate immunity and is devoid of adaptive immunity. Hence it is used
  as a model organism to study the innate immune response to pathogens.
• As a model organism, C. elegans has advantages including having all the physiological
  properties of an animal, the ability to replicate human diseases and a fast life cycle. C.
  elegans has been used as a model organism to study human diseases ranging from
  Parkinson’s disease to mitochondrial diseases, as well as studying the immune system.

Maintenance

C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar which has
been aseptically poured into petri plates (Brenner, 1974).
Several sizes of petri plates are available and can be purchased from companies such as Nunc
or Falcon. Smaller plates (35 mm diameter) are useful for matings or when using expensive
drugs. Medium size plates (60 mm diameter) are useful for general strain maintenance, and
larger plates (100 mm diameter) are useful for growing larger quantities of worms, such as for
certain mutant screens.

Preparation of NGM plates -

• Mix 3 g NaCl, 17 g agar, and 2.5 g peptone in a 2 liter Erlenmeyer flask. Add 975 ml
  H2O.

• Cover the mouth of the flask with aluminum foil. Autoclave for 50 min. Cool flask in 55°C
  water bath for 15 min. 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.

• Using sterile procedures, dispense the NGM solution into petri plates using a peristaltic
  pump. Fill plates 2/3 full of agar.
• 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 sterile technique, apply approximately 0.05 ml of E. coli OP50 liquid culture to
  small or medium NGM plates or 0.1 ml to large NGM plates using a pipet.

• If desired, the drop can be spread using the pipet tip or a glass rod. Spreading will create
  a larger lawn, which can aid in visualizing the worms. Take care not to spread the lawn
  all the way to the edges of the plate; keep the lawn in the center.

• The worms tend to spend most of the time in the bacteria. If the lawn extends to the
  edges of the plate the worms may crawl up the sides of the plate, dry out and die.
• Allow the E. coli OP50 lawn to grow overnight at room temperature or at 37°C for 8
  hours

C. elegans visualization -

• C. elegans is transparent and can be visualized using a dissecting stereo microscope
  equipped with a transmitted light source.
• The CGC uses Wild Leitz model M5A or Zeiss model SV6. Standard 10X eyepieces and
  objectives which range from 0.6X to 5X (total magnification of 6X to 50X) are widely
  used.

Transferring C. elegans from one plate to another -

Chunking -

• A sterilized scalpel or spatula is used to move a chunk of agar from an old plate to a
  fresh plate. There will usually be hundreds of worms in the chunk of agar.

• The worms will crawl out of the chunk and spread out onto the bacterial lawn of the new
  plate.

• This method works well for transferring worms that have burrowed into the agar or are
  difficult to pick individually.

• The chunking method is fine for transferring homozygous stocks but it is not advisable if
  the population is heterozygous or if a stock must be maintained by mating.

Single animal transfer by worm picker-

• A worm picker can be made by mounting a 1-inch piece of 32 gauge platinum wire into
  either the tip of a pasteur pipet or in a bacteriological loop holder.

• Platinum wire heats and cools quickly and can be flamed often (between transfers) to
  avoid contaminating the worm stocks.

• The end of the wire, used for picking up worms, can be flattened slightly with a hammer
  and then filled with an emery cloth to remove sharp edges; sharp points can poke holes
  in the worms and kill them or make holes in the agar.
• The tip of the wire can be fashioned to your liking. Some people prefer a flattened end,
  while others prefer a slight bend that forms a hook. It takes a bit of experience with a
  worm picker to avoid poking holes in the agar. Worms crawl into the holes, making it
  difficult to see or pick them.
• General stock maintenance tips C. elegans stocks can best be maintained between
  16°C and 25°C, most typically at 20°C. C. elegans grows 2.1 times faster at 25°C than at
  16°C, and 1.3 times faster at 20°C than at 16°C. This variation in growth periods can be
  useful when planning experiments.

Growth of C. elegans in liquid medium-

• Large quantities of C. elegans can be grown in liquid medium. Liquid cultures of C.
  elegans are usually grown on S Medium using concentrated E. coli OP50 as a food
  source (Lewis and Fleming, 1995). It is often best to grow just one generation of worms
  in liquid before the worms are harvested.
• When growing worms for more than one generation, overcrowding can often lead to
  dauer formation despite the presence of food.
• Overnight cultures of E. coli OP50 grown in LB or other rich broth medium should be
  used to make a concentrated pellet of bacteria.
• The concentrated pellet can be stored at 4°C for several weeks or in a -70°C freezer
  indefinitely.
• It is a good idea to have a large amount of concentrated E. coli OP50 available so that
  food can be added as needed.
• The amount of food needed will depend on the starting inoculum of worms and the
  length of time the worms are grown.
• As a point of reference, you can grow a 250 ml batch of liquid culture by inoculating with
  worms recovered from 4 large (100 mm) petri plates and growing for 4-5 days.
• Initially, packed E. coli OP50 from a 2 liter culture is provided. The worm culture is
  monitored, and additional E. coli OP50 is added as needed.

Methods-

1. Add 250 ml S Medium to a sterilized 1-2 liter flask. Inoculate the S Medium with a
   concentrated E. coli OP50 pellet made from 2-3 liters of an overnight culture.
2. Wash each of 4 large plates of C. elegans (just cleared of bacteria) with 5 ml S Medium
   and add to the 250 ml flask.
3. Put the flask on a shaker at 20°C. Use fairly vigorous shaking so that the culture is well
   oxygenated.
4. Cultures should be monitored by checking a drop of the culture under the microscope. If
   the food supply is depleted (the solution is no longer visibly cloudy) add more
   concentrated E. coli OP50 suspended in S Medium.
5. When there are many adult animals in each drop, the culture is ready to be harvested.
   This is usually on the 4th or 5th day.
6. Put the flask on ice for 15 minutes to allow the worms to settle. Aspirate most of the
   liquid from the flask.
7. Transfer the remaining liquid to a 50 ml sterile conical centrifuge tube and spin for at
   least 2 min at 1150 × g to pellet the worms. Young larvae may take longer than 2 min to
   pellet.
8. Aspirate the remaining liquid.

Freezing and recovery of C. elegans stocks -

Caenorhabditis elegans can be frozen and stored indefinitely in liquid nitrogen (−196 °C)
(Brenner, 1974). The keys to a successful freeze are using animals at the correct stage of
development, the addition of glycerol to the freezing media, and a gradual cooling to -80°C.
Freshly starved young larvae (L1-L2 stage) survive freezing best. Well-fed animals, adults, eggs
and dauers do not survive well. It is best to use several plates of worms that have just
exhausted the E. coli OP50 lawn and that contain lots of L1-L2 animals. A 15% final volume of
glycerol in the freezing solution is used. A 1°C decrease in temperature per minute is desirable
during freezing.
This can be achieved by placing the worms (in freezer vials) in a styrofoam container at -80°C.
The styrofoam container can be either a commercial shipping box (with walls at least ¾ inch
thick) or a small styrofoam box with slots for holding vials. After 12 or more hours at -80°C, the
freezer vials should be transferred to their permanent freezer location for long term storage.

Freezing C. elegans using Liquid Freezing Solution Methods -

• Use one large, 2-3 medium, or 5-6 small NGM plates that have lots of freshly starved
  L1-L2 animals. Wash the plates with 0.6 ml S Buffer for each vial you will freeze. Collect
  liquid in a sterile test tube.
• Add an equal volume of S Buffer + 30% glycerin. Mix well.
• Aliquot 1.0 ml of mixture into 1.8 ml cryovials labeled with strain name and date.
• Pack the cryovials in a small styrofoam box with slots for holding microtubes or use a
  commercial styrofoam shipping box.
• Place the box in a −80°C freezer overnight (or for at least 12 hours).
• The next day transfer the vials to their permanent freezer locations.
• Thaw one vial as a tester to check how well the worms survived the freezing.

Thawing C. elegans frozen using Liquid Freezing Solution Methods-

• Melt Soft Agar Freezing Solution in an autoclave or microwave and place in a 50°C
  water bath for at least 15 minutes.
• Use one large, 2-3 medium, or 5-6 small NGM plates that have lots of freshly starved
  L1-L2 animals. Wash the plates with 0.6 ml S Buffer for each vial you will freeze.
• Collect liquid in a covered sterile test tube and place in ice for 15 minutes.
• Add an equal volume of Soft Agar Freezing Solution to the test tube. Mix well.
• Aliquot 1 ml of mixture into 1.8 ml cryovials labeled with strain name and date.
• Pack the cryovials in a small styrofoam box with slots for holding microtubes or use a
  commercial styrofoam shipping box.
• Place the box in a −80°C freezer overnight (or for at least 12 hours).
• The next day transfer the vials to their permanent freezer locations. Take a scoop of
  frozen mixture from one vial as a tester to check how well the worms survived the
  freezing (see Protocol 10).

Freezing C. elegans using Soft Agar Freezing Solution Methods -

• Melt Soft Agar Freezing Solution in an autoclave or microwave and place in a 50°C
  water bath for at least 15 minutes.
• Use one large, 2-3 medium, or 5-6 small NGM plates that have lots of freshly starved
  L1-L2 animals. Wash the plates with 0.6 ml S Buffer for each vial you will freeze. Collect
  liquid in a covered sterile test tube and place in ice for 15 minutes.
• Add an equal volume of Soft Agar Freezing Solution to the test tube. Mix well.
• Aliquot 1 ml of mixture into 1.8 ml cryovials labeled with strain name and date.
• Pack the cryovials in a small styrofoam box with slots for holding microtubes or use a
  commercial styrofoam shipping box.
• Place the box in a −80°C freezer overnight (or for at least 12 hours).
• The next day transfer the vials to their permanent freezer locations. Take a scoop of
  frozen mixture from one vial as a tester to check how well the worms survived the
  freezing (see Protocol 10).
  24
• Thawing C. elegans frozen using Soft Agar Freezing Solution Methods Remove a vial
  from −80°C freezer and transfer to a small styrofoam box with slots for microtubes. Work
  quickly so that the solution in the vial does not thaw.
• Flame a small scoop or spatula and use it to remove 1/4 - 1/3 ml of the frozen solution.
  Place solution on a NGM plate with E. coli OP50 lawn. Return vial to −80°C freezer as
  quickly as possible.
• You should see worms wiggling after just a few minutes. After 2-3 days, transfer 10-15
  animals individually to separate plates.
• Allow the animals to reproduce for one generation and score the progeny for correct
  phenotypes

Genome

https://www.ncbi.nlm.nih.gov/datasets/taxonomy/6239/