线虫培养--Culturing Worms

Culturing Worms

 

<center> <font>by Michael Koelle</font></center>

 

1. Bacterial strain for feeding worms: OP50, a uracil auxotroph. Streak OP50 out on a 9 cm NGM agar plate , grow overnight at 37°. Can then parafilm the plate and keep it at 4° for months. To make bacteria for seeding plates, use a flamed wire loop to pick a single OP50 colony into a 100 ml bottle of B broth. Set the bottle (no shaking) at 37° overnight. It should be cloudy the next day. This bottle can be stored at 4° and used for ~3 months.

2. Seeding plates. NGM plates should be allowed to sit out at room temperature in a closed box for a couple of days after pouring to allow them to dry, and so that plates with bacterial or fungal contaminants become obvious. Using a sterile pipette, drop 1-2 drops OP50 suspension on the middle of each 5 cm NGM plate. It is best if the bacterial lawn does not touch the side wall, as worms then tend to crawl up the wall and die. (Don't move the plates soon after seeding, or the puddle of bacteria will definitely slosh against the wall). Take care not to damage the surface of the agar with the pipette tip; worms will then burrow under the surface. Let the plates sit overnight to form a bacterial lawn before using. Plates can be used for about 7 days after seeding, and are best 2-4 days after seeding.

To seed large (9cm) NGM plates: drop 5 drops OP50 in the middle of a plate. Use a sterile pipette at a very shallow angle to spread the OP50 liquid around so that a thin pool of liquid covers the middle 2/3 of the plate. Set the seeded plates at 37° overnight, or at room temp. for 2 days. (Many people try to seed large plates by dropping a huge amount of OP50 on the plates, until the thick pool of liquid covers a large portion of the plate; this results in a wet mess that takes several days to dry into a mealy-looking lawn.)

3. Transferring worms. Use a platinum wire pick. To construct this, take a hammer and bang on a piece of ~32 gauge platinum wire to flatten it. Others use pliers to flatten the end of the wire. Then cut the wire so that only a few millimeters of flat wire are left attached to a longer section of round wire. Can use a razor blade to slice off the sharp corners of the flat end. Then bend the flat section so that it is at a shallow angle to the rest of the wire. Finally, place the non-flattened end inside of a short Pasteur pipette, and seal into the glass over a Bunsen burner. About 2 cm of wire should protrude from the glass, but personal preferences vary. Eventually the end of the pick wears out, and the end can be cut off and a new end flattened and bent.

To pick up worms, flame the wire to sterilize it, and drag the pick along a bacterial lawn to coat the under side of the pick with bacteria. Goopy, several day old bacterial lawns are the best for this; some keep old plates next to the microscope especially for coating their picks. Gently brush against the worm to be picked up so that it sticks to the bacteria on the pick. To set the worm down, gently brush against the new plate, and allow the worm to crawl off. The worm should be immediately active on the new plate if it is undamaged. It is possible to pick up at least ten worms at a time, however, you don't want the worms to spend too much time on the pick, as they will dry out.

4. Worms are typically cultured in a 20° room, with the plates upside down, stored in old scintillation vial boxes. The dividers in the boxes can be reconfigured to fit the 5 cm worm plates. The boxes and dividers are baked at 65° for >1 hour before use to sterilize . Worms can be cultured between 15° and 25°, and will grow slower or faster, respectively, at these temperatures. To keep a stock available for a long time without maintenance, parafilm the plate and set it at 15°. Can then recover the worms for ~5 months afterwards. The limiting factor is that the worms die eventually of dessication when the plate dries.

If you're leaving town for a while a good way to keep your worms is to put the plates in a 12.5° C incubator. The worms will be almost in stasis, and will show only barely perceptable development after 1 week. Apparently prolonged incubation at 12.5° kills worm stocks by sterilizing them. One week is certainly safe, and after 2-3 weeks you'll probably still have some fertile worms.

5. To transfer a stock to a new plate, typically you "chunk it out" instead of transferring worms with a pick. Keep small capped bottle of EtOH by your microscope, and a small spatula. Dip the spatula in EtOH, then place the spatula in the bunsen burner flame for ~15 seconds: the EtOH will burn off and then the spatula should get almost red hot. (If you just quickly let the EtOH burn off this won't sterilize the spatula, and you will always end up contaminating your worms). Dip the hot spatula back in the EtOH until the spattering and fizzling stops- this cools it off so the heat won't kill the worms. Then quickly pass the spatula through the flame to ignite the EtOH, which will burn off without heating the spatula much. Use the now sterile spatula to cut a ~1 cm square chunk of the old plate and place it upside down on the new plate. Worms will crawl out onto the fresh bacterial lawn.

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NGM agar: 3 g NaCl

17 g agar

2.5 g peptone

1 ml cholesterol (5 mg/ml in EtOH)

975 ml H20

Autoclave, and then sterilly add the following, mixing after each addition:

1 ml 1 M CaCl2

1 ml 1 M MgSO4

25 ml 1 M potassium phosphate pH 6

Typically pour 5 and 9 cm plates. Store in plastic boxes with covers at room temperature for a couple days before use to allow the plates to dry.

B broth: 10 gm bactotryptone

5 gm NaCl

1 liter dH20

dissolve over bunsen burner.

Then distribute to 100 ml bottles and autoclave.

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