Bacteria for Worm Food/Seeding Plates

Starting a Bacterial Culture to use as Worm Food

• 100 ml bottle of sterile LB broth
• LB plate spread with bacteria (Note: the standard E. coli strain used as worm food is OP50)
• bacterial loop
• flame source (e.g., bunsen burner or alcohol lamp)

1. Light flame source, flame bacterial loop.

2. Pick large part or all of a single isolated colony from bacterial plate; inoculate bottle of LB broth by swishing loop into the liquid. Re-parafilm bacterial plate and return to refrigerator.

Note: these stock plates last for a few weeks up to a few months in the refrigerator if covered with parafilm. For this reason, the bacteria should be respread and grown on a new LB plate about monthly.

3. Allow culture to grow to high density (saturation) by incubating at 37 C overnight or 2-3 days at room temperature. Place in refrigerator.

Seeding Plates to use for Culturing Worms

• NGM plates (determine the number you need to seed, then add a few extra)
• Bacterial culture in LB broth (made as above)
• 5 ml or 10 ml sterile disposable pipet(s)
• pipet 'pump' (large black/red pipet bulb)

1. Remove bacterial culture from refrigerator, swirl to resuspend bacterial culture evenly.

2. Take up culture into sterile 5 ml or 10 ml pipet. Drop 2 - 4 drops into the center of a small NGM plate from a height of 4-6 inches - this gives a good spread of bacteria. Large NGM plates need 6 - 10 drops, distributed about the center of the plate. Ideally they should merge into a single central lawn.

Try to avoid getting bacteria all the way to the edges of the plate. Worms are harder to see under the microscope at the edge of the plate. Avoid moving plates immediately after adding liquid as this will cause the bacterial culture to run to the edge.

3. Allow plates to incubate on the bench (room temp) at least ON. If in a rush, plates may be dry enough to use by late afternoon if seeded first thing in the morning, however, the lawn will be sparse (with limited worm "carrying capacity"); see Notes below.

Notes: Ideally, plate should be seeded 1 - 3 days before needed, but typically are still fine for about a week or so after seeding. (Older, drier plates make good "picking plates," as long as they're no too dry.) Note that the time necessary for drying plates depends on the temperature and humidity of the lab, which can be quite variable. A thin lawn may be ready in the afternoon for plates seeded in the morning, but don't count on it. Plates can be rushed by putting in 37 C incubator for a few hours. Be sure to allow them to cool to room temperature before adding worms.