Seeding/Spotting NGM Plates with Bacteria

Materials

  • 6 cm or 10 cm plates in the sliding-door 4°C incubator (pre-dried 48 hr.)
  • OP50 culture in a 2 L flask stored at 4°C (refer to Growing OP50 protocol)
  • 50 mL sterile disposable conical tube
  • Repeat pipette
    • 5 mL tips to seed 6 cm plates
    • 25 mL tips to seed 10 cm plates
    • Note: you will need the red adapter piece for 25 mL tip!

Protocol

  1. Transfer the OP50 from the Stock Flask to the conical tube.

    • ONLY POUR AS MUCH AS YOU NEED FOR YOUR NUMBER OF PLATES (Once dispensed, extra bacteria should not be used later, but bleached and disposed of).

    Note: Use a flame for sterile technique- sterilize the Glass Flask edge before and after dispensing into the tube, But DON'T flame the plastic tube!

  2. Work where plates are normally poured. Set up the plates in stacks that are not too high, about 4 plates high.

  3. Load the pipette tip in the repeat pipettor handle (using sterile technique to prevent contamination of the tip).

  4. Set the pipet to dispense 100 μL for 6 cm plates or 1000 μL for 10 cm plates.

  5. Seed 6 cm plates by dispensing one spot to the middle of the plate. Seed 10 cm plates by dispensing one spot to the middle of the plate, then after seeding the stack, shake the plates slightly (left to right, then up to down) to spread the bacteria (ask for a DEMO!).

    Note: Take care not to spatter the bacteria onto the edges of the plate when dispensing and spreading the bacteria.

  6. Leave plates stacked lid side up for at least 24 hr. for bacteria to soak in and grow. Make sure plates grow at least 2 days at room temperature before storing in the 4°C room.

    Note: Seeded plates should not be dried overnight with the Clean Hood on, since this will over-dry the bacteria.

  7. Finally, put the plates in pre-bleached plastic bins, and store them at 4°C.

Na22 bacteria

  • You can use Na22 E. coli as a food source alternative to OP50, if you desire larger numbers of gravid adult worms. Na22 requires liquid 2x YT media. 2X YT Media:

    Reagent Amount
    Tryptone 16 g
    Yeast Extract 10 g
    NaCl 5 g
    dH2O up to 1 L

    Final pH 7.0 ± 0.2 at 25°C

    Note: No antibiotic is used in 2x YT media. Do not contaminate the bacterial culture at this point. Use a Bunsen burner during transfer of the media to the flask where bacteria will be grown and keep a frozen sterile glycerol stock of Na22 bacteria. Grow Na22 overnight in 2x YT media, and seed the bacteria over the entirety of the plate, not just the center. Seed onto HGMA plates.