Haynes:LBagarAmp

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LB Agar Plates

by Karmella Haynes, 2012

MATERIALS

  • Personal Protective Equipment (PPE) - lab coat, disposable gloves
  • ~40 clean, sterile petri dishes (pr "plates"), packed in plastic "sleeves" per 1 liter of LB agar
  • LB Broth powder (Sigma L3022)
  • Bacteriological Agar
  • 1N NaOH
  • Two clean and sterile 1000 mL glass flasks
  • 100 mg/mL Ampicillin from the -20°C freezer (if you are making amp plates)


PROCEDURE

Make LB Agar - these instructions are for 1 liter

  1. Put on your PPE.
  2. Fill a 1000 mL graduate cylinder with deionized water (from the sink)
  3. Weigh out 20 g of LB Broth powder on the balance.
  4. Add the LB powder to the water in the cylinder.
  5. Weigh out 15 g of Bacteriological Agar.
  6. Add the Bacteriological Agar to the mixture in the cylinder.
  7. Use a 1000 μL micropipette to add 1000 μL 1N NaOH to the mixture.
  8. Swirl the mixture for a few seconds.
  9. Fill the cylinder up to 1000 mL with deionized water (from the sink).
  10. Get two clean, sterile 1000 mL glass flasks from the shelf. Remove the foil caps and set them aside
  11. Carefully cover the cylinder with a large square of parafilm. Make sure the seal is tight.
  12. Cover the opening with your gloved hand and invert the cylinder several times to mix the solution.
  13. Quickly and carefully fill each flask up to the 300 mL mark. Cover the cylinder again and invert to mix.
  14. Quickly and carefully fill each flask up to the 400 mL mark. Swirl the cylinder to mix.
  15. Quickly and carefully fill each flask up to the 500 mL mark.

Melt the LB Agar

  1. OPTION 1 - Microwave
    1. Replace the foil caps with Kimwipe-covered black foam plugs.
    2. Carry the flasks (in a tray) to the large white microwave (Wang Lab).
    3. Microwave the flasks for 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).
    4. Microwave the flasks for another 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).
    5. Microwave the flasks for10 minutes. Use a rubber mitt to put the flasks back into the tray and bring them back to your bench
  2. OPTION 2 - Autoclave
    1. Put the foil caps on the flasks.
    2. Put the flasks in an autoclave tray. Fill the bottom of the tray about 1 inch deep with tap water (this trick prevents boil-over).
    3. Take the tray with flasks up to the 3rd floor autoclave.
    4. Choose the "Liquid 15" program/cycle. Put the tray with flasks into the autoclave, close the door, and hit "start".

Set up the empty plates - complete this part while the LB agar is melting in the microwave or autoclave

  1. Cover the work area with bench paper
  2. Get a stack of plates and cut open the very top of the sleeve. Do not rip apart the sleeve. You will need this to store the finished plates.
  3. Take the plates out and stack them in sets of five, lid-side-up.
  4. If you are making Amp plates...
    1. Take a tube of 100 mg/mL ampicillin from the -20°C freezer and thaw it in your hand or at room temperature.
    2. Take a red marker and mark the sides of the plates with a red stripe.


Pour the agar

  1. Using proper PPE, bring the flasks to the bench.
  2. Use the temperature gun to measure the temperature of the flask of agar. When the temperature cools to 60°C, proceed to step 3 or 4.
  3. If you are making Amp plates, add 500 μL of thawed ampicillin to 500 mL melted agar. Swirl the flask(s) gently for at least 30 seconds to mix. Avoid making bubbles.
  4. Pouring plates like a pro: with the uncapped flask of agar in your dominant hand, and a stack of 5 empty plates by your less dominant hand...
    1. Lift the top four plates plus the lid of the bottom-most plate so that only the bottom part of the bottom-most plate is on the bench. Carefully tilt the agar flask and pour the agar into the empty plate just until the surface is covered with agar. Do not over-pour!
    2. Place the lid + four plates onto the filled plate.
    3. Lift the top three plates plus the lid of the second-to-the-bottom plate. Carefully tilt the agar flask and pour the agar into the empty plate just until the surface is covered with agar. Do not over-pour!
    4. Place the lid + three plates onto the filled plate.
    5. Repeat this procedure in a similar fashion for the remaining plates.
  5. When the agar flask is empty, immediately rinse it in the sink so that the agar does not solidify in the bottom of the flask.
  6. Allow the plates to cool and solidify on the bench at room temperature for at least 6 hours or overnight .
  7. Store the plates agar-side-up in a plastic sleeve.
  8. Use lab tape to label the whole sleeve of plates: antibiotic & concentration, your initials, date
  9. Store the plates in the 4°C cold room.


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