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LB Agar Plates
by Karmella Haynes, 2012
- 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)
Make LB Agar - these instructions are for 1 liter
- Put on your PPE.
- Fill a 1000 mL graduate cylinder with deionized water (from the sink)
- Weigh out 20 g of LB Broth powder on the balance.
- Add the LB powder to the water in the cylinder.
- Weigh out 15 g of Bacteriological Agar.
- Add the Bacteriological Agar to the mixture in the cylinder.
- Use a 1000 μL micropipette to add 1000 μL 1N NaOH to the mixture.
- Swirl the mixture for a few seconds.
- Fill the cylinder up to 1000 mL with deionized water (from the sink).
- Get two clean, sterile 1000 mL glass flasks from the shelf. Remove the foil caps and set them aside
- Carefully cover the cylinder with a large square of parafilm. Make sure the seal is tight.
- Cover the opening with your gloved hand and invert the cylinder several times to mix the solution.
- Quickly and carefully fill each flask up to the 300 mL mark. Cover the cylinder again and invert to mix.
- Quickly and carefully fill each flask up to the 400 mL mark. Swirl the cylinder to mix.
- Quickly and carefully fill each flask up to the 500 mL mark.
Melt the LB Agar
- OPTION 1 - Microwave
- Replace the foil caps with Kimwipe-covered black foam plugs.
- Carry the flasks (in a tray) to the large white microwave (Wang Lab).
- Microwave the flasks for 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).
- Microwave the flasks for another 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).
- Microwave the flasks for10 minutes. Use a rubber mitt to put the flasks back into the tray and bring them back to your bench
- OPTION 2 - Autoclave
- Put the foil caps on the flasks.
- 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).
- Take the tray with flasks up to the 3rd floor autoclave.
- 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
- Cover the work area with bench paper
- 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.
- Take the plates out and stack them in sets of five, lid-side-up.
- If you are making Amp plates...
- Take a tube of 100 mg/mL ampicillin from the -20°C freezer and thaw it in your hand or at room temperature.
- Take a red marker and mark the sides of the plates with a red stripe.
Pour the agar
- Using proper PPE, bring the flasks to the bench.
- 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.
- 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.
- 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...
- 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!
- Place the lid + four plates onto the filled plate.
- 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!
- Place the lid + three plates onto the filled plate.
- Repeat this procedure in a similar fashion for the remaining plates.
- 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.
- Allow the plates to cool and solidify on the bench at room temperature for at least 6 hours or overnight .
- Store the plates agar-side-up in a plastic sleeve.
- Use lab tape to label the whole sleeve of plates: antibiotic & concentration, your initials, date
- Store the plates in the 4°C cold room.