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

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.

Set up the plates to be filled

1. Cover the bench top 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