Making LB Agar Plates: Batch makes about 6 plates.

Making the LB Agar

  1. Mass out ______of LB Agar powder (VWR DF0445-17) in a weigh boat
  2. Add 125 mL of dH2O to a ______mL Erlenmeyer flask
  3. Mix powder well into water to bring into solution
  4. Cover the flask with aluminum foil and wrap the neck with autoclave tape.
  5. Label your flask with your name and date and what is in the flask.
  6. Autoclave for 15 minutes at 121 ºC.
  7. Let agar cool to ~55C

.

Pouring the Plates

1.Make sure bench top has wiped down with bleach/EtOH

2.Remove sterile Petri dishes (VWR 25384-208)

3.Pour a thin layer (5mm) of LB Agar (~15mL) into 6 plates being careful to not

lift the cover off excessively (you should be able to just open up enough to pour).

4.Let each plate cool until it is solid (~20 minutes) then flip it over so as to avoid

condensation on the agar.

5.Store plates wrapped in parafilm in fridge with: name, date and contents.

Answer the following questions:

  1. What is LB agar? What is in it?
  2. Why do you autoclave the agar? What is an autoclave and how does it work?
  3. Why do you only open the dish slightly when you pour the plates?
  4. What is the difference between agar and broth?
  5. Obtain the MSDS for LB Agar and LB Broth and describe any precautions and disposal procedures.

Making LB Broth: Batch makes about 20mL.

Making the LB Broth

  1. Mass out ______of LB Broth in a weigh boat
  2. Add 20 mL of dH2O to a ______mL Erlenmeyer flask
  3. Mix powder well into water to bring into solution
  4. Cover the flask with aluminum foil and wrap the neck with autoclave tape
  5. Label your flask with your name and date and what is in the flask.
  6. Autoclave for 15 minutes at 121 ºC.
  7. Let broth cool to ~55C

.

Aliquoting the Broth

  1. Make sure bench top has wiped down with bleach/EtOH
  2. Obtain sterile 15mL centrifuge tubes
  3. Pour approximately 10 mL into each tube being careful to not

keep the cap off an excessive amount of time.

  1. Store tubes in fridge with: name, date and contents.