QB3 Berkeley / Education / Lab Fundamentals Bootcamp / Bootcamp Manual / Molecular Cloning Overview / Making media: autoclaving & pouring plates

Making media: autoclaving & pouring plates

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Key Concepts

Luria Broth (LB) media is a generic, rich growth medium used to grow bacteria such as E. coli.  To ensure that only our bacteria grow, we need to first sterilize the medium to kill any contaminating organisms. We will be using an autoclave to sterilize our LB media. 

An autoclave is an instrument that subjects items to high-pressure saturated steam at high temperatures, killing any microorganisms that may be present. Because the autoclave reaches high temperatures and pressures, be sure to use heat-protective gloves when adding or removing items from the autoclave.  Never open a running or pressurized autoclave and always check that the drain trap is clear before starting a cycle.

Materials

  • LB powder
  • Agar 
  • Weigh-boat
  • 125mL autoclave-safe pyrex flask (2 flasks per group) 
  • 50mg/mL kanamycin (kan), 1000X antibiotic stock

Procedure

LB media contains (per 1L water):

* 10g Tryptone (tryptic digest of proteins to release peptides and amino acids that bacteria can use for food and protein synthesis)
* 5g Yeast Extract (source of additional proteins, vitamins, minerals, and sugars necessary for growth)
* 10g NaCl 

 

To make liquid LB media from a pre-mixed powder: 25 g LB powder/1 L water

If making solid media, also add: 15g agar per 1L

 

We have purchased pre-mixed LB powder (contains the tryptone, yeast extract, and salt).  All we need to do is add water.  Each group will need 50mL of LB, and 50mL of LB-agar.   

  1. In an Erlenmeyer flask, add ____ grams of LB and 50mL of water.
  2. In another flask , add ____ grams of LB, ____ grams of agar, and 50mL of water. 
  3. Mark your flask and bottle with autoclave tape. The tape changes color once it has been autoclaved so that you can keep track of whether or not the media has been sterilized. Autoclave tape with white stripes indicates non-sterile media and tape with dark stripes indicates media that has been sterilized.
  4. Autoclave the media. Usage instructions:
    • Check the pressure and temperature gauges to ensure that the autoclave is not running before opening the door.
    • Turn the wheel to open the door slowly, so that any steam inside does not blast out.
    • Use the autoclave-specific cart to move the interior autoclave cart out on the rails using heat-protective gloves.
    • Put the items in an autoclave-safe secondary containment, like an autoclave-safe plastic bin. This will keep your media from clogging the drain if the flask or bottle breaks. 
    • Loosen the caps of any bottle so that pressure can equalize between the inside of the bottles and the autoclave during the cycle.  This prevents the bottles from exploding in the autoclave.
    • Once you’ve added the items to be autoclaved to the bin, add ~1/2 inch of water to the bottom of the bin. This will help to heat your items evenly during the autoclave cycle.
    • Slide the cart into the autoclave chamber; remove the autoclave rails cart. 
    • Close and latch the door as firmly as you can. A good seal must be made with the door to prevent steam from leaking out and scalding you and to allow the interior to reach high pressure.
    • Check that the jacket pressure is on and reset it if it is not.
    • Set the cycle to “Liquid” and the sterilizing time to 20 minutes. Sterilizing time depends on the volume of the containers being autoclaved, and larger volumes will require longer sterilizing times. Set the drying time to zero.
    • Start the cycle and watch the autoclave to make sure the program reaches the set pressure and temperatures without issue.
    • Return to the autoclave once the cycle has finished and ensure that the temperature and pressure have returned to normal. Remove your items in the same way that you put them in. Make sure that the cycle has finished before removing your items. Since autoclaves are often in high demand, it is best to remove your items promptly when the autoclave cycle has finished.
    • Handle your bottles and flasks with care, as they are very hot. Remove them from the autoclave bin to cool and discard the water in the bin.
  5. If the media contains agar, use oven-safe mitts to gently swirl the bottle while it is cooling down.  This should be done every 5-10 minutes to redistribute the ingredients within the flask.  DO NOT agitate the solution too much as this will promote the formation of bubbles near the surface.
  6. For LB-agar, the antibiotic must be added before the media solidifies, but after the media is cool enough that the hot temperatures will not destroy the antibiotic.  Wait until the media has cooled to ~60°C.  Add the antibiotic, in this case kanamycin, (1000x or 1ul per mL of LB-agar) and swirl to mix. Our vector (pET28b) contains a gene that confers resistance to kanamycin. By adding kanamycin to our plates, we will select for bacterial cells that have taken up the pET28b plasmid.
  7. Prepare your plates. Use oven-safe mitts on one hand to hold the autoclaved bottle containing the media.  Use your other hand to lift the lids of plates.  Pour each plate individually, pouring enough media into each plate so that it is ~1/2 full, or about 25mL per plate. It is best to do this near a flame (a lit Bunsen burner) to prevent microorganisms in the air from contaminating the media in your plates. If your LB-agar solidifies before pouring, use a microwave to melt it.