User:Kalkao/notebook/Making T-broth Agar Plates

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  1. Use weighing plates to measure out:
    • 10g/L Bacto-Tryptone (10g for 1 L T-broth)
    • 5g/L NaCl (5g for 1 L T-broth)
    • 1.5% Bacto-Agar (15g for 1 L T-broth)
    • Be careful when measuring out the Bacto-Tryptone and Bacto-Agar
  2. Use a graduated cylinder to obtain distilled "MilliQ" water (1 L for 1 L T-broth)
  3. Use a sterile flask to mix the T-broth and agar (2 L flask for 1 L T-broth)
    • Replace the tin foil cap over the top of the flask
    • Pour Bacto-Tryptone, NaCl, and Bacto-Agar into flask
      • Be careful when pouring the Bacto-Tryptone and Bacto-Agar
    • Pour distilled water into flask
    • Swirl the flask to mix
  4. Use Autoclave to sterilize T-broth Agar:
    • Put the flask in a plastic autoclave bin before placing it in the autoclave
    • Slighty loosen the tin foil cap on the top of the flask
    • Check to make sure the seal of the autoclave door is clean
    • Set autoclave settings to "fluid"
    • Set pressure to 22.0 psi
    • Set temperature to 250.0 *F
    • Set time to 60 minutes
    • Run the autoclave, but wait until the pressure and temperature reach the set levels before you leave
  5. Remove the flask from the autoclave after autoclaving is finished
    • Tighten the tin foil cap on the top of the flask
    • Pour out the fluid/boil-over from the autoclave bin
    • Remember to use heat mittens when handling
  6. Place the flask in a 65*C water bath and allow it to cool
  7. Obtain sterile plates (40 plates for 1 L T-broth Agar)
  8. After T-broth Agar has cooled, pour agar into the plates
    • Use a bunsen burner to sterilize the lip of the flask between plates
    • Fill each plate with slightly more than is required to cover the bottom of the plate
    • Wave a flame over the plate with a bunsen burner to eliminate bubbles from the agar.
    • Remember to use sterile technique
    • Allow plates to cool overnight
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