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

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