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


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