User:Kalkao/notebook/Making T-broth Agar Plates
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< User:Kalkao | notebook
- 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
- Use a graduated cylinder to obtain distilled "MilliQ" water (1 L for 1 L T-broth)
- 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
- 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
- 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
- Place the flask in a 65*C water bath and allow it to cool
- Obtain sterile plates (40 plates for 1 L T-broth Agar)
- 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