3 December 2008

Date: 3 December 2008; Time: 1100; Agenda: Preparation of Ecoli Inoculum

1.Preparation of culture medium:

• 14g of LB Agar was weighed using a weighing machine and poured into a 400ml bottle.
• 400ml of distilled water was added to the bottle.
• An autoclave sticky tape was attached to the bottle.
• 2 such bottles were prepared for LB Agar.
  • Repeat this procedure for LB Broth.
• The 4 bottles were put into an autoclave machine and autoclaved at 121 degrees and for 15 minutes.
• The autoclave process, however, lasted about 2 hours due to both temperature and pressure fluctuation in the autoclave.
• The bottles were removed from the autoclave and the 2 bottles of LB Broth medium was put into a 70 degrees incubator for storage.
• The 2 bottles LB Agar medium were used to fill 40 petri dishes in the fume hood.
  • The LB Agar was poured out slowly from the bottle such that it covered the entire surface of the petri dish. This requires about 10-15ml of LB Agar per dish.
  • The petri dishes were left in the fume hood for about 10 minutes to let them harden.
  • They are then parafilmed and then placed in the 4 degrees fridge for storage.

2.Preparation of Ecoli (K12 W3110) Inoculum:

• 1ml of Ecoli was added to 9ml LB Broth in a test tube using pipettes in the fume hood.
• The test tube was then placed in an incubator at 30rpm for 16hr for 30 degrees.