5 December 2008

Time: 0945; Agenda: Preparation of Ecoli Inoculum and Quadrant Streaking of Petri Dishes

• We had to re-prepare the medium because we incorrectly prepared it on 3rd December and hence no colonies were seen growing on the petri dishes. This is because pure agar was accidentally placed in the petri dish instead of LB Agar! Hence, LB Agar needs to be prepared.

1.Preparation of LB Agar:

• We prepared the LB Agar medium with the following composition in 1L of water:

25g LB Broth + 15g Agar

• • 25g of LB Broth and 15g Agar weighed using a weighing machine and added into a 1L flask. The flask was then topped up to 1L with distilled water. A glass rod was used to stir the mixture to try to distribute the solids more evenly.
  • The 1L mixture is distributed into 3 500ml bottles each containing about 300ml of the mixture. This ensures that we autoclave bottles which are not too fully filled.
  • Autoclave sticky tapes are attached to the bottles (with loosened caps) and the 3 bottles autoclaved at 121 degrees for 15 minutes.

This composition will be regularly prepared for subsequent use.

2.Preparation of Ecoli (K12 W3110) Inoculum:

• We also learnt that a ratio of 1:9 (Bacteria:Broth) is too high. Hence a ratio of 0.1:9 is used instead.
  • 0.1ml 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 252rpm for 6 hours for 35.2 degrees.

3. Quadrant Streaking of Petri Dishes

• After 6 hours of incubation, the inoculum was used to streak on the petri dishes.
  • See 4 December for protocol on quadrant streaking and subsequent storage.