Chang Lab:Notebook/CBE/08/148/2008/12/18

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  • Took 5 ml from 10-ml overnight E coli culture into 150 ml broth for 5-6 hr incubation at 37 C (inoculum for biofilm optimization trial III)

Biofilm Optimization Trial III

  • 3 CBDs were prepared to be incubated at 21, 30 and 37 degree (30rpm for 24 hours).

(Temperature at 42 degree was not used as based on the previous trial, the medium in the well gets evaporated)

  • Procedure is the same as the previous 2 trials
  • Adjustment according to McFarland Standard
    • For LB:9X (0.3ml broth culture to 2.7ml broth) dilution to match standard.
    • For M9:LB which match the McFarland Standard is prepared for centrifugation. The supernatant LB was then discarded and M9 medium was added.
  • Using a mivropippete,200 uL of adjusted inoculum pipetted into each well of CBD.
    • Columns 1-6: rich M9 Medium
    • Columns 7-12: minimal LB Medium
  • CBD covered with pegged lid. Incubated at desired temperatures at 30 rpm. Will check on 12/19 to score for biofilm growth.

Serial Plating

  • Plated LB-based 1.0 McFarland E coli inoculum at 10^-10 to 10^-20 dilutions to verify cell number.
  • Plated M9-based 1.0 McFarland E coli inoculum at 10^-6 to 10^-10 dilutions to verify cell number.
  • Plated planktonic cells from CBD wells at 10^-2, 10^-4, 10^-6 dilutions for both LB and M9 wells of 21, 30 and 37 C CBDs.

To be checked after ~24 hrs for colony counting