Chang Lab:Notebook/CBE/08/148/2008/12/16

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Biofilm Optimization Trial II

  • 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 II)
  • Autoclave new M9 medium
  • 3 CBDs were prepared for incubation at 30, 37 and 42 degress at 30rpm for 48 hours
  • The procedures are as written in Trial I
  • The 5 hour incubated Ecoli is adjusted to follow the McFarland standard(A600 = 0.257).
  • Approximatedly 6X dilution was needed (0.45ml broth culture to 2.55ml broth) to match standard.
  • For minimal medium M9, E coli broth centrifuged at 3000g for 10 minutes, supernatant LB Broth discarded through careful pipetting, without disturbing sedimented E coli cells. Previously prepared M9 medium used to resuspend E coli cells. *Absorbance at 600nm adjusted until it matched 1.0 McFarland Std.
  • Approximately 5.4X dilution (0.55ml to 2.45ml broth) to match standard.
  • Using multipipette (8-pronged), 200 uL of adjusted inoculum pipetted into each well of CBD.
    • Columns 1-6: rich LB Medium
    • Columns 7-12: minimal M9 Medium
  • CBD covered with pegged lid. Incubated at 4:00 PM at 37C, 150 rpm. Will check on 12/18 to score for biofilm growth.

Serial Plating

  • Plated 10^-10, 10^-12, and 10^-14 dilutions to verify cell number after 5-hr incubation.