Chang Lab:Notebook/CBE/08/146/2008/12/18

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Time start: 915 AM

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

Biofilm Optimization (Trial III)

  • 3 CBDs were incubated at 21, 30 and 37 C at 30 rpm for 24 hours. (All procedures are same as the first 2 trials only the M9 inoculum suspension method was changed to matching the broth with 1.0 McFarland before centrifuging.
  • 5-hr incubated E coli broth was adjusted to the 1.0 McFarland Std (Absorbance@ 0.257). About 0.3ml broth culture to 2.7ml broth was required.
  • For minimal medium M9, absorbance of E coli broth at 600nm adjusted until it matched 1.0 McFarland Std. E coli 1.0 McFarland inoculum centrifuged at 3000g for 10 minutes, supernatant LB Broth discarded through careful pipetting, without disturbing sedimented E coli cells. M9 medium was then used to resuspend E coli cells.
  • About 200 uL of adjusted inoculum pipetted into each well of CBD.
    • Columns 1-6: rich M9 Medium
    • Columns 7-12: minimal LB Medium
  • CBD were covered with pegged lid, parafilmed and incubated at desired temperatures at 30 rpm.

Serial Plating

  • LB-based 1.0 McFarland E coli inoculum was done at 10^-10 to 10^-20 dilutions.
  • M9-based 1.0 McFarland E coli inoculum was done at 10^-6 to 10^-10 dilutions.
  • Planktonic cells from CBD wells were also done 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.