Chang Lab:Notebook/CBE/08/146/2008/12/16

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Time start:820am

  • Took 5 ml from 10-ml overnight E coli culture into 150 ml broth for 5hr incubation at 37 C (inoculum for biofilm optimization trial II)
  • Autoclave new M9 medium (1 liter)

Biofilm Optimization (Trial II)

  • 3 CBDs were prepared to be incubated at 3 temperatures: 30, 37 and 42 C at 30 rpm for 48 hours. The procedures are same as Trial I.
  • 5-hr incubated E coli broth adjusted to get 1.0 McFarland Std (Abs = 0.257 at 600nm). 0.45ml broth culture : 2.55ml broth to get 0.267 reading.
  • For minimal medium M9, E coli broth centrifuged at 3000g for 10 minutes, supernatant LB Broth discarded (without disturbing sedimented E coli cells). Previously prepared M9 medium added to the E coli cells. Absorbance at 600nm adjusted until it matched 1.0 McFarland Std. 0.55ml broth culture : 2.45ml broth to get 0.25 reading.
  • 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 18/12/08 to score for biofilm growth.

Serial Plating

  • Plated 10^-10, 10^-12, and 10^-14 dilutions to verify cell number after 5-hr incubation. To be checked after ~24 hrs for colony counting.

Note

  • CBD incubated at 30 C was transferred to static incubator (in used by others)
  • Inoculum volume in wells reduced to 200 ul to prevent spillage of liquid upon replacement of pegged lid.



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