Chang Lab:Notebook/CBE/08/146/2008/12/13

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Entry title

Time started:915am Bacteria Culture:

  • 5ml taken from the overnight 10ml E.coli culture and added to 150ml of broth.
  • Incubated this for 5 hour.

Time started:215pm Biofilm Optimization Preparation:

  • After 5 hours, the incubated Ecoli culture was diluted to McFarland standard (Abs:0.257 at 600nm).
    • A reading of 0.258 was obtained for 0.22ml E.coli culture : 2.78ml LB broth.
  • The Ecoli culture was centrifuge for 10minutes (3000g).
  • Pippeted out the supernatant (LB Broth), the E.coli residue was mixed with M9 medium and adjusted to Mc Farland Standard.
    • A reading of 0.265 was obtained for 0.5ml of E.coli culture: 2.5ml of M9 medium
  • 0.230 ml of adjusted(according to Mcfarland standard) inoculum was pipetted into each well of CBD using a multipipette.
    • 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.

Serial Plating:

  • Plated 10^-8, 10^-10, and 10^-12 dilutions to verify cell number after 5hr incubation. To be checked after ~24 hrs for colony counting.

Notes

  • M9 medium not homogeneous (could be due to saturation or missing of autoclaving step).
  • Need to check with RPM value. 30 rpm from Tre-hardy et al. 150 RPM used was too fast, may have caused the spillage during biofilm growth .