Chang Lab:Notebook/CBE/08/149/2008/12/13

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Antimicrobial efficacy testing of antibiotic-containing biodegradable nanopolymers against biofilm and planktonic cells
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Logs

  • Start 9:15 AM
  • 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 )
  • Prepared LB Broth (400 mL)

Biofilm Optimization (Trial I)

  • Following 1.0 McFarland Std (A600 = 0.257), 5-hr incubated E coli broth adjusted to match A600 value. A 13.6X dilution was required to match standard.
    E. coli in LB dilutions
    E. coli in LB dilutions
  • 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 6X dilution to match standard.
    E. coli in M9 dilutions
    E. coli in M9 dilutions
  • Further dilution was again carried out (i.e. 1 inoculum : 29 medium)
  • Using multipipette (8-pronged), 230 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 Monday to score for biofilm growth.

Serial Plating

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

Notes

  • M9 medium not homogeneous, may be due to saturation or skipping of autoclaving step. Must recalculate and redo later.
  • Unsure of second step above, regarding minimal medium. Do we culture a separate inoculum using M9 medium?? If so, starting cell number is not consistent....
  • Need to check with RPM value from literature.... 30 RPM from Tre-hardy et al. 150 RPM used was too fast, may have caused the spillage during biofilm growth


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