Chang Lab:Notebook/CBE/08/149/2008/12/16

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

  • Start 8:20 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 trial II)
  • Autoclave new M9 medium (1 liter)

Biofilm Optimization (Trial II)

  • 3 CBDs are prepared to be incubated at 3 different temperatures: 30, 37 and 42 C at 30 rpm for 48 hours. The procedures below follow from Trial I.
  • Following 1.0 McFarland Std (A600 = 0.257), 5-hr incubated E coli broth adjusted to match A600 value. Approximately 6X (0.45ml broth culture to 2.55ml broth) dilution 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. To be checked after ~24 hrs for colony counting.

Notes

  • CBD incubated at 30 C was transferred to static incubator. Cannot do 3 CBDs next time, number of incubators limited.
  • Inoculum volume in wells changed to 200 ul due to overflow of liquid upon replacement of pegged lid.


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