Chang Lab:Notebook/CBE/08/146/2008/12/09

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  • Stock culture preparation
    • Single colony from stored culture plates was transferred onto 10-ml broth, and incubated at 9:50am to 3:50pm (28 C, 250 rpm).
    • After which 40% glycerol stock was diluted to achieve 15% glycerol in 1-ml stock culture solution (0.625 ml from broth to 0.375 ml 40%-glycerol). Stored at -80 C.
  • Log-phase determination (set 1)
  • Inoculum preparation
    • 150 ml LB broth inoculated with 5 colonies (taken from culture plates on 6 Dec ). Rationale was for 1 loop every 30 ml.
    • 150 ml inoculated broth distributed into 12X 10-ml test tubes.
  • Incubation at 37 C, 10:10 AM.
  • Temporal OD Measurement and Viable cell count via serial dilution method
    • Time points: every 30 mins for 1st 3 hours; every hour for 4th - 11th hour and 24th hour.
    • Absorbance Reading at 600 nm was taken twice.
    • Serial dilution using LB broth until 10^-5. 0.1 ml inocula from four dilutions (10^-2 to 10^-5) mixed with warm LB agar upon plating (overnight incubation for plate counting the next day).
  • Notes
  • Growth curve based on absorbance
    Absorbance vs Time
    • Based on log plot, mid-log phase for E. coli is around 5-6 hours (similar to thoe from expected from literature).
    • Absorbance includes both viable and dead cell debris.
    • Log phase to be repeated to check growth curve.
  • Serial plating results
    • Cell size variable and too numerous to count!!
    • Lack of oxygen within agar may have prevented size.
    • Starting number of loops to be reduced to 1 for next growth curve experiment
    • Increase serial dilution increments for the innocula there are incubated for longer time..