Chang Lab:Notebook/CBE/08/148/2008/12/09

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Entry title

Stock Culture Protocol:

1. A single colony from the culture disk is obtained and transfer to a 10ml broth in a test tube. 2. The bacteris culture is then incubated from 9.50am till 3.50am at 30 degree, 250 rpm. 3. To prepare for a stock culture that can be stored for a long period of time, glycerol is added (0.625 ml inoculum from broth: 0.375 ml 40%-glycerol). Stored at -80 C.

Log Phase Trail 1:

1. 5 single colonies was obtained and transfered to 150ml of broth. 2. The mixture is then transfered to twelth 10ml test tube. 3. These test tube were then left in the incubator at 37 degree.

OD Measurement:

  • Time point: every 30 min for the 1st 3 hour; hourly from the 4th to 11th hour and 24th hour.
  • Absorbance reading is at 600nm

Serial Dilution:

  • For the
    • 1st hour - no dilution was done
    • 2nd hour - 10^-1 dilution
    • 3rd - 6th hour - up to 10^-5 dilution
    • subsequest hour - up to 10^-6 dilution

Plate counting (Pour method)

  • 0.1ml is obtained from the diluted test tube and then pipitted into the petri disk, LB Agar is then poured (swirl to allow equal mixing).
  • The bactaris plates is then stored overnight at the 37 degress incubator

Notes

  • Growth curve based on absorbance as shown
    Absorbance vs Time
    • Based on log plot, mid-log phase for E. coli is around 5-6 hours -- as expected from literature.
    • Lack of time points around 12th - 18th hour. Require data at these time points for next trial.
    • Absorbance includes both viable and dead cell debris.
      • Suggestion: Reproduce results with next trial.
  • Serial plating results
    • Cell size variable and too numerous to count
    • Lack of oxygen within agar may have prevented size(too small to count confortably)
      • Suggestion: Try spread-plate technique.
    • Decrease starting inoculum concentration AND/OR increase serial dilution increments.
      • Suggestion: Suspend 1 colony only in 150 ml broth, plate 10^-6, 10^-8 and 10^-10 for < 8-hr incubation; while plate 10^-8, 10^-10, and 10^-12 for > 8 hr. Qualitative observation 10^-8 and 10^-10 plates may be done instead of actual cell count.