- 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).
- Growth curve based on absorbance
- 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..