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
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..