- As per procedure, single colony obtained from stored culture plates, transferred onto 10-ml broth, and incubated at 9:50 AM to 3:50 PM (30 C, 250 rpm).
- 40% glycerol stock diluted to achieve 15% glycerol in 1-ml stock culture solution (0.625 ml inoculum from broth; 0.375 ml 40%-glycerol). Stored at -80 C.
Log-phase determination (trial I)
- Inoculum preparation
- 150 ml LB broth inoculated with 5 colonies (taken from 12/06 culture plates). Reason: based on 1 loop every 30 ml .
- 150 ml inoculated broth distributed into 12 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; 24th hour.
- Absorbance Reading at 600 nm, done at two replicates.
- Serial dilution until 10^-5. 0.1 ml inocula from four dilutions (10^-2 to 10^-5) mixed with warm LB agar upon plating (for overnight incubation).
- Growth curve based on absorbance
- 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;
- 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.