Growth Curve

Contact: Dingding An

1. Streak frozen culture onto agar plate with appropriate antibiotic selection.
2. Inoculate 5-mL fresh growth medium from a single colony grown on plates; grow it to log phase at proper temperature.
3. Prepare 250-mL flask with 50 mL of medium. Once the culture has reached log phase, measure and record the OD₆₀₀, then dilute the culture with fresh medium to make OD₆₀₀ equal to 0.1. Subculture 1 mL into the 250-mL flask.
4. Place the flask in a shaking incubator at the appropriate temperature.
5. Measure OD₆₀₀, and fluorescence if necessary. For each measurement, withdraw 200 μL from the flask, and transfer it to a clear-bottom, black microtitre plate. Also put the same amount of fresh medium in one well as a blank.
6. Continue to take data points until the growth has leveled off.

Notes

1. Since the log phase tells important information about growth kinetics, it is important to have numerous data points in this range. Normally, you can take data points every 2-5 hours during the initial lag phase.  Once you notice the culture starting to enter log phase, start taking data points every hour or so.
2. When using plasmid strains, such as AT with GFP, you should add anti-microbial selection from the very beginning of culturing, but not in the final growth curve medium. It should be added to the molten agar before plating as 1 μL/mL, using the appropriate antibiotic stock concentration.