Before you start
- Grow a starter culture overnight from a single colony.
Reagents and materials
Totals are for 3 cultures (+10% extra). Per-culture (50 mL) amounts are in the middle column.
| Reagent | Per culture (50 mL) | Total |
|---|---|---|
| Fresh medium | 50 mL | 50 mL |
| Overnight starter culture1:100 dilution | 0.5 mL | 0.5 mL |
Buffers and recipes
Doubling time per culture
| Growth rate μ (per hour) | slope of ln(OD₆₀₀) vs time in exponential phase |
| Doubling time | ln 2 ÷ μ |
| Linear range | dilute samples above OD₆₀₀ ≈ 0.8–1.0 and multiply back |
Equipment
- Shaking incubator
- Spectrophotometer and cuvettes (or plate reader)
- Baffled flask (volume ≥ 5× culture)
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Inoculate
Dilute the overnight culture 1:100 into pre-warmed medium (target OD₆₀₀ ≈ 0.05).
Blank
Blank the spectrophotometer with sterile medium.
Time zero
Measure OD₆₀₀ immediately.
Sample regularly
Incubate with shaking and measure every 30–60 min.
Keep readings linear
Dilute samples with medium once OD₆₀₀ exceeds ~0.8 and multiply by the dilution factor.
Plot and calculate
Plot ln(OD₆₀₀) against time, identify lag, exponential and stationary phases, and calculate the doubling time from the exponential slope.
Critical points
- OD-to-cell-number conversion depends on the organism and instrument; calibrate with CFU counts if you need cell numbers.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Noisy readings | Mix before sampling; clean cuvettes; use the same instrument. |
| No growth | Check medium, antibiotic and temperature. |
Safety
Work at the organism’s biosafety level.
Further reading
- Monod J. The growth of bacterial cultures. Annual Review of Microbiology (1949).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.