Low-inoculum overnight culture (strategy 1)

Use this workflow when you want a low-inoculum overnight culture that reaches late exponential or early stationary phase by a defined timepoint.

Concept

You identify which cell concentration to use to inoculate an overnight culture to achieve a High-quality culture at the time when you bacteria are needed for an experiment.

When to use

Use this strategy when:

• You want a culture that is ready at a fixed time next day (for example after ~ 18 hours).

Step-by-step

1. Identify inoculum concentration of overnight culture

   • Run our Get high-quality overnight cultures from cryo stocks protocol to identify how you must inoculate your overnight culture in the workflow.

2. Inoculate and incubate

   • Inoculate the overnight culture using the identified cell concentration.

   • Incubate with shaking for the time that you plan to use.

3. Measure the overnight culture just before dosing

   • Take a well‑mixed sample from the overnight culture at the planned harvest time.

   • Chill the sample on ice to halt growth if you will also plate it (see use case validation tips on the High-accuracy microbial input page).

   • Prepare dilutions and measure cells/mL with BactoBox.

4. Calculate the dosing volume Use the approach outlined on the High-accuracy microbial input page to calculate the dosing volume.

Often the calculated volume is too small to pipette accurately. In these cases, prepare an intermediate dilution of the seed suspension instead of attempting to transfer very small volumes.

Example: If $V_{add}$ is 0.2 µL, dilute the seed suspension 1:100 and then add 20 µL instead.