# Low-inoculum overnight culture (strategy 1)

## Concept

You identify which cell concentration to use to inoculate an overnight culture to achieve a [high-quality-culture](https://help.sbtinstruments.com/encyclopedia/bacterial-growth/high-quality-culture "mention") 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 [Broken link](https://help.sbtinstruments.com/protocols/high-accuracy-microbial-input/broken-reference "mention") 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 [](https://help.sbtinstruments.com/protocols/high-accuracy-microbial-input "mention") page).
   * Prepare dilutions and measure **cells/mL** with BactoBox.
4. **Calculate the dosing volume**\
   Use the approach outlined on the [](https://help.sbtinstruments.com/protocols/high-accuracy-microbial-input "mention") page to calculate the dosing volume.

{% hint style="warning" %}
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.
{% endhint %}