# Check lowest culture

After removing irrelevant cultures, it's time to measure the concentration of the remaining overnight cultures. Begin with the culture having the lowest turbidity, which in this case is the vial <kbd>-8</kbd> . We will use a 1:1 000 dilution in two sequential dilution steps: An initial 1:100 dilution followed by a additional 1:10 dilution.&#x20;

<figure><img src="https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2F19Mw4e2yD31gitNYOJ2r%2FOptiseeding_overnight_-8_workup.avif?alt=media&#x26;token=20337294-cd71-4211-8cb8-b1b44bf07474" alt=""><figcaption></figcaption></figure>

{% stepper %}
{% step %}

### Disaggregate cell clumps

Vortex the vial 1 minute at maximum speed to disaggregate cell clumps. See [break-up-clumps-and-chains](https://help.sbtinstruments.com/protocols/prepare-your-sample/break-up-clumps-and-chains "mention") if your bacterium needs more aggressive disaggregation.
{% endstep %}

{% step %}

### Dilute 1:100&#x20;

Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}

{% step %}

### Dilute 1:1 000

Remove 1 mL diluent from a 10 mL diluent vial for a residual volume of 9 mL.

Transfer 1 mL of the 1:100 dilution to the this vial. Vortex 10 seconds at maximum speed.
{% endstep %}

{% step %}

### Measure on the diluted sample&#x20;

Transfer the tubing kit to the 1:1 000 vial.&#x20;

Press <kbd><mark style="background-color:purple;">Measure<mark style="background-color:purple;"></kbd>  in Access to start a new measurement.&#x20;

* Dilution: <kbd>1 000</kbd>
* Label: <kbd>overnight\_-8</kbd>
  {% endstep %}
  {% endstepper %}

{% hint style="info" %}

## Pro tip: 1-step 1:1 001 dilution.&#x20;

The described 2-step procedure for a 1:1,000 dilution is reliable with high precision. However, it demands a longer sample preparation time. If you have good pipetting skills, you can save time, sample and diluent by doing a 1-step dilution. Simply transfer 10 µL of sample to 10 mL of diluent to [get a 1:1 001 dilution](https://help.sbtinstruments.com/protocols/prepare-your-sample/hit-the-right-concentration).
{% endhint %}

## Summary

Of the remaining vials, the culture with the lowest turbidity has a concentration of 1.4 × 10<sup>9</sup> cells/mL. Next, we will [check-remaining-cultures](https://help.sbtinstruments.com/protocols/optimize-bioprocesses/identify-optimal-inoculum-size/check-remaining-cultures "mention") to identify the culture that has just reached onset of stationary phase.