> For the complete documentation index, see [llms.txt](https://help.sbtinstruments.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.sbtinstruments.com/tutorials/measure-overnight-culture/disaggregate-bacteria.md).

# Prepare sample

After \~16 hours of incubation, the overnight culture is at a high cell concentration and we can use it to demonstrate BactoBox® measurements on concentrated bacterial suspensions.

{% hint style="success" icon="bacteria" %}

## Vortex thoroughly and vigorously

BactoBox is a flow cytometer and works best when cells are present in a single-cell suspension.

Cells stick together and you need thorough disaggregation to prepare a single-cell suspension.

One minute of vortexing may sound excessive, but trust us. You need it!
{% endhint %}

## Step by step

{% stepper %}
{% step %}

### Get a sample of the culture

Make figure-eight swirls to suspend the culture

Use a serological pipette to aspirate 2 mL culture.

Transfer to a 5 or 15 mL centrifuge vial.

<figure><img src="/files/M3juGNcP54s33QubdYaY" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% step %}

### Disaggregate clumps of bacteria

Vortex the sample 1 minute at max speed.

Ensure that a proper vortex is seen inside the tube when vortexing.&#x20;
{% endstep %}
{% endstepper %}

## Summary

You retrieved a sample of the overnight culture and used vortex mixing to disaggregate clumps of bacteria. Proceed to [Dilute and measure](/tutorials/measure-overnight-culture/dilute-and-measure.md) to determine cell concentration with BactoBox®.


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