> 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/not-in-the-new-structure/measure-vitroid-tm-growth-curve/disaggregate-bacteria.md).

# Prepare sample

After \~5 hours of incubation, the cell concentration should be sufficient for BactoBox® measurements. The step-by-step guide below describes the sample preparation for all growth curve measurements.

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

## Vortex thoroughly and vigorously

BactoBox® is a flow cytometer and performs best when cells are present as a single-cell suspension.

Because bacterial cells can stick together, thorough disaggregation[^1] is essential. One minute of vortexing may seem excessive, but it is necessary to achieve reliable measurements.
{% endhint %}

## Step by step

{% stepper %}
{% step %}

### Get a sample of the culture

Make 10 figure-eight swirls to resuspend the culture prior to sampling.

Use a serological pipette to aspirate 2 mL culture and transfer it to a 5 or 15 mL centrifuge vial.

<figure><img src="https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2FHCaGCXDddXIWlTsyno0W%2Fvitroid-LB-sampling.avif?alt=media&amp;token=131af017-cdb5-4d52-a3e5-4b4f33b59181" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% step %}

### Disaggregate clumps of bacteria

Vortex the sample at maximum speed for 1 minute.

Ensure that a proper vortex forms inside the tube during mixing.
{% endstep %}

{% step %}

### Set a countdown for the next measurement

Set a 30-minute countdown to remind yourself to collect the next sample for the growth curve.
{% endstep %}
{% endstepper %}

## Summary

You have now collected a sample after \~5 hours of incubation and used vortex mixing to disaggregate[^1] bacterial clumps. Proceed to [1:100 dilutions](/not-in-the-new-structure/measure-vitroid-tm-growth-curve/1-100-dilutions.md) to determine the cell concentration using BactoBox®.

[^1]: Break up clumps or chains of cells


---

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