# Centrifuge your sample

{% hint style="success" %}

### Filter-centrifuge your sample

Use the [filter-centrifugation method](/protocols/prepare-your-sample/hit-the-right-concentration/concentrate-your-sample/filter-centrifuge-your-sample.md) to *consistently* increase the concentration of your sample. Unlike simple centrifugation, filter-centrifugation gives consistent results.

In addition, filter-centrifugation is more gentle for the bacteria. The force required to drive liquid through the membrane filter is relatively subtle.

See [Filter-centrifuge your sample](/protocols/prepare-your-sample/hit-the-right-concentration/concentrate-your-sample/filter-centrifuge-your-sample.md).
{% endhint %}

{% hint style="danger" %}

### Do not expect consistent results with this method

There are too many pitfalls that each lead to inconsistent results.

For dilute bacterial samples, the pellet will be brittle and very difficult to see with the naked eye. When you remove the supernatant, you may remove (parts of) the pellet as well.

Instead, [filter-centrifuge your sample](/protocols/prepare-your-sample/hit-the-right-concentration/concentrate-your-sample/filter-centrifuge-your-sample.md) to get consistent results.
{% endhint %}

<div><figure><img src="/files/VmdzG2Hx5yIj3qyNKEft" alt="" width="375"><figcaption><p>Centrifuge for 15 mL conical tubes</p></figcaption></figure> <figure><img src="/files/viH7fxJ8RZjnwSp8m9SO" alt="" width="304"><figcaption><p>Centrifuged liquid. Notice the pellet<br>at the bottom of the vial.</p></figcaption></figure></div>

Use a centrifuge to concentrate your sample. Here is the overall procedure:

1. Centrifuge your [sample vial](/encyclopedia/item-register/vials-flasks-and-liquids/sample-vial.md)
2. Remove the [supernatant](https://en.wikipedia.org/wiki/Precipitation_\(chemistry\))
3. Resuspend the pellet in [diluent](/encyclopedia/item-register/vials-flasks-and-liquids/dilution-liquid-5-l.md).

Repeat as necessary to increase the concentration further.

{% hint style="info" %}
All our [vials](/encyclopedia/item-register/vials-flasks-and-liquids.md) are 15 mL conical centrifuge tubes. They fit inside standard centrifuges.
{% endhint %}

{% hint style="warning" %}

### The pellet may be difficult to see

The pellet may be transparent and difficult to see with the naked eye. In turn, there is a high risk that you unintentionally remove bacteria from your sample when you try to remove the supernatant.
{% endhint %}

## How to resuspend bacteria

The centrifugation process puts the bacteria close together. In turn, the bacteria may form clumps (aggregates). This is something to avoid. See [Break up clumps and chains](/protocols/prepare-your-sample/break-up-clumps-and-chains.md) for more information.

Here is a short how-to guide for proper resuspension:

1. Resuspend the pellet in [diluent](/encyclopedia/item-register/vials-flasks-and-liquids/dilution-liquid-5-l.md). Use an [empty vial](/encyclopedia/item-register/vials-flasks-and-liquids/empty-vial.md) for this purpose. Target the 10 mL mark.
2. Coarse disaggregation. Use a pipette to break apart the cells. Repeatedly aspirate and dispense the liquid.
3. Fine disaggregation. Use one of the mechanical disaggregation methods (See [Disaggregate mechanically](/protocols/prepare-your-sample/break-up-clumps-and-chains/disaggregate-mechanically.md)). For example:
   1. Ultrasound bath (20 khz, 5 min)
   2. Vortex with 3 mm glass beads. Add beads to increase the liquid level by 1 mL and vortex (1 min, max RPM. Decant liquid to empty vial before you measure on BactoBox®.


---

# Agent Instructions: Querying This Documentation

If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter:

```
GET https://help.sbtinstruments.com/protocols/prepare-your-sample/hit-the-right-concentration/concentrate-your-sample/centrifuge-your-sample.md?ask=<question>
```

The question should be specific, self-contained, and written in natural language.
The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.