# Make diluent

This page describes how you make [dilution liquid](https://help.sbtinstruments.com/encyclopedia/item-register/vials-flasks-and-liquids/dilution-liquid-5-l) (aka diluent).

It is essential that the diluent:

* Has the right conductivity
* Is both pure and sterile

Follow the steps below closely to ensure that your diluent meets the above-mentioned requirements.

## Diluent (1-in-9 PBS)

This is our standard diluent.

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## Diluent (1-in-9 PBS) is the standard diluent

Start with the standard diluent. It uses 1-in-9 PBS. Only customize it if you run into conductivity-related issues. See [](https://help.sbtinstruments.com/protocols/prepare-your-sample/hit-the-right-conductivity "mention").
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We target a conductivity of approximately 1850 µS/cm. This is right in the middle of the [<code class="expression">space.vars.BB\_LIMIT\_EC</code>](https://help.sbtinstruments.com/encyclopedia/measurements-explained/what-can-i-measure-on#electrical-conductivity) allowed range.

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## Use SBT [dilution vial](https://help.sbtinstruments.com/encyclopedia/item-register/vials-flasks-and-liquids/dilution-vial) or SBT [dilution liquid](https://help.sbtinstruments.com/encyclopedia/item-register/vials-flasks-and-liquids/dilution-liquid-5-l)

Use an SBT [dilution vial](https://help.sbtinstruments.com/encyclopedia/item-register/vials-flasks-and-liquids/dilution-vial) or SBT [dilution liquid](https://help.sbtinstruments.com/encyclopedia/item-register/vials-flasks-and-liquids/dilution-liquid-5-l). Both have the right electrical conductivity already.

When you [dilute your sample](https://help.sbtinstruments.com/protocols/prepare-your-sample/hit-the-right-concentration) (1:1 000 or more), the conductivity of the diluent dominates over that of your sample. This puts the conductivity within the [allowed range](https://help.sbtinstruments.com/encyclopedia/measurements-explained/what-can-i-measure-on#electrical-conductivity).
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### Get things ready

You need these items:

* 10 mL of [PBS (phosphate-buffered saline)](https://en.wikipedia.org/wiki/Phosphate-buffered_saline). Sometimes sold as *1X PBS*.
* 80 mL of [ultrapure water](https://en.wikipedia.org/wiki/Ultrapure_water). E.g., [Milli-Q®](https://www.sigmaaldrich.com/DK/en/life-science/milliq) or [water for injection (WFI)](https://en.wikipedia.org/wiki/Water_for_injection).
* 1 × sterile container. E.g., an [empty vial](https://help.sbtinstruments.com/encyclopedia/item-register/vials-flasks-and-liquids/empty-vial).
* Either of:
  * 1 × 0.2 µm syringe filter
  * 1 × [vacuum filtration](https://en.wikipedia.org/wiki/Suction_filtration) setup with 0.2 µm&#x20;

The above is enough to make 90 mL of diluent. This corresponds to 9 [dilution vials](https://help.sbtinstruments.com/encyclopedia/item-register/vials-flasks-and-liquids/dilution-vial).

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## Use ready-made PBS and WFI

We recommend that you buy ready-made PBS and WFI. PBS and WFI are not expensive to buy ready-made and it reduces the risk of errors and surprises substantially.
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### Step by step

![How to make diluent (1-in-9 PBS). This is the standard diluent.](https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2FJYIgM8hcjabvjiYY1qLg%2F0.png?alt=media)

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### Mix together

Add 10 mL of PBS to 80 mL of ultrapure water. Mix.

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## Alternative approach

Dispense 11.1 mL of PBS to a container. Add ultrapure water till you reach the 100 mL mark. Mix.
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### Filter your diluent

Filter your diluent through a 0.2 µm syringe filter into a sterile container.

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## Alternative approach

Filter your diluent with a vacuum filtration setup.
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## Autoclave is not a good approach

If you need to remove bacteria and/or particles from your diluent, then you must sterile-filter it. An [autoclave](https://en.wikipedia.org/wiki/Autoclave) evaporates water, which changes the conductivity of the diluent. Moreover, an autoclove does *not* remove particles (does not make the diluent *pure*).
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### Measure the conductivity of your diluent

**Wait until your diluent is at room temperature*****.*** Electrical conductivity changes with temperature so we want to avoid temperature changes.

Use the *Conductivity* program on BactoBox® to measure the electrical conductivity of your diluent. See [measure-conductivity-with-bactobox-r](https://help.sbtinstruments.com/protocols/prepare-your-sample/hit-the-right-conductivity/measure-conductivity-with-bactobox-r "mention"). Note that this uses *two* vials.

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## Alternative approach

Use an [electrical conductivity meter](https://en.wikipedia.org/wiki/Electrical_conductivity_meter) (EC meter) to measure the conductivity of your diluent.
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### That is it!

For diluent (1-in-9 PBS), the acceptable electrical conductivity is 1850±50 µS/cm.

Did you get another electrical conductivity? It may change slightly with the brand of PBS that you use. Try with [another dilution factor](#diluent-1-in-x-pbs) until your diluent is within the acceptable range.
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## Diluent (1-in-10 PBS)

Follow the [steps above](#step-by-step) for diluent (1-in-9) but change the volumes to:

* 10 mL of [PBS (phosphate-buffered saline)](https://en.wikipedia.org/wiki/Phosphate-buffered_saline)
* 90 mL of [ultrapure water](https://en.wikipedia.org/wiki/Ultrapure_water)

See the recipe above for the details.

![How to make diluent (1-in-10 PBS)](https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2FXVUuhzOwIziH0TTJpYyB%2F1.png?alt=media)

## Diluent (1-in-X PBS)

Follow the [steps above](#step-by-step) for diluent (1-in-9) but change the volumes to:

* 10 mL of [PBS (phosphate-buffered saline)](https://en.wikipedia.org/wiki/Phosphate-buffered_saline)
* $$10 \cdot (x-1)$$ mL of [ultrapure water](https://en.wikipedia.org/wiki/Ultrapure_water)

where $$x$$ is the "1-in-$$x$$ PBS" that you aim for.