# 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.
{% hint style="success" %}
## 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").
{% endhint %}
We target a conductivity of approximately 1850 µS/cm. This is right in the middle of the [space.vars.BB\_LIMIT\_EC](https://help.sbtinstruments.com/encyclopedia/measurements-explained/what-can-i-measure-on#electrical-conductivity) allowed range.
{% hint style="success" %}
## 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).
{% endhint %}
### 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
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).
{% hint style="info" %}
## 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.
{% endhint %}
### Step by step
{% stepper %}
{% step %}
### Mix together
Add 10 mL of PBS to 80 mL of ultrapure water. Mix.
{% hint style="info" %}
## Alternative approach
Dispense 11.1 mL of PBS to a container. Add ultrapure water till you reach the 100 mL mark. Mix.
{% endhint %}
{% endstep %}
{% step %}
### Filter your diluent
Filter your diluent through a 0.2 µm syringe filter into a sterile container.
{% hint style="info" %}
## Alternative approach
Filter your diluent with a vacuum filtration setup.
{% endhint %}
{% hint style="warning" %}
## 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*).
{% endhint %}
{% endstep %}
{% step %}
### 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.
{% hint style="info" %}
## Alternative approach
Use an [electrical conductivity meter](https://en.wikipedia.org/wiki/Electrical_conductivity_meter) (EC meter) to measure the conductivity of your diluent.
{% endhint %}
{% endstep %}
{% step %}
### 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.
{% endstep %}
{% endstepper %}
## 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.
## 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.
---
# 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-conductivity/make-diluent.md?ask=
```
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.