Make diluent

The Do-It-Yourself (DIY) guide to make diluent

This page describes how you make dilution liquid (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.

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 Hit the right conductivity.

We target a conductivity of approximately 1850 µS/cm. This is right in the middle of the 1500–2200 µS/cm allowed range.

Use SBT dilution vial or SBT dilution liquid

Use an SBT dilution vial or SBT dilution liquid. Both have the right electrical conductivity already.

When you dilute your sample (1:1000 or more), the conductivity of the diluent dominates over that of your sample. This puts the conductivity within the allowed range.

Get things ready

You need these items:

• 10 mL of PBS (phosphate-buffered saline). Sometimes sold as 1X PBS.

• 80 mL of ultrapure water. E.g., Milli-Q® or water for injection (WFI).

• 1 × sterile container. E.g., an empty vial.

• Either of:

  • 1 × 0.2 µm syringe filter

  • 1 × vacuum filtration setup with 0.2 µm

The above is enough to make 90 mL of diluent. This corresponds to 9 dilution vials.

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.

Step by step

How to make diluent (1-in-9 PBS). This is the standard diluent.

1

Mix together

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

Alternative approach

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

2

Filter your diluent

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

Alternative approach

Filter your diluent with a vacuum filtration setup.

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 evaporates water, which changes the conductivity of the diluent. Moreover, an autoclove does not remove particles (does not make the diluent pure).

3

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®. Note that this uses two vials.

Alternative approach

Use an electrical conductivity meter (EC meter) to measure the conductivity of your diluent.

4

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 until your diluent is within the acceptable range.

Diluent (1-in-10 PBS)

Follow the steps above for diluent (1-in-9) but change the volumes to:

• 10 mL of PBS (phosphate-buffered saline)

• 90 mL of ultrapure water

See the recipe above for the details.

How to make diluent (1-in-10 PBS)

Diluent (1-in-X PBS)

Follow the steps above for diluent (1-in-9) but change the volumes to:

• 10 mL of PBS (phosphate-buffered saline)

• $10 \cdot (x-1)$ mL of ultrapure water

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