# Hit the right concentration

Samples with high concentration are usually turbid.

Samples with low concentration are usually transparent.

If the concentration in your sample is too high, we dilute it. If the concentration is too low, we concentrate your sample. In either case, we try to hit the [target of 0.5–4 million cells/mL](https://help.sbtinstruments.com/encyclopedia/measurements-explained/what-can-i-measure-on#concentration). See the table below for our recommendations:

Concentration in your sample	Dilution	How-to guide(s)
5 · 10¹¹ cells/mL	10^-6	Three dilute-1-100 in sequence.
5 · 10¹⁰ cells/mL	10^-5	dilute-1-100-000.
5 · 10⁹ cells/mL	10^-4	dilute-1-10-000
5 · 10⁸ cells/mL	1 0^-3	dilute-1-1-000
5 · 10⁷ cells/mL	10^-2	Preferred dilute-1-100, alternatively dilute-1-1-000 if measurement gives a conductivity error.
5 · 10⁶ cells/mL	1:10	dilute-1-100. Contact SBT for guidance or review hit-the-right-conductivity if you receive conductivity errors.
5 · 10⁵ cells/mL	N/A	dilute-1-10 ⚠️ You must use custom diluent.
≤5 · 10⁴ cells/mL	N/A	Generally it is not recommended to use BactoBox for very dilute samples. For workarounds, see concentrate-your-sample.

You can [do combinations](https://help.sbtinstruments.com/protocols/prepare-your-sample/dilute-1-100#need-to-dilute-1-10-000-or-1-1-000-000) to get even high dilution factors. Just like how we do three [dilute-1-100](https://help.sbtinstruments.com/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-100 "mention") steps in a row to get a 1:1 000 000 dilution overall. ## Quick 1-step dilutions The above dilution principles rely on integers divisible by 10. This straightforward method typically works well for samples with high bacterial concentrations. However, there are situations where these dilutions may not be optimal. * For highly conductive broths, some cultivation media may require a dilution greater than 1:100 to achieve acceptable conductivity levels. In such cases, performing a 1:201 dilution by adding 50 µL of the sample to 10 mL of diluent might be beneficial. * For rapid and rough qualitative assessments, a 1-step dilution is efficient and conserves consumables. For example, you can mix 10 µL of your sample with 10 mL of diluent to achieve a 1:1 001 dilution. Ensure precise pipetting to maintain accuracy, especially when handling small volumes. | Sample volume | Diluent volume | Dilution | | :-----------: | :------------: | :------: | | 80 µL | 10 mL | 126 | | 50 µL | 10 mL | 201 | | 40 µL | 10 mL | 251 | | 25 µL | 10 mL | 501 | | 10 µL | 10 mL | 1 001 | | 8 µL | 10 mL | 1 251 | {% hint style="success" %} ## Target 0.5–4 million cells/mL for fast results The more things to count, the faster we are certain about the concentration. Give BactoBox® something to work with and it triggers the *Fast results* feature :fast\_forward:. Make a dilution series and [**measure the most diluted sample first**](https://help.sbtinstruments.com/protocols/prepare-your-sample/dilute-1-100#measure-the-most-diluted-sample-first). Aim for 0.5–4 million cells/mL. {% endhint %} {% hint style="info" %} ## Very low bacterial concentration? BactoBox® is generally not suitable for very low bacterial concentrations. {% endhint %} ## Motivation BactoBox® counts individual particles. If there are too *few* particles, the concentration estimate is too uncertain. If there are too *many* particles, we can't tell them apart. Therefore, we have limit of detection (LoD). BactoBox® outputs concentrations in these ranges: * 30 000–5 000 000 **cells/mL** (3·10⁴ to 5·10⁵ cells/mL) * 30 000–5 000 000 **total/mL** (3·10⁴ to 5·10⁵ total/mL) See [limits of detection](https://help.sbtinstruments.com/encyclopedia/measurements-explained/limits-of-detection) for more details. BactoBox® uses an electric field to measure individual cells.
Below the LoD (top), next-to-nothing enters the electric field and the cells/mL concentration is uncertain.
Within the LoD (middle), individual cells enter the electric field so we can tell them apart and count them.
Above the LoD (bottom), multiple cells enter the electric field at the same time and we can't tell them apart.

BactoBox® uses an electric field to measure individual cells.
Below the LoD (top), next-to-nothing enters the electric field and the cells/mL concentration is uncertain.
Within the LoD (middle), individual cells enter the electric field so we can tell them apart and count them.
Above the LoD (bottom), multiple cells enter the electric field at the same time and we can't tell them apart.

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