Use BactoBox® to find the *cells/g* concentration in [probiotics](https://en.wikipedia.org/wiki/Probiotic), live biotherapeutic products (LBPs), [postbiotics](https://en.wikipedia.org/wiki/Postbiotic), etc.
Also note that the bacterial concentration in freeze-dried products can be extremely high and therefore it may be necessary to dilute sample more than 10-5.
## Detailed protocol
{% stepper %}
{% step %}
#### Prepare your sample
1. Homogenize the dry bacterial product as much as possible. E.g. massage, invert, and stir the powder with a spoon or similar.
2. Weigh approximately 3 g of powder into a [Stomacher® bag](https://www.seward.co.uk/stomacher-400-classic/). Note down the weight.
3. **1st dilution (total 1:10)**\
Add [diluent](/encyclopedia/item-register/vials-flasks-and-liquids/dilution-liquid-5-l.md) to the Stomacher® bag. Target a 1:10 dilution based on weight. If the initial weight is exactly 3 g, the weight of powder plus medium is 30 g.
4. Homogenize the Stomacher bag® with a [Stomacher® Lab Blender](https://www.seward.co.uk/stomacher-lab-blenders/) at 250 ± 10 RPM for 4 minutes
5. Allow the bacteria to rehydrate (wake up) for 30 minutes at room temperature
{% hint style="info" %}
### Proper resuspension and gentle homogenization is essential
Notice that some media protect cells better than others upon resuspension. Buffered peptone water is a good general-purpose medium applicable to many species of bacteria. However, you may need special media for some cases. For example, lactic acid bacteria need MRS to protect cellular integrity during resuspension.
{% endhint %}
{% endstep %}
{% step %}
#### Dilute your sample
Prepare three [dilution vials](/encyclopedia/item-register/vials-flasks-and-liquids/dilution-vial.md): Take three [empty vials](/encyclopedia/item-register/vials-flasks-and-liquids/empty-vial.md) and add 10 mL of [diluent](/encyclopedia/item-register/vials-flasks-and-liquids/dilution-liquid-5-l.md) to each.
**2nd dilution (total 10****-3****)**
[Dilute 1:100](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-100.md): Transfer 101 μL from your prepared sample into the *first* dilution vial. Vortex (15 sec., Max RPM).
**3rd dilution (total 10****-5****)**
[Dilute 1:100](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-100.md): Transfer 101 μL from your *first* dilution vial into your *second* dilution vial. Vortex.
**4th dilution (total 10****-7****)**
[Dilute 1:100](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-100.md): Transfer 101 μL from your *second* dilution vial into your *third* dilution vial. Vortex.
{% endstep %}
{% step %}
#### Measure your sample
Measure with BactoBox®. Start with the *least* concentrated sample and move towards higher concentrations.
First, clean your BactoBox® setup with a fresh disinfection vial.
**Measure on the 10****-7****-diluted sample**
If there result is above the limit of detection (above 30 000 cells/mL) then you are done! Otherwise, move on.
**Measure on the 10****-5****-diluted sample**
If there result is above the limit of detection (above 30 000 cells/mL) then you are done! Otherwise, move on.
**Measure on the 10****-3****-diluted sample**
Is even the most-concentrated sample below the limit of detection? See [Concentrate your sample](/protocols/prepare-your-sample/hit-the-right-concentration/concentrate-your-sample.md) for next steps.
{% endstep %}
{% endstepper %}
Multiply the *cells/mL* concentration with the dilution factor to calculate the *cells/mL* concentration in your sample stock.
---
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