# Mitigate high background
High particulate background can lead to confusing results. For example, particulates in the growth medium can cause a pronounced offset between BactoBox® cells/mL and plate count results during the lag phase, when bacterial concentration is still low.
If the background particulates in the 1:100 diluted sample exceed 30,000 cells/mL, consider these strategies.
| Strategy | Illustration |
| ----------------------------------------------------------------------------------------------------------------------------------------------- | ------------------------------------------------------------------- |
|
Can the particulates be removed prior to adding bacteria? See #filter-growth-medium.
| |
## Filter growth medium
Yeast extracts and similar ingredients may contain bacteria-sized particulates from dead cells and other insoluble substances. Usually these are not necessary for cultivation of the bacteria and - once filtered - the medium will be background-free . This results in fewer pitfalls and simpler data interpretation.
1. Prepare the culture medium according to the manufacturer's instructions.
2. Use a magnetic stirrer to completely dissolve the powder before moving on to the next step.
3. Allow the medium to cool to room temperature before proceeding.
4. Use vacuum filters with 0.2 µm cutoff to remove particulates from the culture medium.
5. Autoclave according to the manufacturer's instructions.
{% hint style="warning" %}
### Particulates may be important for promoting growth
In certain situations, particulates in formulated growth media should not be filtered out. This includes media containing serum and lipids, as these components are crucial for the growth of the target organism.
In these conditions it may still be relevant to filter some of the components but the final formulation should not be filtered.
{% endhint %}
{% hint style="info" %}
### Skip the autoclave step by using a 0.1 µm filter
Use a [0.1 µm vacuum filter](https://shop.cobetter.com/products/disposable-vacuum-filter-unit-0-1%CE%BCm-double-layer-pes-membrane-sterile?srsltid=AfmBOor6veoAPma1X_7-C-tKZHYoRK8ob3nAwNTlk8dgstnKyEi-By5d) in step 4 to ensure a sterile growth medium Hereby you skip the autoclave step. Ensure the receiving vessel is pre-sterilized. Keep in mind that 0.1 µm filters will clog faster than 0.2 µm filters.
{% endhint %}
## Subtract background
You can simply subtract the *cells/mL x Dil* from the individual measurements if the background contribution is consistent and constant over time.
Below growth curve is for *Mycoplasma gallisepticum*. The mycoplasma growth medium has a background of \~4 x 106 cells/mL. During the lag phase, there's a noticeable offset between the uncorrected BactoBox® measurements (blue) and the color-changing units (CCUs, grey). Once the "media blank" background is subtracted (shown in orange), the CCUs and BactoBox® results align almost perfectly. The particulate background from the growth medium becomes negligible after mid-exponential phase, as the Mycoplasma concentration far exceeds background levels.
## Investigate stricter gating
BacTotal\_v2024-10 is a broad ruleset for differentiation of bacteria and non-bacterial substances. You can often use stricter gating to increase contrast between these objects. [Contact SBT](/troubleshooting/contact-our-support-team.md) for further assistance.
{% hint style="info" %}
## Power user feature 🥊
This feature is for BactoBox® power users. Use this feature for added value with your BactoBox®, though it's optional for regular use.
{% endhint %}
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