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 .
Is the background constant? See Subtract background.
None of the above? See Investigate stricter gating

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.

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.

Skip the autoclave step by using a 0.1 µm filter

Use a 0.1 µm vacuum filter 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.

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 for further assistance.

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.