Is background substantial?

High levels of background particulates can affect measurement accuracy. In growth media, these particulates may cause a significant discrepancy between BactoBox® cell counts (cells/mL) and plate counts, particularly during the lag phase when bacterial concentrations are low. In practice, however, the particulate background contributed by the growth medium is typically much lower than the signal from bacterial cells and therefore has little impact on the results.

Relevant versus negligible contribution

Start with a measurement of the bacteria-free growth medium or another source of background particulates.

• If the 1:100 result is below 30,000 cells/mL, the background is negligible. Ignore it.
• If the 1:100 result is above 30,000 cells/mL, compare it to the bacterial concentration in the culture you care about. Use the rule-of-thumb in the info box to distinguish between relevant and negligible contribution.

Rule of thumb for significant versus negligible contribution of background particulates

Background is negligible if the cells/mL×dil. for the growth medium makes up <1% of the cells/mL×dil. of the measured bacterial cultures. If the background exceeds 1%, you should use a mitigation strategy.

Example of a use case where particulate background is negligible

Let's take an example where you want to quantify the cell concentration at the end-of-fermentation.

• A non-inoculated growth medium measured at 1:100 gives 100,000 cells/mL. This corresponds to 1×10⁷ cells/mL in the undiluted growth medium.
• The end-of-fermentation culture measured at 1:10,000 gives 1,000,000 cells/mL. This corresponds to 1×10¹⁰ cells/mL in the undiluted culture.

Here, the background contributes 0.1% of the measured concentration (1×10⁷ / 1×10¹⁰ cells/mL).

In this example, background from the growth medium accounts for less than 1% of the bacterial culture you care about. As defined in the rule-of-thumb the background can be ignored.

Example of a use case where particulate background is significant

Another calculation example could be the preparation of a liquid starter culture from a colony on an agar plate.

• A non-inoculated growth medium in a capture tube measured at 1:100 gives 100,000 cells/mL. This corresponds to 1 × 10⁷ cells/mL in the undiluted growth medium.
• The cell suspension in the capture tube measured at 1:201 gives 1,000,000 cells/mL. This corresponds to ~1 × 10⁸ cells/mL in the undiluted culture.

Here, the background contributes ~10% of the measured concentration (1×10⁷ / ~1×10⁸ cells/mL).

In this example, background from the growth medium accounts for more than 1% of the bacterial culture you care about. As defined in the rule-of-thumb a mitigation strategy should be used.

Summary

Here we discussed how to distinguish between relevant and negligible contribution of background particulates from growth media and other sources. In other words; can the background be ignored. In the next page Mitigate high background we will detail strategies when the background in non-negligible.