BactoBox® pitfalls

BactoBox® is robust and usually just works. When results go wrong, the root cause is often sample workup or third-party consumables.

We call these issues pitfalls because the device may still operate as intended while the measurements become misleading. The table below lists common examples. It is not exhaustive.

Sin	Why this matters	Do this instead
Using third-party vials	Some third-party vials release microplastic particles. This creates false particulate background in the measurement.	Use SBT-provided Empty vial and Dilution vial. These vials are routine quality-tested.
Using third-party chemicals	Alcohol and deep-clean chemicals may contain a high particulate background. After a clean or deep clean, these particles may carry over into your measurements.	Always use the SBT-provided Disinfection vial and Deep clean vial.
Storing filtered DIY diluent	A 0.2 µm filter reduces bioburden, but it does not guarantee sterility. If bacteria pass through the filter, they may multiply and create high, unpredictable background.	Do not store filtered DIY diluent. Prepare it immediately before use. Switch to the Dilution vial if you want a simpler option. It is gamma-sterilized and can be stored without risk of bacterial growth.
Forgetting to change the filter on the dilution dispenser	The 0.2 µm filter will eventually become contaminated on the filtrate side. This may add bacterial background to your measurements.	Replace the 0.2 µm filter on the dilution dispenser at the start of each day of use.
Letting diluted samples sit before measurement	BactoBox® diluent has relatively low salt concentration. Longer exposure may lyse cells and shift the measured cell concentration.	Measure the diluted sample immediately on BactoBox®.
Repeating measurements on the same vial	The final part of a measurement cycle is the empty step. It uses high flow rates. Combined with small flow channels and hypotonic diluent, this may damage cells.	Measure each diluted vial only once. Prepare a fresh dilution if you need replicates.
Using an unsuitable vortex platform attachment	Poor vortexing is a common source of variability. Poor mixing leads to inconsistent dilution series and noisy results.	Make sure you see a proper vortex in the vial. Use a platform attachment that fits 15 mL vials.
Using too much surfactant	Surfactants such as Tween may form micelle aggregates. Electrically, these soap bubbles may look like bacteria and falsely elevate the measured concentration.	Avoid surfactants when possible. If needed, use a sample workup that does not create excessive foam. For example, use repeated pipette aspiration.
Not disaggregating chains or clumps	Objects with sphere-equivalent diameter above 5 µm do not enter the measurement channel. Clumps and chains are therefore likely to be underestimated.	Use suitable sample workup to create a single-cell suspension. Use microscopy to verify the result. See Break up clumps and chains for advanced sample workup.
Ignoring background particulates	Background particulates may come from growth media, cryoprotectants, excipients, or other process materials. This may inflate the measured bacterial concentration.	Check the materials in your workflow for particulate background. If the background is non-negligible, use a mitigation strategy.
Skipping the QC test	If you skip the QC test, you do not know whether your setup works as intended. This increases the risk of useless measurements and repeated errors.	Run a QC test at least once per week when the BactoBox® setup is in use. This also adjusts the flow rate for more robust measurements.
Using poor incubation vessels	Poor agitation and poor aeration stress the bacteria. This may increase the fraction of stressed or dead cells and may manifest as Divergence from plate counts.	Use suitable incubation vessels such as shake flasks, bioreactors, or suitable multiwell plates.
Sampling too infrequently	Endpoint measurements alone often hide what happened in between. If BactoBox® and plate counts disagree, it becomes difficult to interpret the result.	Endpoint measurements are fine for workflows such as End-of-fermentation cross-check and Screen growth media. If you need to troubleshoot a bioprocess, sample across the full time course. See Track growth curve for a head-to-head comparison between plate counts and BactoBox® across the classical Growth phases.
Using one-plicates for critical conclusions	It is impossible to avoid noise, i.e. inherent variability in sample workup and measurements. Sometimes a higher cell concentration is not statistically significant. This may lead to wrong decisions on e.g. harvest timing.	A single replicate for each measurement point is fine for a regression-based determination of growth rate where you are fitting to multiple data points. But it is best to use triplicate repeats if you want to determine subtle changes in concentration e.g. to determine onset of stationary phase.