Summary

You have now completed an EoF cross-check between BactoBox® and CFU.

See the full picture

To complement the EoF cross-check we cheated a bit and did a full growth comparison of BactoBox® and CFU methods. The two column charts after 13.5 and 35.8 hours are shown on the left. The growth curve on the right shows the impact of changing the harvest time.

For the 13.5 h EoF cross-check it is beneficial to harvest later. In fact, the CFU value would be ~6 times higher if the incubation was extended to ~23 or ~29 hours.

For the 35.8 h EoF cross-check it is beneficial to harvest earlier. In fact, the CFU value at 35.8 h is ~7.5 times lower than the peak concentration at ~23 or ~29 hours.

P. fluorescens end-of-fermentation cross-check. BactoBox® (lavender) and plate counts (yellow) measured in triplicate repeats. The first column chart is the cross-check after 13.5 hours post-inoculation where BactoBox ~ CFU/mL. The second column chart is the cross-check after 25.8 hours post-inoculation where BactoBox® > CFU. Cyan box highlights where cells and CFUs are within 50%. Grey box highlights region where cell concentration is significantly higher than CFUs. All measurements were done by three individual dilution series prior to measurements. Error bars reflect standard deviation.

Possible outcomes

The possible outcomes and their recommendations are summarized in the below table. There will always be some variability and slight offsets between the methods. As a rule-of-thumb we suggest that 50% difference is used as a thresholds for determining if BactoBox® is higher or lower than CFU.

Occasionally (but rarely) BactoBox® is substantially lower than CFU at EoF. This is usually due to clumping or chaining of cells. Large objects are excluded from the BactoBox® flow cell measurement channel, while a clump of e.g. 100 cells will still form 1 colony on an agar plate. See Objects are too big for more information.

With improved sample workup you will likely get better insights for increasing CFU values. See Break up clumps and chains for more information.

BactoBox® ~ CFU (within 50%)	BactoBox® > CFU	BactoBox® < CFU
If BactoBox® cell concentrations is within 50% of the plate counts results, then you are likely within the cyan region. This could either mean that harvest time is perfect or that higher cell concentrations can be obtained by extending the incubation time.	If BactoBox® cells/mL is substantially higher than CFU/mL then you are likely in the grey region. This indicates that earlier harvest could get you closer to the peak CFU concentrations.	If BactoBox® cells/mL is substantially lower than CFU/mL, then this indicates that the culture is prone to clumping. Improved disaggregation could increase the max CFU/mL and help perfect harvest timing.
Try longer incubation time	Try shorter incubation	Try improved sample workup

Confused about the results? More information is available in Correlation with plate countsand Divergence from plate counts.

Not finding a suitable answers to your questions? Contact SBT for application support.

Next steps

The presented workflow is a quick and simple end-of-fermentation cross-check. The obvious next step is to see the full picture by doing a full Track growth curve experiment. This will show if your harvest timing can be improved to unlock higher CFUs.

To save work, this can be done with BactoBox® only, but it is also possible to do the investigation as a correlation group type with BactoBox®, CFU, and optical density.