Summary

In this workflow, MPD-1, you tracked the deceleration and stationary phase to identify a suitable harvest point. In the S. epidermidis growth curve examples, we obtained the below growth curve. The fully drawn line is the cell concentration, while the dashed line represents CIZE.

Key parameter for harvesting

The below plot shows the S. epidermidis example. The harvest time (dashed line with arrow) is 11.8 hours as assessed by BactoBox® and CFU data (the CFU arithmetic mean for the 15 hour data point is slightly higher, but the change is not statistically significant).

We obtained the below harvest time parameters.

Harvest time	Harvest time concentration	Harvest time CIZE
11.8 hours	7.2×109 cells/mL	0.83 µm

These values are of particular interest for subsequent production runs. The 11.8 hours will get the culture close to the onset of stationary phase with maximal cell concentration, but we recommend that you also do BactoBox® measurements when approaching the defined incubation time for your production runs.

• If the harvest time concentration is lower than in the engineering run, it may be beneficial to extend the incubation time.

• If CIZE is higher than in the engineering run, it may be beneficial to extend the incubation time.

In practice this means that - for production runs - the harvest time is actually a harvest window. Find more information on this in the explainer Harvest window.

Key assumptions and verification tests

To use BactoBox® as a proxy for maximum CFU/mL, we rely on assumptions that have generally been proven valid. But there may be exceptions. Use our additional workflows to verify these assumptions for your cultures.

Assumption	Verification test
The sample is suitable for flow cytometry with predominantly single free-flowing cells	A quick check in a microscope is always a good idea. Also, check Break up clumps and chains for how to get a single-cell suspension.
BactoBox® and plate counts correlates closely in stationary stage.	Run a head-to-head experiment using the Correlation group type to verify correlation between the methods.

Summary

You have now completed the MPD-1 workflow on determining harvest time for batch processes with bacteria as the product. The engineering run serves as a decision-making basis for future production runs.

We recommend that you also use BactoBox® for tracking the production runs. This will enable fine-tuning of the harvest point within the harvest window.

We have added an Optional: rigorous approach section to this workflow. It uses a more involved statistical test for evaluating the best harvest time.