Plan experimental timeline

Since your goal is to identify the optimal harvest time, you should avoid the lag and exponential growth phases and begin measurements at the onset of the deceleration phase. This requires careful planning of when to inoculate the growth vessel. Working in shifts is often an effective strategy:

• Team 1: Inoculates the culture in the morning and monitors the bioprocess to ensure it is progressing as expected.

• Team 2: Takes over once the culture enters the deceleration phase and performs the required measurements.

If you already have an experimental timeline in place, you can skip to Get things ready.

When to inoculate

You can use historical information, and/or knowledge on growth parameters to determine when to inoculate.

Use historical information

If you have previously performed a similar engineering run, you may already know the incubation time required to reach the deceleration phase. In that case, simply work backwards from the desired experiment start time to determine the appropriate inoculation time.

Calculate inoculation time based on historical information

In this calculation example, we assume that the process has previously been characterized using the same vessel, starter culture, seed concentration, growth medium, and other critical process parameters. Under these conditions, the culture reached the deceleration phase after 6 hours.

If the experiment is planned to start at 12:00 PM, the inoculation should be performed 6 hours earlier:

12:00 PM − 6 hours = 06:00 AM in the morning.

Use knowledge on growth parameters

If you already know lag phase duration, λ, and growth rate, µ you can use an exponential growth model to determine the incubation time needed to reach the deceleration phase.

Calculate inoculation time based on λ, and µ

In this calculation example, we assume λ, and µ is known in advance and that the parameters for the engineering run lead to similar growth profiles. Deceleration phase typically kicks in after ~1×10⁹ cells/mL.

• λ = 2 hours | µ = 0.5 h^-1

• Growth needed: 1×10⁷ cells/mL→ 1×10⁹ cells/mL. 10⁹ / 10⁷ = 100

• Number of generations: log₂(100) = 6.64

• Growth-phase time: 6.64 × 0.5 h^-1 = 3.32 h

• Total incubation time: 3.32 h + 2.00 h = 5.32 h

If the experiment is planned to start at 12:00 PM, the inoculation should be performed 5.32 hours earlier:

12:00 - 5:32 AM = 6:28 AM in the morning.

Use BactoBox® to hit target seeding concentration

An accurate seeding concentration is essential for both approaches to work effectively. BactoBox® is great for this purpose. Follow the guides in Get starter culture to hit a given target concentration, e.g. 1×10⁷ cells/mL. Specifically - if you have a liquid starter culture - follow the subworkflow From liquid culture.

Summary

Once you've calculated when to inoculate, next step is Get things ready.