Conclude engineering run

Continue hourly tracking until the concentration is stable. Here, stable means the relative change is within 5% per hour. Use the arithmetic mean of the triplicate repeats for the calculations.

For now, the easiest approach is to read the average concentration from the growth curve plot in Access and enter the incubation time and cell concentration into a spreadsheet template. The template calculates the relative change per hour.

The data used in this example are available below as an Excel file. You may notice that the interval between the last measurements is longer than the recommended one hour. We did not have the luxury of a tag team, and sleep is precious. Still, the S. epidermidis growth curve is a useful example for deciding when to conclude the experiment.

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mpd1-excel-example.xlsx

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We are working on an Access feature that will automatically determine when to conclude the engineering run. Stay tuned for updates.

Step by step

1

Get the arithmetic mean

Open the measurement group and drag the slider to see the growth curve.

Move the mouse cursor over the plot. A tooltip appears with the time and arithmetic mean of cell concentration. Identify data points with similar, high arithmetic means.

For these data points note down the incubation time and cell concentration in a spreadsheet.

2

Calculate relative change per hour

Use the Excel file as a template for your calculations. A step-by-step example is provided in the Data example section.

• Add the incubation time (calcHPI) data to column K.

• Add the mean concentration data to column L.

• Column M calculates relative change per hour automatically. The first field stays empty because a preceding measurement is required.

3

Conclude or extend

Conclude the experiment when two consecutive data points show less than 5% relative change per hour. In this example, that happens at 22.6 hours. The value at 14.9 hours is -0.5%, and the value at 22.6 hours is -4%.

If this condition is not met, continue hourly sampling until it is.

Data example

The table below shows incubation time, mean concentration, and relative change for the S. epidermidis dataset. Relative change per hour is the slope between two consecutive data points. Here, we use the 14.9-hour data point as an example.

The Excel template does these calculations automatically

This example is for explanatory purposes. The Excel template does the calculation for you.

• Change in concentration: 7.07×10⁹ cells/mL - 7.17×10⁹ cells/mL = -0.1 ×10⁹ cells/mL

• Relative change in concentration: -0.1 ×10⁹ cells/mL / 7.07×10⁹ cells/mL = -1.4×10^-2.

• Change in time: 14.9 - 11.8 hours = 3.1 hours

• Relative change per hour: -1.4×10^-2 / 3.1 hours * 100% = -0.5%

The plots below visualize the data. The right plot shows relative change per hour approaching zero through deceleration and stationary stage. By definition, stationary stage begins when growth rate is zero, meaning the concentration no longer changes. In this example, onset of stationary phase occurs after ~15 hours of incubation.

S. epidermidis growth curve data. Left plot is the growth curve with concentration as a function of incubation time. Right plot is the relative change per hour.

Rule: Two consecutive stable data points suggest onset of stationary stage

One data point with low relative change is not enough. Bacterial growth profiles can be unpredictable. Phenomena such as diauxic growth may cause a temporary pause before cell division resumes. Requiring at least two consecutive stable measurements reduces the risk of mistaking a transient pause for true stabilization.

The 5% threshold is a rule of thumb. Your bioprocess may need a different threshold, especially for slow-growing cultures.

Diauxic lag can lead to a premature stop

A transient pseudo-stationary stage — also called diauxic lag — can make it harder to choose the right harvest point. This is shown in the figure below with the arrow. At this stage, the cells have likely depleted the first substrate and need time to adapt before growth resumes and the culture reaches the true stationary phase with the best harvest time (green data point).

If you want to be certain that the culture has reached stationary stage, let the vessel incubate overnight after the last hourly sampling point. Recheck the concentration in the morning as described below.

Illustration of a transient pseudo-stationary stage (arrow) caused by diauxic lag. If the engineering run ends too soon, max cell concentration may be underestimated.

Extend the incubation overnight to ensure the culture has reached true stationary stage

Some cultures need longer incubation to reach stationary stage than the basic rule of thumb allows.

After the hourly sampling is complete, extend the incubation overnight and measure again in the morning. If the cell concentration has increased substantially, the culture had not yet reached the true stationary stage and the engineering run needs to be repeated.

In that case, use a stricter rule of thumb than two consecutive data points below 5% relative change per hour. Try four in a row instead.

Summary

In this step, you tracked the culture with hourly triplicate measurements. Conclude the experiment when the relative change in cell concentration per hour is stable. Next, inspect data for harvest time.