Interpretation of OD600 Calibration

Hi everyone,

I calibrated 4 Pioreactors with 5 mL dilution volume (blue lines) and 2 Pioreactors with 2 mL dilution volume (yellow and orange lines). However, when I exported the calibration data and plotted, two different clusters emerged as it can be seen in the picture below.

From the plot, I can see that 5 mL dilutions generated an almost linear match with the spectrophotometer measurements, but 2 mL dilutions were 2x higher than spectrophotometer measurements.

So my question is, what exactly the y-axis indicates?

Does this plot means that using lower dilution volume (in this case, 2 mL) enables Pioreactors to estimate greater OD levels, or does this means it makes the measurements overestimated?

Hi @sharknaro, to answer your bolded question: the y-axis is measured in arbitrary values. Each Pioreactor has slightly different optics (based on component variability), so comparing the y-axis between Pioreactors doesn’t mean much. However, it’s interesting that there is some “clustering” going on here. I’m going to investigate our OD600 calibration to see if there could be a problem in the algorithm.

Does this plot means that using lower dilution volume (in this case, 2 mL) enables Pioreactors to estimate greater OD levels,

No, that’s generally not true,

or does this means it makes the measurements overestimated?

possibly, but I can’t see where at first thought. I’ll return after investigating.

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Hi @sharknaro

We repeated your test in our office, and didn’t see any significant differences between using 2ml as a dilution versus using 4ml.

This was tested on the same Pioreactor, using software 24.2.11 (though the version shouldn’t matter much). I can’t explain why you saw the effect in your post.

However, after doing only four OD calibrations, we were so fed up with the process, we plan on making OD calibrations significantly better for users. Expect some simple user-experience improvements in the next release, and newer, faster, calibrations in the near future.

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Hi again Cameron,

First if all thank you for your help and patience of going through the OD calibration with 2mL. I can say that it is a laboursom process. I just recalibrated 10 pioreactors with 2mL setup which took ~4 hours. This being said, what I learned:

1- We use parallel calibration of all Pios by opening separate command line interface. This way we can use one vial for all the Pios before diluting and reduce the number of steps. (I am not sure if this is helpful but happy to share)

2- I realized that the clustering must have to to with the with the LED manufacturing setu, so to say, some LEDs require higher/lower voltage input, thus resulting in the variation of Pioreactor measurements.

I have tested the same Pios (all the ones from previous calibration) and I can say that the clusterimg occured again, which makes sense considering it is caused by the condition 2.

Soon I will change the LEDs and run some tests to see my assumption is correct.

Certainly there is variation in the LEDs (clear bulb), but also the photodiodes (black bulb).

Note that the Pioreactor measurement is a ratio of the 90° sensor to the REF sensor, that is:

Pioreactor measurement =  90° signal / REF signal

So if the REF signal between Pioreactors is different, then this will affect the Pioreactor measurement. Why might the REF signal vary? Positioning of the IR LED and REF PD is a major reason, but also the manufacturing variability in the size of the light channel between REF PD and IR LED.

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