Hi everyone,
I noticed something strange during my Pioreactor experiments and I would like to ask for your advice.
Whenever I take my vial out of the Pioreactor to take a photo and then put it back in, I see a clear step change in the OD readings on my graph (see attached image). I expected the OD values to remain the same, since I just take the vial out briefly and then put it back in without changing anything else. But instead, there is always a noticeable jump or drop in the OD value after I return the vial.
Why does this happen? Is this normal?
Is there any way to prevent or minimize these sudden changes in OD readings after removing and returning the vial?
Thank you for your help!
hi @starzeus,
hm, these jumps do seem dramatic! We can expect some change after removing it, mostly due to some variation in the glass vial (so if the vial is rotated, the OD can change). But these seem larger than that variation.
How long is the vial out of position for? And what is being done while the vial is outside?
Hi, thanks for your reply!
Every time I take the vial out, it’s usually for about 1 minute, just for taking a photo. There might be some slight shaking during handling, but I don’t change the location or temperature, and I don’t do anything else.
I’m curious about the angle issue: since the OD detector is fixed, does the angle of the vial really matter? Do I need to pay attention to always putting the vial back in the same orientation? I hadn’t considered this before, but I can try marking the vial to keep the direction consistent, if that helps!
Thanks for the suggestion.
The rotation variance is small, typically 1-2%, and due to changes in the borosilicate glass thickness, etc. But in the new Pioreactor 40ml, we have a system that fixes the rotation each time, so it’s not an issue in the 40mls.
Returning to your issue: I’m not why this is happening. Can you replicate it? What if you removed the vial out, and then instantly in again - do you see the same issue?
Oh one idea: could there be biofilm on the glass?
Follow up on this with a similar but more acute issue. For me, the OD read out changes extremely rapidly when the vial is disturbed, even when the vial is not removed. I used a needle to extract 50 ul of culture from a 15 ml vial, tilting the device maybe 40 degrees to access the top. The OD went from reading steady between 0.92-0.94 to reading stead between 0.35-0.40. Extensive checking of the sensors makes the values move around between 0.30 and 0.45 but nothing similar to the 0.9 readings from the 48 hours before the small subsampling. Any idea what causes these huge shifts?
Hi @agrossman, can you share with me the “IR LED intensities” and “Optical density” datasets from your Export Data webpage for this experiment? You can email them to me at cam@pioreactor.com
Also, can you tell me what version of Pioreactor software you have?
So, looking at your relative IR intensities (which is basically what the REF photodiode sees), I see some instability at 25h and 70h, which correspond to your jumps.
As explained in this other post, the displayed OD is actually a ratio of [90deg signal] / [REF signal]. So if the REF changes significantly independently, it will effect the reported OD.
What I suspect happened was that the REF was slightly moved during the sampling, and this caused the shift. (In our next version of the Pioreactor, we fix this. For now, you can try to fix the photodiode in place with glue or sticky tac).
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Thank you so much, I’ll give that a try and see how it goes.
