eVOLVER Forum

Some vials stop back-diluting mid experiment

Hi,

I’ve been running an experiment for ~850 hours, and for the last ~100 hours about half of the vials stopped back-diluting. I had not touched the machine for over 50 hours at that point. I’ve turned off-and-on the turbidostat and router, and restarted the python script multiple times, but only one of those vials began back-diluting again. Has anyone encountered this? If so, do you know what might have gone wrong/what helped you fix the problem? I’m totally stumped.

Thanks,
Melanie

P.S. This question is unrelated to my question about media tubing hardware posted a week ago, since I haven’t yet implemented the change in media containers from that post.

Can you provide more information? Like if you have any OD traces from the last 100 hours, or what the OD set values are.

If you run just the efflux pumps like during setup, do those pumps turn ON? That would tell us where there might be an error.

Sorry to hear that!
Assuming you’re running turbidostat mode, if the cells are very dense, and OD values are NaN (likely the case if they haven’t been diluted for 50 hours), then dilutions will not trigger even when restarted. You can manually dilute (using fluid_test or electron app to run pumps) and once OD values return to measurable levels, then automated dilutions will kick in (often over diluting due to backed up pump commands).

Not sure why dilutions stopped in the first place however, would need some more data as Brandon mentioned.

Thank you both! Here’s some more information on the experiment:

This is an OD trace of one of the vials that stopped back-diluting:


There’s a sudden spiky drop, which looks like it happened within the last day (I’m guessing one of the times I re-started). If I look at the OD text files, they also look like 0.6, not NaN values. The sudden spiky drop at ~830 hours makes me think that the pumps are still working.

Here’s another one of these problem vials - I thought it started working again yesterday after turning the machine off and on, but the OD’s started to climb again.


Yesterday, I lifted that one out of its smart sleeve to look at it, and the cells were in clumps rather than suspended throughout the media. I can imagine that adding to noise in OD measurements (it does), and/or clogging pumps. It didn’t look like efflux had been a problem up to that point though, since the liquid level was below the efflux tubing outlet. However, as I held the vial media flowed in all the way to the top of the vial - I put it back quickly since I was worried I had tripped the OD sensors by removing the vial from the smart sleeve, but that indicated to me that inflow, as well as outflow, is working.

For contrast, here is a vial which had no problems:
54%20AM

Thanks again,
Melanie

Thanks for all the details! This really helps debug the situation. I just want to rule out one more thing. Can you take a picture of the vial? Is there heavy biofilm? That might obstruct the sensor from reading properly.

I think I figured out the problem, actually! It seems that the tubing was fraying around the connectors from multiple media bottles, and those were the bottles corresponding to vials which had stopped back-diluting. I cut off the frayed part of the tubing and re-inserted the connector barb, and the back-dilutions resumed.

Thanks again,

Melanie

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