maximum time frame for an experiment

[Katja]

hello, is there a maximum time frame for chi.bio experiments?
We started an experiment and unfortunately the system stopped it after 72 h, I guess.
I don’t know, is there any limitation right from chi.bio, or was something wrong with my computer or so.

Thanks a lot for your comments

Katja

[harrison]

Hi Katja,

No, there is no reason it should stop in particular. The software is designed to run indefinitely and we have done experiments of a month or more.
Do you know what error was shown in the PuTTY window when you saw it had stopped? In order to diagnose what went wrong we would need to know what message it displayed.
If I were to guess, the most common failure mode is some liquid (or condensation) reaching the moisture sensing tracks on the top of the device and causing it to cut out (since it would then think the liquid might have overflowed).

Best
Harrison

[Katja]

Hi Harrison,
thanks for your fast answer last time, unfortunately we don’t know the error shown in PuTTY, because the PC was shut down. But we startet the experiment new.
At this new trial we had also issues with the system: it was not connected anymore, after around 3 days.
Today I have the PuTTY comments, lukily. You find them below

2021-11-28 22:49:14.813805 Failed Pumps comms 11 times on device M0
2021-11-28 22:49:14.835333Failed to communicate to a device 10 times. Disabling hardware and software!
[2021-11-28 22:49:14 +0000] [1800] [INFO] Shutting down: Master
[2021-11-28 22:49:14 +0000] [1800] [INFO] Reason: App failed to load.

Can you help us, why does the system stopped this time?

And another point we had: in our experiment, the OD is kept at a certain value and the medium is pumped in accordingly. Suddenly, however, the OD has increased and we do not know why.

The PuTTY report was:
2021-11-27 04:55:55.289723Failed to disconnect multiplexer on device M0
2021-11-27 07:15:02.953718Failed to disconnect multiplexer on device M0

Thanks a lot for your help!

Katja

[harrison]

Hi Katja
This error is consistent with either a hardware fault, OR there getting liquid on the moiusture sensor. In fact the later is much more likely since as you said it ran for 3 days first before failing randomly.

Did you see any liquid on it? Did you see even any dried liquid/residue around the top of the device? What kind of lid are you using? (is it 3D printed, or a rubber washer one?)

I would have to say this is by far the most likely issue if it is randomly crashing with that error during an experiment.

As to the OD jump -can you send me the data file (csv) so I can look at this? If it is just for ONE time point in the experiment it may be just an error in digital transmission of data somewhere.

[Katja]

Hi Harrison,

thanks a lot for your answer!
Actually, I don’t know, if I have found the moisture sensor, is it the part, which is called sensor aperture in chapter 2.1 in the mechanical assembly guide?
If there is so, then no liquid is visible on the sensor, also no dried one. As well as on the device itself, there is no residue. The lid we use is the 3D printed one.
Thanks for your offer to have a look on the data! How can I send you an attachment with the csv file? It is an Adaptive Laboratory Evolution Experiment.
Thank you for the support, best regards

Katja

[harrison]

The moisture sensor/s are the parallel silver tracks around the main hole in the device where the test tueb goes, and also around the outside of that top face. They are silver lines approximately 1mm thick and 1mm gap between them. It senses moisture by looking at the conducitivty between those tracks – so if a droplet spans the two it will trigger the sensor.

For the csv, could email it to me at harrison [at] chi.bio

[Shalni]

Hello,

I just tried my first unattended run and had a similar problem occur.
About 3 hours into an experiment the OD spiked from 0.3 where it was being maintained to 0.8. This triggered the input pump to spike and caused the tube to overflow and wet the reactor. I’m assuming the overflow is what stopped the run via moisture sensors etc but I was wondering how to prevent the OD spike from occurring again. Ideally I would need things to run smoothly for 4-5 days in Turbidostat mode.

Thanks for the help!

Shalni

[harrison]

Hello Shalni,
It should be almost impossible for the automated control algorithm to overflow the reactor, even if the OD readings are wrong, unless the tubing or pump is malfunctioning.
In particular, have you made sure that the liquid level in the reactor is always significantly above that in the fresh media and waste media containers? Furthermore, have you tested the pumps to check they make a good seal, and if not, added tape to the pump heads as shown in the troubleshooting guide?

• This reply was modified 2 years, 8 months ago by harrison.

[Shalni]

Hi Harrison,

Thanks for the advice.
I pushed down the tubing on the output pump and double checked the seal.
I reran my experiment and this time got no errors however about 10 hours in the OD regulation stopped working.
It held at my target of 0.4 for quite awhile but then both the input and output pump stopped and the OD saturated.
When I came back this morning I manually diluted the culture by running the pumps and the OD regulation started working again.
Any ideas?
This link has the output plots for OD, and pump rates from my experiment.
https://drive.google.com/drive/folders/1fpZo-hMuZzVzpBVAuyRMYalJvrMojHnO?usp=sharing

Also to clarify, the top height of liquid for my fresh media and waste should be a lot above the top height of liquid in my reactor? Do you mind explaining why this is the case? (especially for the waste side) Does the actual tube end need to be at a specific height as well?
I have attached an image of my current setup in the link above as well. Right now my media and pump/reactor is on a lab bench with a large amount of fresh media and some waste media to start. The tubing rests at the bottom of each container.

Thank you so much for your help
Shalni

[harrison]

Hello,

– In your graphs, I agree with you at some point it seems that the OD regulation algorithm decided it was going to stop running. But, it seems the input pump rate didn’t spike, i.e. the system didn’t THINK it ran out of media or got contaminated or such. This indeed seems like the OD regulation code simply stopped running – but it is very unclear to me how this could happen whilst the system keeps running as a whole.
I suppose you or anybody else didn’t press any buttons around this time that might have changed things? When you checked in the morning did the OD algorithm SAY it was running (ie.. the regulate OD button is blue)? If that was selected/blue then you shouldn’t be able to control the pumps manually since the OD Regulation algorithm blocks the user from changing pump rates (since they are being used to regulate OD).

– The top height of liquid in your fresh media and waste should be BELOW the top height of liquid in your reactor. Can you tell me where you read that it should be above? Is there some part of the manual that is ambiguous that I might fix? The reason they should be BELOW the reactor height is so that if a pump breaks the liquid won’t flow into the reactor. To achieve this in your current setup you need to put your reactor on a small raised surface (e.g. a used tip box or something) so it is higher than the media.

Harrison

[Shalni]

-Sadly I stopped the experiment before manually changing the pumps and I don’t remember if the switch was still blue before. I’m not sure if the switch state has a log record anywhere I can check. The od set point did change to 0 in the data files.
I will remember to check the switch if it happens again.

-Oh I see thanks for clarifying. I was just referencing your above comment “have you made sure that the liquid level in the reactor is always significantly below that in the fresh media and waste media containers” but not any part of the manual!

Thanks, Shalni

• This reply was modified 2 years, 8 months ago by Shalni.

[Shalni]

Resolved! haha
I did not realize everytime you stop the experiment (to change media etc) the OD regulator switches off and doesn’t switch back on when you start again.
Even though my exponential growth happened a few hours after the switch the od setpoint went to zero exactly when I restarted the experiment, I just didn’t see the effects till some time later.
No mystery turn off!
Sorry for the trouble
Potential recommendation to keep the switch on unless the user resets the experiment but other than that it was a human issue.

[eliza_]

Hi!

We are also trying to use Chi.Bio in our lab to do the long-term evolution experiments. I am searching here for people who succeeded with this, because it seems to me from the discussions that we are facing all the same troubles.
Did you finally figure out how to operate the system for your purposes?
What do you think about sharing an experience?
My email is mitichev@post.bgu.ac.il , just in case.
I hope that sharing email is not prohibited by this forum.
Everybody who is reading this forum and using chi.bio are also welcome.
Especially I am searching for Chi.Bio users in Israel!

Best,
Liza

[harrison]

Hi Liza,

If you are having issues with crashing then 95% probability the issue is the seal of the lid onto the glass for the vial. It can be solved by adding a rubber washer in there which is mentioned in the operation manual troubleshooting guide. This issue arose with a change in 3D printing technique for the lid recently, but seems to be resolved for almost everyone by following this fix.

[Author]

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