maintaining very low OD

[anmo...@colorado.edu]

What is the lowest OD anyone has successfully held during a run? I'm running an experiment with a fairly slow growing bacteria (3-4hr doubling time) and trying to maintain an OD of 0.01. I assumed that since it grows so slowly it wouldn't be an issue, but the OD oscillated quite a bit (see attached plot). Does this paragraph from the paper have anything to do with why it is struggling to maintain the OD (attached)? Some relevant info from the run:

Kp = 3

Ki = 0.05

set OD = 0.01

period = first I tried 180s, then went to 120s

Another thing I noticed was the bacteria were forming small planktonic "clumps." No biofilms on the side of the culture tubes, but I suppose the clumps could be causing an increase in OD if they are not be getting sucked out the effluent port.

I initially attributed the inability to maintain the OD to the fact that the OD readings are highly variable at that low of an OD. Or the strain is growing faster than expected. But after re-reading that paragraph of the paper, maybe I should be adjusting the Kp and Ki to prevent oscillations? Or am I misunderstanding equation 6?

-Andrew

[anmo...@colorado.edu]

Note: around day 2 is when I switched the dilution period to 2min from the initial 3min. You can see it caused the OD to drop initially, but then it jumped back up again. I'm starting to think more and more that it is an issue with the clumps that were forming as growth progressed...

[Chris Takahashi]

This isn't the kind of oscillations you'd see if you had the gains wrong.  Such a low OD is near the noise floor of the device though.  If there's a positive offset to the OD then dilution would eventually exceed the washout rate and you'd just end up plotting noise.  This is my best guess.  My second best guess is that your gains are too low for such a small OD, but increasing gains makes the system more susceptible to noise.

Personally I think 0.1 is the lowest i'd attempt.  Remember though that this is OD 650 at about 2cm path length so to adjust to "standard" OD600 1cm you multiply by about 0.7 for e. coli.  Shorter wave lengths scatter more so if you tried a blue laser (make sure its dim enough!) that might also allow you to run lower cell densities for the same apparent OD.

Also on a side note, if you have slow growing cells you'll probably need to also make sure you humidfy your air (pass it through a bubbler before the chamber).  Otherwise evaporation can be non-trivial at those low dilution rates.

Flocs are an unfortunate fact of life too :(

Taking effluent from the top of the culture selects for them eventually.  If it's an issue the only thing you can do is take effluent from the middle of the culture, but then you'd need a pump for effluent and a liquid level sensor.  Still, that can be rigged up without modifying any of the existing electronics if you're determined.

Chris

[anmo...@colorado.edu]

Yeah the more I think about it, the more I think the issue is the flocs. I'm working with a pretty unique bacterium in this experiment so I really had no idea flocs would form or how to get rid of them. I think we should still be able to make the experiment work with the increase in OD.

That is a great idea about humidifying the air before it hits the chamber. I work with lots of slow growing strains and in the past I've just manually had to add media to make sure it doesn't completely evaporate.

Thanks again for the tips Chris!

-Andrew