Variable OD measurements

[Neythen]

Hey,

We are calibrating the OD measurements and noticed that the measured OD seems to vary significantly depending on the angle of rotation of the vials. To test this we sequentially took 200 OD measurements, rotating the vial randomly by hand between each measurement. You can clearly see how the OD measurement changes for each rotation and it is significant enough that it could make calibration difficult. We are using the recommended vials and have repeated with fresh vials, both empty and filled with distilled water, to negate the possibility that the vials got dirty. Is it possible the curved glass refracts the laser differently at different angles?

Thanks in advance for any help!

[harrison]

Hi Neythen,

Yes – you are right. I think the answer is that there are small differences in the glass (i.e. thickness) and also slight positional differences as you rotate/move it. There are two primary ways to address this:

1. Install a diffuser – this is a small rectangular piece of platic which goes in front of the spectrometer and spreads out its receiving area to reduce noise (by about a factor of 10!). If you email me your postal address and tell me how many reactors you have I can mail you some. They are meant to be installed on future purchased Chi.BIos but I guess you bought yours a while ago.

2. Another way, which I commonly do, is put my test tube + fresh media into the reactor, screw on the lid, THEN calibrate the zero OD, and only then pippette in the cells that you want to grow through a hole in the lid. This way you never move the tube post-calibration so there is minimal error.

I should also say, the errors you are seeing are generally mitigated as cells grows since they act as a natural diffuser (spreading out the light). THe zero-OD point is the worst from that perspective…

[Nikako]

Hello to both of you,

I am facing a similar issue of variability in the OD values but at higher ODs as well (OD=0.4 using the turbidostat functionality of the bioreactor, which results in an OD of ~0.2 [more precisely OD is contained between 0.16 and 0.19] when culture’s OD is measured in a cuvette and at 600nm wavelength). I am not sure if the variability comes from the cells growing and then getting diluted when OD gets above 0.4, but in some cases, I could see a notable difference depending on the orientation of the culture tube. I also observe variability in OD with fresh medium, but if left untouched, the OD values are quite stable. The issue is that I am taking samples out of the culture tube when my bacterial population reaches OD=0.4, which leads to some displacements, I assume. Could you let me know the amount of variability you observed in OD values at ODs around 0.4 (or 0.2) if you have the numbers?

To clarify, the culture tube’s surface is cleaned with isopropanol before placing it in the ChiBio module.

@Neythen, were you able to solve your issue? And if yes, how did you manage?

@Harrison, regarding the plastic diffuser, would it be possible to get some of these plastic pieces? I checked and the spectrometers of my ChiBio reactors don’t have them. I was planning to go with 2mm thick opaque acrylic sheets otherwise. Also, I am not sure to understand how the light diffuser can improve the OD readings as it would be located after the glass surface of the culture tube.

Thank you in advance for your reply.

Niko

[harrison]

Hi Niko,

Usually if it is regulating at 0.4 OD it should be something like 0.36 to 0.44. Some due to variability in measurements and some due to actual variation in the OD due to pumping in and out.
Indeed taking the reactor in and out of the device will change the geometry slightly and this will change the OD. If you look in the operation manual it talks about how to get OD measurments as reliable as possible – in short start the experiment with blank media (no cells), then let it arrive at correct temperature, then zero OD, then add cells through lid. This will typically reduce variability by more than 50%.

For sampling, IMO the best approach is a long GC needle or other blunt needle that you can put through the lid WITHOUT taking the tube out of the device, and thus maintain alignment as above.

If you want diffusers please email me your mailing address and I can send a few.

[Author]

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