OD600 calibration

[schmma]

Hello! We just discovered Chi.Bio and bought one for our laboratory. We are very excited about its capabilities and can imagine so many terrific experiments that we’ve been wanting to do but didn’t have the infrastructure to perform. Thanks for making it happen!

When I did my first trial experiment just to see how things worked, I was able to maintain a Chi.Bio OD set point of 0.5 for several hours after entering a raw value for OD0 to use as a blank. However, when I checked the OD of the same culture using our plate reader, my measured OD there was only 0.332.

So I’ve been digging into the code to identify the relevant calculations happening to turn my laser measurements into typical “1 cm pathlength cuvette” measurements. I found the relevant sections of the code starting at lines 1542. And I also identified that the code I’m using has values of a = 0.226 and b = 1.833 for solving the quadratic function relating ODi to 1 cm.

This led me to some questions:
1. In the SI for the Chi.Bio paper, the curve fit in Figure S10 uses a = 0.397 and b = 1.374. Why are these values different than those implemented in the code? Do I need to manually calculate them myself for every reactor? (If I interpreted Figure S10 a) and b) correctly, I thought that the setting of OD0 using the raw read for just blank media was supposed to account for differences between lasers/reactor set-ups)
2. I just want to confirm that the coefficients a and b shown in Fig S10c are correct and that they were transposed when writing equation S2 — that would match what’s coded more closely.

If there’s anything I’m missing doing or not understanding properly that is likely to explain why my Chi.Bio measurement (where I’m very new to using and understanding the code) is so much different than the one from my plate reader (that I’ve been using for a long time), I would really appreciate your time explaining it to me. I am *not* an expert coder so I apologize if the answer is very clear to experienced coders.

Thanks for your thoughts.

[harrison]

Hello
Yes I think the FigS10C is fine. THose parameters in both cases were found by calibrating against different spectrophotometers in our lab.

In practice, if you take bacterial samples and measure them in different instruments you will IN GENERAL find quite significant disagreement. E.g. even between plate readers of different manufactures, plate reader vs cuvette-based OD meter, or nanodrop type methods, etc. This is particularly true at “High” ODs (i.e. >0.1 or so) which is the multiple-scattering regime where light may bounce of multiple particles (cells) when passing through the sample.

So as a conclusion – IMO I wouldn’t worry about it. There are big differences between machines and measurement modalities even if they “claim” to be doing the same thing. If you are really caring about such things (e.g. the differences between OD = 0.35 and OD = 0.5) because your science requires it then you will need to be very careful and make standard calibration solutions and collect a range of concentrations across all machines and then use this to convert between them. But even then – I think you will be lulling yourself into a false sense of security since there will be still batch- and time-variation in all these measurements.

[schmma]

Hello!

Thank you so much for this reply.

I am relieved by your reassurance. We will probably decide not to worry about 0.35 vs 0.5 as I am most concerned about numbers of doubling times for this particular experiment so those I should be able to get fully from the Chi.Bio readouts. Being so un-unsed to coding, I was mostly concerned that *I* was the problem and was just missing something very obvious.

Really appreciate your response. We are loving the Chi.Bio — I just want to make sure I’m not being an idiot! 🙂

[harrison]

Perfect – and if that is your goal you don’t care AT ALL about the absolute OD number, just however long it takes to double. So it should very much be fine.

[schmma]

Yep. As long as I am in a log phase situation, I just need to maintain a constant OD and wait for them to keep growing and dividing happily. (Or unhappily, as the case may be. Hee hee)

[harrison]

FOr this, look at the Chi.BIo paper and consider using the Zigzag / dither OD mode. It is exactly for this kind of thing.

[schmma]

yes! I saw that ability and was happy to see the code to make it happen is already there! thanks again!

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