Questions regarding OD measurement

[jeff8838]

Hi guys,

In your “Supplementary Information” paper, you guys note that the LED measurement of OD (scattering) can be computed using OD_i = (R / Po) – Mo. I have a few questions regarding this equation:

1. What steps would I need to take to measure Po, the raw power level? Is it the “raw” value measured when there is no media in the reactor?

2. What steps would I need to take to measure Mo, the media compensation factor?

Thanks for your help!

[harrison]

Dear Jeff,

P0 is meant to reflect the “raw power” of the excitation LED – i.e. to compensate for the fact that a LED in one reactor might be slightly brighter than that in another reactor.
M0 is how much scattering there is from the media itself – this is conceptually similar to the OD0 value from the Laser measurement approach (i.e. eqn s1 in the supplementary of the paper), which in that case reflects the absorption of the media.

You can see what these factors do by looking at Figure S10 middle or right column. In the top row you have the raw data – the transformation in eqn S3 is then used to get the second row. So, divide by approximately 1000 (ballpark value of P0), which takes our values of ~10000 to ~10. In the top row you see there is a non-zero baseline (i.e. the measured intensities are approaching some positive asymptote ~500 or there abouts) – this corresponds to M0. So, this is then removed in the second row and we see the asymptote is now the x axis (M0 ~0.5 in this case since we are subtracting it _after_ division by P0).

I think the approach I used was: To get P0 measure the amount of light when you just have a blank test tube in the reactor (and the appropriate LED is on) to give us an approximate raw power level of the LED. Then, put blank media into the reactor and measure this to give M0.

[harrison]

Let me know if any of that is unclear!

[jeff8838]

Great, thanks for the explanations Harrison!

I’ll let you know if I have any further questions.

[jeff8838]

Hi Harrison,

Another question: what OD range does the Chi.Bio platform support?

[harrison]

In principle – whatever you calibrate it for. Very high ODs will go beyond the _linear_ range of the normal setup, but you can fit a curve to compensate for this if you are mostly interested in that regime.

[jeff8838]

Hi Harrison,

Some follow-up questions:

1. We’ve noticed that most OD600 meters seem to only support an OD up to 2.5A or 3.0A. What would you suggest doing if we want to calibrate above 2.5 or 3.0?

2. In the supplemental information document, you mention that one calibration solution you could use is evaporated milk. What OD value should evaporated milk have?

Thanks for your help!

[harrison]

Hi Jeff,

Usually when people want to measure higher ODs what they would do is dilute it down into the instrument’s linear range. The reason they struggle with high ODs is because of the light bouncing around on many cells in the solution, and hence the measurement ceases to be a “linear” measurement of absorption.

So, I would make a sample high-OD solution. Dilute this to OD<1 to measure it in the spectrophotometer, then (knowing the dilution factor you used) calculate the true OD. Then you can put it in the Chi.BIo and calibrate it against this (true/high) OD.

For the evaporated milk I did something similar - made a bunch of sequential dilutions, measured their "true" ODs in a spectrophotometer, then measured their ODs in the Chi.Bio to calibrate.

WBW
Harrison

[jeff8838]

Hi Harrison,

Would an example of “blank media” for the computation of Mo be water?

Thanks

[harrison]

No, just fresh media with no cells (i.e. LB just out of the autoclave…)

[Author]

Posts