spectrophotometry for biomass quantification

[Brecht]

Hello,

 

For my current thesis research I need to quantify the biomass of a bioreactor 5 x each day,

therefor i use as a quick method a spectrophotometer.

 

Now to make a standard curve, i dried a certain amount of biomass.

The problem starts with the dilution: when i dilute this biomass it will not give a homogenous solution.

Can I use sonification to make this homogenous?

I know the cells will be broken but we still have all the biomass?

Is this correct?

 

[John Cuthber]

To a degree, you can do what you like, as long as you do the same thing to the samples as to the test calibration mixtures.

 

Sonication might work, so might adding a little sodium hydroxide.

But you need to make sure that you do two things

Get some "stuff" from the bio-reactor and split it into two sub samples. Measure the biomass in the "official" way on one sample and make a spectrophotometric measurement of the other sub sample.

Then do that for lots of samples so you get a spectrophotometric result for a range of different concentrations of biomass.

 

That will give you a calibration which you can then use to measure biomass.

[CharonY]

I am not sure whether you can do that. Typically cell count using optical density is based on scattering of light on the cells. Thus, you want to prevent lysis during the measurement. Sonication is usually too harsh for many cells and you would be better off by just vortexing (carefully). I am actually also not sure how well it works once you dried samples. I have only either measured dry weight or did a quick OD measurement, but never tried drying, then resuspending. My feeling is that drying down and then resuspending would skew results quite a bit (though a calibration using e.g. dry weight could take of that).

[John Cuthber]

If you are measuring scattering then you are barely doing spectrophotometry, you are doing nephlometry.

It's not going to give a linear calibration over much of a range of concentrations.

[CharonY]

That is absolutely true. That is why commonly it is only used within a narrow range of cell concentrations (usually not much more than one order of magnitude) and you use dilutions to get into that range. Turbidimetry is sometimes used, which is technically superior. But since a photometer is more versatile more people stick to photometers.

There are other approaches, too, including measurement of protein content as proxy for cell mass.