I am measuring growth of H. mediterranei at OD 600 nm in order to construct growth curves.
However I just read this useful guide on measuring bacterial cell growth: http://www.mpi-bremen.de/Binaries/Binary13037/Wachstumsversuch.pdf
On page 6 is says that " The proportionality between OD and cell density exists only for OD ≤ 0.4 (approximately). Because higher OD values are no longer proportional to the cell density (Fig. 4), their direct plot versus t yields a wrong (distorted) growth curve in the upper range."
Until now I thought that as long as the OD value did not exceed 1 it would be fine.
Can my measurements with values over 0.4 still be used?
Any answer is highly appreciated, especially with an explanation as to why it is like that :)
Thanks!
In general photometers have their best performance around 0.1. The further you are away from that region the worse the linear relationship in the Lambert Beer law fits the true behavior. You have to decide how precise you need your results to be. To get an idea about the behavior of your instrument at your wavelength do a dilution curve with a highly concentrated bacterial solution and check your dilution curve to see in which area the slope of the curve is linear enough for you to rely on it.
In general photometers have their best performance around 0.1. The further you are away from that region the worse the linear relationship in the Lambert Beer law fits the true behavior. You have to decide how precise you need your results to be. To get an idea about the behavior of your instrument at your wavelength do a dilution curve with a highly concentrated bacterial solution and check your dilution curve to see in which area the slope of the curve is linear enough for you to rely on it.
I agree withe above answers. Besides cell concentration, optical density may be influenced by size, shape & sedementing of particle (bacteria) involved. Constructing a dilution curve (as advised above) with your bacteria, is a great idea to find out a ideal linear relationship between cell density and OD for your organism.
actually, when you made that calibration curve with small enough intervalls, you can create a function (polynom of a high degree) to fit your data or a linear function for every small intervall and use this function even outside the linear range since you now have a function to predict the nonlinear behavior. The only restriction is that your optical density must still increase significantly, your function must of course have a good adjusted R^2 and you cannot trust results outside your calibration region
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