Im culturing Chlamydomonas on test tubes with constant agitation. We take the tube directly into the spectrophotometer and measure the absorbance at 750nm. As the time passes cells and pigments seem to settle down in the bottom. Should I be shaking the tube to reach an homogeneous mix before measurement?
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As mentioned in the article recommended by Dr Girma Hailemichael, measuring the OD of microalgae suspensions is not a precise way to estimate the growth of the biomass (variabilities in cell volume, pigment content and other factors). However, if one needs these data, it is desirable to mix the suspension right before proceeding to the measurement of the OD.
It is also important to mention that, older cultures tend to form flocs due to the increase of pH of the medium (if the pH is not controlled using, for example, CO2 injection). And obviously, flocs can also form if the growth medium contains coagulants, flocculants or alkali. This makes it even harder to obtain a precise assessment of microalgal growth by spectrophotometry.
Here is a more practical answer. I assume your light path is vertical (as in a plate reader for 96-well plates). Then you could argue, that it doesn't matter at all, because seen from the top, the density of particles remains the same, irrespective of whether they are settled or not. This argument might probably upset some physicists that would come up with some reasons why this is wrong, but hey, we are sloppy biologists... So: why don't you take a sample of your culture and pipette 96 replicates into a 96 well-plate. Then you measure them in your plate reader right away. Then you wait 5 minutes and measure them again. And again after 10 minutes. Then plot the data: if the average values remain the same, no worries, you are fine. If the values change from 0 to 5 to 10 minutes, you are in trouble: settling seems to matter. Then, have a look at the first 96 samples of the t0 measurement: do the values even change when you plot them in the order in which they were measured? If yes, then there is a good reason to shake before every single measurement.
If you do this: report your results here! Curious to see them.
About the discussion pro/con OD: yes, OD is not the most reliable measurement for grwoth. But it does the job very well, is sufficiently precise for most use cases and it is much faster than any other method. Just be aware of its shortcomings.
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