I am running some lab-scale experiments comparing different algal strains in different growth conditions. To measure algal cell number I am using a cell Coulter counter (Multisizer II, Beckman Coulter), since my cultures' volume is around 75 mL and it's impossible to take samples for dry biomass determination (which then I measure at the end of cultivation).
When I measure cell concentrations with single-cell strains, like Chlorella, I have single peaks on my spectra and I can draw very nice growth curves with a good estimation of cell concentration.
The problem comes with strains like Scenedesmus, which forms colonies of 2, 4 or even 8 cells, and other strains which tend to form cell clumpings. Here the spectra present different peaks corresponding to different diameters, and the cell concentration is under-estimated because more cells are counted as one bigger cell. The underestimation is confirmed by the dry weight yield measured at harvesting.
Does anybody has faced this problem yet? Do you "manually" correct the cell concentration to get it reliable? Actually I was thinking to analyse each spectrum after checking cell morphology under the microscope. In this way I could associate the correct number of cells to each peak in the spectrum, and then recalculate the cell concentration on this basis.
Do you think it would be a good way? Or do you suggest anything else?
Thanks in advance.
Lorenza
Dear Lorenza,
You can check this paper. The authors describe two different methods (packed cell volume and loss of weight by dissimilation) to quantify the biomass in cell suspension cultures. As you know, in this type of cultures cells form groups of different sizes and it is very difficult to count then using light microscope.
Dear Lorenza,
In the past I have been working a lot on colony formation in Scenedesmaceae so I recognize the problem.
As Gustavo suggested, if you are interested in actual cell numbers (per colony), you need to do a visual side count of a representative number of cells or colonies, typically 200-250. This gives an estimate of the size distribution (unicells, 2-, 4-, 8-celled colonies) and the mean number of cells per colony. This can be related to the peaks obtained by Coulter counting (mean particle volume). They do not match linearly but are of course correlated, see also the link to the paper.
If you are actually only interested in biomass (dry weight), the total particle volume as obtained by Coulter counts is a fairly good measure of that, provided that you have relatively clean cultures. For different Scenedesmus cultures you can make dilution series for which you determine both total particle volume (biovolume) and dry weight concentration.
Good luck.
Article Inducible colony formation within the Scenedesmaceae: Adapti...
Dear Lorenza.
Algae cell counting for species that tends to form colonies, filaments or groups is a challenge even for optical counting techniques. It´s complex defining the unit when different groups or colonies have different number of individuals. I don´t have further experience in this topic, but I faced a similar problem the answer is count the individuals in several colonies and obtain a mean of individuals/colony to do an extrapolation in function of colony counting. Then, you can repeat measurements with the cell counter do an estimation and validate these results respect to optic microscopy count results.
Best regards.
So, if the machine measures the volume of cells passing through the aperture, why in my Scenedesmus spectrum do I have a peak in the same region where I find the peak of Chlorella? The cells are considerably different in size, so it is not possible to have particles so small. I checked my cultures under the microscope and they are not contaminated. I don't understand how to analyze the spectra then if I have non-spherical cells which also form colonies.
I "solved" by using the biovolume instead of number of particles, which is much more correct in my opinion, since this is the actually measurement of the machine, and especially if a growth curve has to be drawn.
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