I would suggest you read the following paper: D. J. S. Montagnes and Berges, J. A. and Harrison, P.J. and Taylor, F. J. R., 1994. Estimating carbon, nitrogen, protein, and chlorohyll a from volume in marine phytoplankton. Limnology and Oceanography, 1994, 39: 1044--1060. The paper discussed how fixatives affect cell volume and biomass estimation.

Thank you Dr Mei.

The excellent paper you cite shows the effect of fixative on cell size, but not on the C content of the cells (there is indeed an indirect effect, as fixation affects the cell volume vs. carbon relationship). Many thanks for your insights. -Best Regards.

I would suggest to try own method to estimate the impact of glutaraldehyde on the phytoplankton C content comparing the phytoplankton cells suspension fixed with 0.1% glutaraldehyde and without glutaraldehyde. The next could be GF/C phytoplankton filtered samples with rinsed out glutaraldehyde. Then you can burn the sample in the furnace and calculate organic matter content.

I would be most concerned about breaking cells during the freezing phase. So long as you do not break cells you should only have issues with soluble C portions (typically less than 20%).

Hi!

You might want to check this reference: W.J. Kimmerer and A. D. McKinnon. Glutaraldehyde fixation to maintain biomass of preserved plankton. J. Plankton Res. (1986) 8 (5): 1003-1008.

Regards,

I have no experience dealing with the preservation of phytoplankton for carbon determination, but maybe some of our experience with zooplankton might be helpful. We found that the combination of two rather poor methods, aldehyde fixation and freezing, provided consistently good results (Salonen and Sarvala 1985, Arch. Hydrobiol. 103: 217-230). The secret of this surprising result is that 1) aldehydes work as a cryoprotectants and 2) as they have their first contact with the surface of tissues or cells, they also strengthen them to better withstand mechanical stress due to freezing. Whether results similar to zooplankton can be obtained with phytoplankton might be affected by smaller size of phytoplankton. If so the preservation process could be shortened by cooling of water before aldehyde addition and freezing.

Using liquid nitrogen is not necessarily a good idea. The functioning of cryopreservatives is based on minimizing the amount of water in cells and hence adequate time is needed for water to escape before cells are internally frozen. Boersma and Vijverberg (1994, Archiv für Hydrobiologie, 130(2), 241-247) concluded that for zooplankton freezing in aldehyhydes is no better than other methods, but they did not realize that in our method we used -18-20 oC temperature instead of liquid nitrogen. Cryopreservation is widely used in medicine and also its basic concepts have been thoroughly studied.

Finally I would emphasize that it is not freezing, but melting, where main damages (losses of carbon) probably occur. In melting the mean size of ice crystals increases and it is most damaging. Consequently, rapid melting leads to the best results.

I am convinced that glutaraldehyde is much better than formaldehyde. However, 0.1% concentration is probably too low to act as a cryopreservative.

One precision: fixing phytoplankton cells with glutaraldehyde and storing them in liquid nitrogen is the standard sample preservation technique for subsequent flow cytometric analysis.

The question is: glutaraldehyde being an organic molecule, how much carbon does it add to a phytoplankton cell when cross-linked to its proteins?

Such preservation is probably OK for flow cytometry, but it is likely different for carbon.

Theoretically glutaraldehyde increases carbon content although in zooplankton samples it remained undetectable (or was compensated by small losses). Due to the smaller size of phytoplankton than zooplankton, relative increase might be higher and therefore I recommended cooling of samples before preservation. Decrease of sample volume also decreases time for the penetration of glutaradehyde into cells.

I notice that you primarily did not seek the best method to preserve phytoplankton carbon but you wanted information about the magnitude of possible losses with your flow cytometry samples. However, in the preservation of future samples, both goals may be reached by minute adjustments of the protocol.

Thanks for your insights Dr. Salonen. Yes, my concern is the gain of carbon caused by glutaraldehyde fixation.

On the basis of zooplankton results, I would propose an educated guess of losses of ca. 25% for phytoplankton, too. It is hardly possible to have increases with such a low glutaraldehyde concentration.