I want to use flow cytometry to distinguish diverse groups of algae. Does anyone know what configuration of the flow cytometer is needed for this?
Pls contact Dr Carole of PML regarding this : [email address deleted]
I havent work with flow cytometer, but i came across this article, may be it is useful. Rogers, C. E., Navas, C., Bush, K. F., Dyble Bressie. A novel, transportable flow cytometer facilitates algal quantification in cultures and environmental samples. BD Biosciences, October 2011. Technical Bulletin
Hi Lee. We studied selectivity of feeding of rotifers on mixtures of microalgae. So we got citograms with a series of distinct clusters corresponding to different algal species. It was amazing. I have a page with some examples but it is in Russian - http://ibssequipment.at.ua/index/issledovanie_selektivnosti_pitanija_kolovratok_s_pomoshhju_protochnoj_citometrii/0-21 Anyway, look at pictures)))
Sorry, I have forgotten that I translated this page!)) http://ibssequipment.at.ua/index/selective_grazing/0-25
First, use a monoculture with middle cell size. Set chlorophyll channel (FL4 on FC 500) as discriminant, start measurement to optimize voltage and amplification settings for FL4 and FS (for algal mixtures it is better to use LOG scale). You finally get a distict cluster in the center of cytogram. After this, prepare polyspecies mixture and test it. Ideally, you get well-isolated clusters but it depends on the species (differences in their cell size and chl content). For our experiments we had to use the species which produce spaced clusters.
as Vladimir Mukhanov explained..but if you use FASCalibur there are many available protocols that can help you a lot.. anyway its useful to read them:
Marie D., Brussaard C., Partensky F. and Vaulot D. Flow cytometric analysis of phytoplankton, bacteria and viruses. 1999. In: Current Protocols in Cytometry. John Wiley & Sons, Inc. 11.11.1-11.11.15.
How to count picoalgae and bacteria with the FACScalibur flow cytometer
by: J. M. Gasol
(1999), pp. 1-51
I've looked at phytoplankton here for researchers from the Botanics.
Need to filter samples through appropriate size mesh for cytometer - I was sorting so had to use 40µm to prevent blocking the nozzle.
Measured fluorescence for phycoerythrin 576/26nm, chlorophyll-a 675/20nm and phycocyanin, chlorophyll-a and -b 670LP.
Also used calibration beads (1, 3, 6 and 10 µm) for size evaluation.
Comparison of fluorescence against SSC is useful
Thanks to you all for your really helpful responses! I have access to a Beckman Coulter CyAn ADP analyzer, which appears to have the capabilities needed to analyze the pigments Martin mentioned. There are a couple of closely related species that I don't think we can distinguish with this method, but otherwise the size and pigment compositions should be easily distinguished.
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