In order to have a true count of the number of cell/mL, we need to disperse all agregates before fixing it with glutaraldehyde and count it with a C6 flow cytometer. Agregates, if not dispersed, could lead to clogged tubing, and filter the culture (benthic diatom, probably gender Navicula) results in loss of precision when we need a true absolute counting.
Many benthic diatoms are simply too large to be counted using flow cytometry.
Nevertheless, if you are interested in the smaller than 20 µm diatom population, you may first sonicate the biofilm and then use a 20 µm or 40 µm cell strainer or a filter in order to remove the larger cells (and avoid cytometer clogging).
How dense are your samples? We usually dilute the samples if needed and sonicate them prior to flow cytometry. As far as I remember there are also differently sized flow cells available from Accuri. Good luck!
Thanks for your answers ! Problem is culture is taken from an erlenmeyer, and following the spot, we take more or less agregates, and in my opinion it can falsifies the counting. I'll try to take 5 mL in a falcon and then sonicate for 15-20 min, then use a 30 µm filter, fix the cells and count it. I think Accuri C6 FCM does not have different sized flow cells unfortunately :( ! Thanks again for the answers anyway !
In case of high density of samples one of the best way is treatment of diatoms in ultrasonic bath (during 20 min) , then stir repeatedly and count in countin chamber under light microscope. This technic provide the most reliable result, and you can identify tha most mass species at least on the genera level.
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