This is the most recent protocol:

Marie, D., Rigaut‐Jalabert, F., & Vaulot, D. (2014). An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples. Cytometry Part A, 85: 962-968.

From my experience, fixation with 0.1% glutaraldehyde, freezing in liquid nitrogen and storage at -80°C works fine.

Hi Nelson,

I have done the following procedure with mammalian cells. You may consider doing the same with microbes. I pellet down the cells, wash them in PBS, cross-linked with 4% Formaldehyde at room temperature for 10mins, and then finally stored in PBS containing 0.1% Sodium Azide.

Hope this helps. 

Good Luck!

Utkarsh

This is the most recent protocol:

Marie, D., Rigaut‐Jalabert, F., & Vaulot, D. (2014). An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples. Cytometry Part A, 85: 962-968.

From my experience, fixation with 0.1% glutaraldehyde, freezing in liquid nitrogen and storage at -80°C works fine.

Thank you very much indeed! I am about to start some sample prep protocols to see what works fine (aldehyde conc.). Fixation to me is ok, what about separation? Should I centrifuge or filtrate it?

Dear Nelson,

You should avoid centrifugation (many cells are very fragile).

You may want to filter the water samples on a 40 µm mesh in order to remove the larger cells that cannot be analysed by flow cytometry but can clog the flow cell.

Regards,

Claude

Many thanks Utkarsh and Erin, in fact i have tons of things to try to get good cytometric signatures (if possible??) from our amazonian microbes. All you pointed out is usefull. Usually it is very important to do filtration/centrifugation steps before fixing/preserving samples for FCM.