Hi, Lana, what kind genera of filamentous are you culturing and can you describe how you've been isolating them? I usually try to start 5-10 cultures in test tubes at once in the event that most do not take to the media or get stuck somewhere and don't actually make it into the media.

Hi dear Matthew, the genera like anabaena, osillatoria, nostoc, and more, but when I culture them in media with agar they aren"n grow, but they grow well in lidqid media, I want to isolate every species alone or to get pure culture of it.

By invert microscopy and your glass made tiny loop, you can isolate your filamentous cyanobacteria.

Hi Lana Ezzdeen

I would suggest that you use the single cell (or filament) technique to start, in liquid media. This medium can be BG-11, ASM-1, WC medium, among other options. I mostly used WC medium for freshwater species (Guillard RRL, Lorenzen CJ (1972) Yellow-green algae with chlorophyllide C 2. J Phycol 8:10–14 doi:https://doi.org/10.1111/j.1529-8817.1972.tb03995.x).

This is the way I used to do it:

• Under the microscope, dissecting microscope, or their inverted versions, use a sterile glass pipette to transfer a small volume of your sample containing your target organisms onto a sterile microscope slide (do not use slide covers)*.
• On that same slide (if you have the space in it) or on another slide, put a drop of newly prepared and sterile medium using another, sterile glass pipette.
• Then, try to very careful aspire into the pipette one filament of your organism at a time from that drop of your sample and transfer it to the drop of the medium. Get a good number of filaments.
• Now, transfer only one filament to a second drop of fresh and sterile medium.
• Check if all looks fine (i.e., no major cell damage) and if you didn't transfer other cells. If there are other organisms in there, repeat the procedure transferring the target organisms to yet another drop of your medium until you have only one healthy filament in the drop.
• Aspire the now isolated filament and transfer to a test tube previously filled with sterile liquid medium (about 10mL in a tube of approximately 20 ml of volume).
• For every organisms you are trying to isolate and culture, you should ideally have between 5 and 10 tubes. Sometimes is very hard to grow cyanobacteria in the lab, so it pays off to have several tubes and test different media, maybe with the different media having 3 or 4 tubes each.

It may not be feasible at the moment, but if you want to obtain monoalgal and axenic cultures contact the Phycology Laboratory at the Federal University of Sao Carlos. They have an impressive freshwater microalgae culture collection and they might be able to ship it to you (CCMA-UFSCar).

I hope that those tips help you isolate your organisms!

All the best,

Guilherme

*We used hydrophilic cotton on the bottom of the pipette to prevent contamination, and also adapted a silicon tube that we would connect to the pipette and would suck with our mouths, with hydrophilic cotton on the "mouth" end. Sounds like it doesn't work, but it is super effective and we didn't have problems with contamination, not even with bacterial contamination when working with axenic cultures.

Thank you dear Guilherme for your tips, I will fellow your tips to get pure filamentous isolates.