I would like to separate from the mixture contains above three algae for the calculaton of individual growth rate and the microalgae separation method is much more essential for my work. N.B. Mixture media aquous.
Regards, Mr. Ghosh.
I don´t know exactly the growth rate of these microalgae but if this growth rate is good in universal medium for Cyanobacteria (BG-11, i. e) for Phormidium l. and Gloeothece r. you can have a partial selection (something like an enrichment culture) and later with a good cell density you can count each and perform serial dilutions of the culture to obtain 1 cell/volume unit in same way with a universal medium for Eukaryotic algae in the case of Chlorococcum i.
Another way is dispose an microtiter plate, invert microscope and pasteur pipettes with elongated tip in flame (like a capilar) and try to take only one cell from cell culture with the capilar and dispense in other well check that this is clean of other cell types or foreign objects, this process is repeated as many times as necessary in order to obtain 10 wells with one cell for each specie (separately, obviusly). Later take the cell and place in eppendorf tube with 100-200 microliter of media and wait, expect to grow. You can 1-10 scale cultivation, ie, next reseeding should be 200 microliters at 2 mL, next 2 mL of media in 20 mL ... This process and other isolations techniques is present in the attached book pp. 83-101 and 117-133
Best regards,
Gustavo.
Have you think about differential centrifugation? If the three different algae have a sufficient different density or size, you could separate your species. Another way could be to dilute your suspension and spread it on a petri dish containing generic medium (like BG-11 maybe?) with 1.75% of agar.
Growing them on selective media, size partitioning by sieve and then isolation by micropipette (like described in above mentioned book Algae culturing techniques)
In case you have access to a flowcytometer with sorting option, that would also be a method to test.
It is possible to separate cells with different densities using Percoll or Ficoll gradients. You need a good quality centrifuge and swing bucket rotors. Warning: it takes a lot of time and fiddling around to get it to work and making up the gradients manually is absolute torture. If you can find or buy a gradient maker that is fine.
your three algae have quite different morphologies and growth dynamics. I would use streak plating on solidified agar medium since Phormidium filaments will easily grow on such a substrate, and the other two algae will form colonies that you will be able to separate under a dissection microscope with the aid of a 26 gauge needle. Or you can seed a microvolume of your mixed sample in a Petri dish and then add melted agarized medium (take care of the temperature of course!). Thh three algae are well protected by wall and mucilagines, hence they will growth in the agar and come to the surface allowing you to pick them up with a needle.
The easiest way is through picking and choosing colonies or filaments from agar plates of mixed inoculum. Do spread plating or pour plating with your mixed inoculum in BG11 agar plates. Observe your plates frequently, i.e. once or twice a day. Different algae will appear gradually in succession on your plate. Each time you pick a colony or filaments from the plate, observe under microscope and then subculture it immediately onto etiher BG11 or any other selective media plates. Also, initially If you inoculate your mixed inoculum in broth, you can easily pick phormidium from the walls of your culture vessel, as soon as they appear and subculture. However for distinguishing Gloethece and Chlorococcum, I feel you need to inoculate them on agar plates as mentioned earlier.
However, if you have a microtiter pasteur pipette assembly, the best way will be to pick and choose with the help of that. Also, density gradient or differential centrifugation is a good option. However, whichever process you follow, you have to be really careful about cross contamination.