I have tried different enrichment and isolation methods. I have also used cereal grass medium recommended for protozoans. But even after a number of successive shifts I am unable to get a pure culture, there is always a complex mixture of different protozoans.
How about Using dilution-plate method? Or you may have a try picking the single protozoan under microscopy and then enrich it?
it is much difficult to maintain the culture pure specially for endosymbiotic protozoa because they mostly carry fungul , bacterial or other protozoa contamination as food, or due to poor separation techniques and specially if you are culturing the protozoa which are parasitic in nature, thn your oculm should be pure to get pure culture and it will take bit more dilution because some protozoan species also use other protozoan as thier food, if u keep it for some days then that protozoa at the top of the food chain will become the most common but will quickly die off as the food supply is exhausted. for Paramecium and amoeba etc, can be get in pure cultur aftr 4 to 5 days by hay infusion methodology.
For the free-living amoebae isolation you can try to grow them on non-nutrient agar plates (0.5% NaCl, 1.5% Agar) adding first 100 microliters of fresh (or heat killed) E. coli. I don't know how do you maintain your samples now but depending on the concentration you can start adding around 10 microliters drops in different places of the NNA plates. Incubating them for 3-5 days you should see that some amoebae already moved from the spot and started to graze the E. coli. Then you can try to isolate them with a loop or a steril toothpick and strike them again on a NNA plates.
On the other hand what you can also do a dilution serie of your sample and transfer around 100 microliters on NNA plates. Incubating them for 3-5 days you should see that some amoebae already moved from the spot and started to graze the E. coli. Then you can try to isolate them with a loop or a steril toothpick and strike them again on a NNA plates.
Good luck!!
The method can done in two parts by 1. Using Water sample nutrient material with antibiotics added to delay or inhibit the growth and then doing the final purification of the culture by allowing the protozoa to swim through a migration tube.
Hope this helps.
You may use non-nutrient amoeba saline agar(NN).It has to be prepared according to recommendations of Page (1988) and Kalinina and Page(1992).It is used for primary isolation and also for subculturing of amoeba.
Other non-free living protozoans can be "purified" using percoll gradients. It will depend on the purpose of your studies, and if you pretend to keep them alive.
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