I have a very promising consortium, which is able to grow almost in pure oil and degrade it as well. I want to isolate cells for further analysis and sequencing, but there is a problem. When I am trying to centrifuge the medium, nothing is happening (no pellet is visible, only some scums).
Does anyone have any experience with such samples and methods of isolation?
Dear Tomas,
It depends on the bacterial strain that you want to isolate from oil. A specific strain is grown to its own culture medium. If you write me which bacterium is, I can suggest you an isolation method.
Just a crazy idea, but perhaps you can try some classical phase separation method: When you have your Oil with the bacteria, try to add some water, mix the two solutions and then let the water settle. Then take off the oil and spin down the water fraction and check if you got a pellet.
Best regards.
Hi. Michael Nikolaou is correct but if you want to do direct isolation so I think you can try magnetic antibodies to isolate your bacterial from oil. first find a polyclonal antibody that can bind to your bacterial cell wall then use second monoclonal magnetic antibody to bind to the polyclonal antibody and finally isolate your interest bacteria.
Dear Michael Nikolaou and Mohsen Ashrafi, at the beginning I want to isolate whole bacteria population for sequencing (I don't need them grow at the beginnind) and investigate what kinds of species are abe to live there. Then, I will choose a particular species for further studies.
Thank you for your help.
Maybe you can try filtering bacteria on 0.2 or 0.45 micron membrane with strong vacuum. And then do DNA extraction from the membrane by bead-beating method.
Yes Tomasz i find i think this articles may be helpful
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0CCcQFjAB&url=http%3A%2F%2Fwww.researchgate.net%2Fprofile%2FAsif_Muhammad%2Fpublication%2F46028221_Extraction_and_Characterization_of_Oil_Degrading_Bacteria%2Flinks%2F0fcfd510c317216ae5000000&ei=VLcbVKrgNpHHuAStxID4AQ&usg=AFQjCNEKPaSaU6Zys12gV7Ph90jOXkfqdQ&bvm=bv.75774317,d.c2E
http://www.hindawi.com/journals/ijmicro/2012/156537/
As alexander suggested, take 50 microlitre of oil (source of bacteria) add 100 mu l of milliq, and do a vortex, and just go ahead with Qiagen DNA extraction kit, you will get your bacterial DNA for sure, but you may need vigorous purification step to remove oil from the extraction protocol.
As Daniel said use a filter.
Agree with mr kurian and mr daniel, use a filter (like capsulated or cartridge filter) for oil. Using aseptic method, place the filter or swab sampling to general media like nutrient agar or blood agar and then incubated 37 degree celcius for 24-48 hours. You can choose aerob or anaerob condition too. If any bacteria grow the next step is to identification what kinds of bacteria. After that, you can purify to selection particular bacteria.
I also agree with the filtering part, but if you want to find out what is in the oil it might be better to filter more oil instead of using a culture medium.Some bacteria might not grow on the culture medium but on oil.
Several sugestions are good, but thinking about filtration and considering you wnt all possible microorganisms, think abou the limitation of the pore size!
Hi!
For filtration consider using serial dilution method and filter each dilution separatly you will get more results; and by the way do not forget using PTF filters instead of cellulose based filters
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