We tried several times to make petri dishes cultures isolation techniques, flat and inclined agar methods, and still appears 2 strains in 16s pcr. We want to contract a service of strain isolation fast and reliable to deposit as soon as possible.
Any suggestions?. Thanks
Antonio,
What is the originating source of the anaerobic strain? You may be faced with a codependency problem where one microbe is dependent upon the other for survival.
Or cultivate on different antibiotics. If one of the strain can make spontaneous mutants, you ll get a pure culture, as probability that both strain make a spontaneous mutant on the same antibiotic at the same location on the petri dish is null.
Have you observed them under the microscope? Do they aggregate?
Did you try to make dilutions to extinction?
In any case, it is known that most bacteria have multiple copies of the rRNA genes and in some cases some of the copies within the same genome can have pairwise similarities below 97% (see linked paper)
Article The Variability of the 16S rRNA Gene in Bacterial Genomes an...
Can you give more information about the medium, the substrate, terminal electron acceptor used etc and Which anaerobic strain do you try to isolate?
I just know company in Europe, for example DSMZ.
https://www.dsmz.de/
Not sure they offer the service though. You could try to contact them anyway.
On the methodological point of view, I could suggest to try a dilution to extinction approach. There are many really nice paper on that.
Thank you everyone for your comments.! . Noticing that is not available an outsourcing isolation service, We will try like this. 1) Use another substrate. Gelatin, agarose or geltrix. and 2) Limiting dilution until extinction. Would be ideal for us to use FACS or optical tweezers to catch just one, because one is a rod and the contaminant is a coco, and we are thinking a kind of symbiosis. We asked to DSMZ but they do not offer the service, neither ATCC. An also I can not reveal more information about the strain because is under patenting process. I will tell you later how was our experience. Regards.
I agree with the answer of Dr. Adward related to codependency. You can try dilution to the extinction method and try to isolate the strain from different dilutions. It might be possible that you can get single strain at highest dilutions with maximum number of cells . Also vortex the Hungate tube or other culture tubes vigorously to separate the cells from one another before plating.
Dear Antonio, I spent quite some time in isolation of anaerobic microorganisms. Could you provide more details about what kind of microorganisms you are working with?
With best regards, Alexander.
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