I performed plate counts (soft agar, 0.5%) for microaerophilic bacteria in some unknown soil samples. I would like to identify some of the bacterial strains. Can anyone recommend a good protocol for the isolation of colonies grown on such plates, in similar condition? Thanks.
Hello,
create micro-aerobic condition in your cultivation environment and your microaerophilic bacteria would grow.
Candles jarrs are a good option for growing microaerophilic microorganisms. In those conditions you will be able to use a regular agar concentration and colonies will be formed onto the surface, easily managable. If yoy don´t have a candle jarr you can impovise one with any hermetic (or semi hermetic) recipient like a pressure pot. if the recipient is not hermetic after you close it with the lighted candle and the plates you can seal it with parafilm or any other film.
I am not sure if I understood the question correctly, but it seems to me you are asking, how to get single, pure colonies from your enrichment rather than asking about the microaerophilic technique.
For the later it is quite obvious to use anaerobic jars; as mentioned candle jars will do for most of the strains.
Separation of pure colonies from soft agar is much trickier. Not being able to streak will leave you with doing a dilution series until distinction to single out your colonies (e. g within microtiter plates). However, doing so will return only the most prominent members of your enrichment. To recover also less dominant members of your community, maybe a technique published by Goodman et al. (2011: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3076821/) would help, which makes use of the underlying Poisson distribution of your population. They distributed their culture/sample onto several mirotiter plates, estimating (and validating) that due to a Poisson distribution one would expect single cells in about 25% of wells if using a sample dilution which gives no growth in about 70% of inoculated wells (the remaining would be mixed cultures). Those single cells can then easily be enriched from the single wells.
Thank you so much for all answers. I'm sorry, I was not clear enough in my question. I was talking about single, pure colonies isolation, as D. Taras said in the answer!!
The methods described earlier are useful.The best method now is to grow your culture in semisolid medium in agar tubes (soft agar) then you can use those in the middle layers which should be microaerophilic. best of luck
Hello,
You can use the anaerobic Jarrs, it is perfect for this test.
good luck!
My experience is on Campylobacter from diarrheatical samples. I use a selective antibiotic media. Incubation in an anaerobic jar with microaerophilic pouch. Alternatively, the extinction flame technique is also useful
Javier J. Flores-Abuxapqui By "extinction flame technique" do you simply mean leaving a lit candle in the jar till it cannot burn any more?
Hi Leonid, extintion flame technique it is just that, you light on the candle inside the jarr or recipient and then close it tight and leave the flame consume oxigen until extintion...
Quite simple, its works fine, just need a glass jar wide enough to get BA plates thru the top
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms
- Bacteria