I work on symbiotic-nitrogen fixing Burkholderia (so there is no problem to isolate Burkholderia sp. from nodules). This time I would like to isolate bacteria of Burkholderia genus from rice rhizosphere and from seeds, where the bacterial diversity can be very high. I wondered if there exists a selective culture medium to get rid of the gram-positives bacteria, or even to specifically select Burkholderia, in order to avoid identification of all bacterial isolates.
A liquid medium containing 2% glucose, 0.1% magnesium chloride, 0.025% magnesium sulfate, 0.1% ammonium sulfate, 0.02% potassium chloride, and with 5 mM potassium phosphate (to induce the phosphatase) and selection of colonies able to solubilise inorganic phosphate on agar. Wish this could help. A paper by Rombola, dos Santos, Martins Pizauro will be published soon on BMC Research Notes, but it does not add much more about selection or screening tricks.
http://www.bd.com/europe/regulatory/Assets/IFU/HB/CE/PA/ES-PA-254481.pdf describe a medium
You can use PCAT medium to isolate Burkholderi, there is described how they used combined with a specific probe to study in Soil.
http://www.ncbi.nlm.nih.gov/pubmed/11566224
hai
currently no commercial media available for isolation of Burkholderia so u can try with media used by this paper
http://www.ajtmh.org/content/80/6/1023.long
Hi
I would not agree with the above suggestion that there are no commercial media for the B. cepacia complex. Apart from large literature on "in house" media, we have used Mast cepacia media for many years for environmental and clinical specimens. However, I would also check PUB MED as there might be other commercial media more suited to soil culture.
PCAT medium will work for field-isolated Burkholderia very well, but it is not the easiest medium to make because it has some unusual ingredients. We also include Congo Red in the medium to help differentiate Burkholderia from other bacteria (see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC242283/).
I use Burkholderia cepacia agar made by Oxoid (oxoid.com) for isolation of Burkholderia cepacia complex from sputum of patients suffering cystic fibrosis. They sell ready plate, agar base and selective supplement. It is rather expensive, but works well to supress growth of Pseudomonas sp. that frequently overgrows the slow-growing Burkholderia sp. in this type of samples. Of course, it does not supress the growth of yeasts and filamentous fungi - most probably you will need to add some antifungal agent when working with plants. Also, closely related Ralstonia sp. (formerly classified as Burkholderia) is able to grow there based on my experience. In addition, I have one patients who caries B. cenocepacia that for some reason never grows on this agar. Therefore, I would also suggest using more media with different mechanisms of selection if you want to be sure to catch all Burkholderia species.
John Govan's answer is correct (he will know better than anyone). We used this (MAST) media in the 1990s to isolate Burkholderia from soil, water and rhizosphere samples. It is not perfect but will work better than other media from our experience using 'spiked' soil samples. The caveat is confirming what you get is Burholderia as other 'pseudomonads' may grow too. I would recommend a molecular test (even 16S) as the metabolic diversity in these bugs makes biochemical methods less reliable.
I agree that ribotyping the isolates is critical. We have found a large number of bacteria in soil samples, and even on selective media, non-Burkholderia species will grow. PCAT (I have not tried MAST medium) plus congo red works fine, but we check everything we get using 16S primers to be absolutely sure. We have isolated B. cepacia from nodules, but I think that they are getting into the root tissue by "hitchhiking" in with either a nodulating Burkholderia or Rhizobium. No evidence for nod genes exists in B. cepacia.
I forgot to mention, our Mexican colleagues use N-free semisolid BAz medium and for agar cultures, BAc medium for isolation of N2-fixing Burkholderia species. see: http://www.ncbi.nlm.nih.gov/pubmed/11375196.
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