We have a task for water samples analysis and need to concentrate /isolate all groups of plant pathogens separately for PCR from the same sample. So far, we apply 3 rounds of filtration. Is there any other options?
Hi Alex! For Phytophthora ramorum, water was filtered through a Buchner funnel with Whatman filter paper and the filter was then plated on selective medium. The medium would only select for oomycetes, however. The filter paper could also be tested by PCR to test for whatever organisms you desired.
Dear Alex,
I am now working on marine viruses. To concentrate viruses and to prepare VCs (viral concentrates), we use TFF columns.
Best regards,
Alireza
Alireza, thank you! we use TFF for bacteriophage concentration, I will try for plant viruses...
Alex
Dear Alex Ignatov
I am not currently working on water viruses
Pls. find the attached files https://www.researchgate.net/publication/269697066_Efficient_purification_and_concentration_of_viruses_from_a_large_body_of_high_turbidity_seawater https://www.sciencedirect.com/science/article/pii/S2215016114000119 http://www.annualreviews.org/doi/full/10.1146/annurev-marine-120709-142805 http://aem.asm.org/content/67/3/1384.full
Hoping this will be helpful to your project, good luck
Best regards
Prof. Houda Kawas
Hi Alex! For Phytophthora ramorum, water was filtered through a Buchner funnel with Whatman filter paper and the filter was then plated on selective medium. The medium would only select for oomycetes, however. The filter paper could also be tested by PCR to test for whatever organisms you desired.
Nina, thank you a lot! Do you think that use of other filters will help with fungi and bacteria? Some recommends 1.2-0.65 mkm membrane filters for fungi and 0.46 mkm for bacteria...
Alex
Here's a good citation (RGate won't let me paste, so this paper cites the others):
https//doi.org/10.1094/PDIS-11-15-1380-RE
A very effective method for collecting, isolating and enumerating oomycete and fungal CFU from water samples is to pass (approx. 1 litre) samples through 3 micron cellulose nitrate membranes (Whatman, diameter 47mm) using a vacuum pump. Nalgene produce a good bottle top filter unit that supports these ,membranes and sits nicely on a 1 litre duran bottle. Once sample has been filtered, lift the membrane out of the funnel with sterile forceps and place in 5 ml of resuspension medium (= dilute agar! about 0.1% w/v) and shake thoroughly. Depending on what you wish to isolate, antibiotics can be added at this stage and aliquots (I usually use 0.5ml for oomycetes) are spread onto appropriate antibiotic agar and left to dry down/soak in with lids off in flow hood for about 5 minutes then incubated at appropriate temperature and time (depending on target, but generally about 36 h) the count and identify/sub the colonies. Check out
BÜTTNER, C, BANDTE, M, PETTITT, TR. (2014). Filtration and centrifugation for detection of plant pathogens in irrigation water. In: Biology, Detection and Management of Plant Pathogens in Irrigation Water. Eds. Hong, CX, Moorman, GW, Wohanka, W, Büttner, C. American Phytopathological Society, St Paul, Minnesota, USA. pp. 139-148.
I can send a pdf if you have difficulty getting hold of this. Happy hunting! Cheers TimP
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