In parallel with a project I am currently working on ("BiodiverCité", the inventory and characterization of the wetlands of the urban area of Bordeaux in France), partners will have to opportunity to collect samples of water over a few dozen streams carefully selected. While they are interested in the hydrological and chemical characteristics of these rivers and their associated fauna, I had the idea to take advantage of this sampling campaign to focus on the microflora and, later, possibily establish a link between the different data produced.
I am interested in two types of microflora : on the one hand, bacteria, archaea and other unicellular organisms (but mainly bacteria I must admit) and, on the other hand, the viral microflora.
Concerning the bacterial flora, I will unfortunately exclude any pasteurian approach, because of lack of time and lack of means.
I started with the idea of centrifuging the collected water samples and carrying out a DNA extraction which would join a DNA library to be analyzed once the financial means have been collected.
For the viral flora, I thought to carry out a precipitation of 1M NaCl, PEG6000 10% on the supernatant to concentrate the viruses in a volume easily handled and storable to have a phagolibrary in which to possibly find phages directed against bacterial species of interest (eg F. psychrophilum or other pathogenic species in aquaculture) and secondly, carry out a phage nucleic acid extraction to constitute a phagoDNAlibrary exploitable in the longer term.
My questions are:
1) What volume of sample should be collected to have enough bacterial DNA to make metabarcoding ? For the soil, the specialists of Genosol (INRA, France) urge me to extract from at least 500 mg of soil for bacterial and 1 g for fungal. But for water ?
2) Is the NaCl / PEG6000 precipitation suitable for a long-term phago-library ? I fear that this step is too brutal for some phages and that even if I concentrate theoretically in number of viral particles, I lose in infectivity (due to damage to viral fibrils). If this technique is suitable, is the use of PEG8000 better recommended to ensure a wider enrichment spectrum (for podoviridae for example, more difficult to precipitate if I understand my literature) ?
3) I know there are publications on viromics but I have not looked into detail: what minimum sample volume (before precipitation) is desirable to have enough DNA for metagenomics (or just qPCR detection of well known virus) ? 200 ml ? 1L ? 10L ??? (2 ml :) ?)
Thank you for your attention.
Hi Guiraud,
About sampling water microbial communities I would suggest you to have a look at Ocean Sampling Day protocols and recommendations (this focus on seawater communities, but the same principles could be applied to freshwater; link: https://www.microb3.eu/sites/default/files/deliverables/MB3_D4_3_PU.pdf). As it was mentioned before, regarding the volume of water to sample properly microbial communities, it depends on several factors. For instance, usually, if you collect water from either highly eutrophicated water bodies, or with high levels of primary producers, such as microalgae, cyanobacteria, you will not need so much water to sample microbial communities, maybe one/two liter(s) could be enough. However, if you sample oligotrophic water bodies, under darkness (e.g., caves), you probably will need much more volume of water to get enough biomass to extract DNA representative of microbial communities. I believe that wetlands fits more the former scenario. Nevertheless, you should keep in mind that high number of cyanobacteria or micro algae as well as organics (matter, in case of eutrophication) represents obstacles in the purification and quality of DNA.
I hope this helps.
Best Regards,
António
Thank you for your answers (and link), now I have a more precise idea of the volume I need to sample (I think I will collect 1 liter or 2 x 1 liter).
I must add some details :
The main part of the project should not initially consider microbial ecology of these water bodies. We don't have money or workforce dedicated for complete analysis ie. culturomics, identification and characterisation of the bacteria we could find in these spots. And the corollary is that we don't have the means to isolate phages targeting these bacteria.
But all of these water bodies and the lands surrounding them will be analysed at the level of the flora, the fauna, the soil properties (including microbial communities via metabarcoding), the possible disturbations (eutrophisation, contaminations, etc.), etc. Some of them will be analysed in different times. A lot of data will be producted by the different teams involved in this project. I think it's a opportunity more interesting to collect some samples in this campaign rather than build another project fully oriented toward ecological microbiology but with probably less diverse information. The isolation of fresh bacteria (and their phages) will be sacrified on the altar of feasability.
Sampling will be done progressively and we have equipment to centrifugate relatively high volumes at sufficiently high speed (we can also use the filtration option...). This timing doesn't seem to endanger my main activities, this is why I considered to collect the pellets for a subsequent analysis of microbial communities and the supernatants in order to precipitate the phages which have the nice capacity to keep their infectivity on the long term at 4 °C. If I don't intend (with regrets...) to characterise the homologous pathosystems possibly observable in these water bodies, these phages samples will be used in a monitoring concerning some pathogenic species we are working on. The use of these phage samples for viromics is only a hope but I would like to ensure to not be limitated by unsufficient phagic DNA concentration.
Ali, do you think there is more phages in the infected bacteria than in supernatant ? Maybe I could use PEG/NaCl precipitation on a crude water sample (rather than on the supernatant) to pull down bacteria and free phages. In the same thematic, I read somewhere (Article Real-time PCR provides improved detection and titer determin...
) that we can add NaCl (1M final) in a crude water sample in order to take off phages adsorbed on organic particles and rise the phages titer. I wonder also if I could use some chloroform to release phages from these infected bacteria before precipitation. Tell me if I said a stupid thing...Antonio, do you think DNA extraction from eutrophic water is more difficult than DNA extraction from soil ? Because of polysaccharides ? In this case, I hope that my experience in plant science will help (I found that strawberry leaf, full of polysaccharides and polyphenols, is one of the most recalcitrant matrix I met).
Thank you again,
Thomas
Hi Thomas,
Maybe you can consider sampling sediments, as well.
Sediments from the bottom of the streams are suitable places for microbiota sticking.
In water and in sediments, you need to consider a mixed sample, with at least eight sampling sites for every stream reach. How are you defining the stream reaches? How long are they? Fluvial geomorphologists use 10 to 12 times stream-width to determine a stream reach for sampling.
Please tell us about the results and sampling design.
Best,
It is also recommended to consult with experts in molecular biology and virology for specific guidance on the best methods and protocols to use for your project.
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