I would tend to make sure the culture is pure before storing in 20% glycerol in cryotubes especially manufactured for such storage. I would avoid too many subcultures and would make as thick a suspension as possible would also recommend suspending the pure culture in PBS not water, to avoid osmotic shock, and leave the cultures in the fridge overnight before transferring to -80 to avoid freezer shock. Of course, 2-3 replicates in separate freezers would be ideal.
@Desmond - can you please make yourself clear what you mean by pure culture, if the question mentioned about the microbiome?
Well, it all depends on whether you are looking at the microbiome as a whole and go down the genetic route and doing a DNA extraction of the whole root microbiome. If THAT is the case, then just extract the DNA (there are oodles of kits out there specifically for that purpose), and store the extracted DNA at -80 before using specific probes for specific genes present in the root microbiome. eg N-fixation, P-solubilisation, and so forth. Obviously use fresh DNA for PCR work and use stored DNA for sequencing work.
99.9999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999% (whew) is naturally unculturable, but if you are looking specifically for microbes that can be cultured, transferred to edible crops and seed-borne, so that you know the (potential) endophyte is durable, transferable and stable, then by all means do it as I said. You could also compare culturable vs non-culturable microbes by simply placing a known weight of soil in PBS, doing serial dilutions and then plating it out, looking for the interesting ones which produce IAAs, phosphatases, involved in N-fixation, and so on and so forth.
Hopefully this is a clear as mud.
Is there any method to store the soil microbiome as a whole without the extraction of DNA ie is there any method ?
The simplest way would be to take a known amount of soil and put it into a liquid broth and let it grow. There are specific pieces of glassware out there that can circulate media through the soil, let it collect and with the use of a fish pump and sterile filter, you can be relatively confident that what grows came from the soil. Now whether you 'bother' to separate bulk soil from root soil from rhizoplane from rhizosphere soil is completely up to you, as well as what you grow it in. I'd go for rich media for the easy-growers, to minimalist media for those that can grow on the sniff of an oily rag. For THOSE microbes, if the liquid is cloudy, centrifuge it, resuspend in minimalist PBS, make multiple backups and store at RT, 4 degrees and -80 degrees. Then have fun playing around with types, numbers, and so forth. Eventually you WILL have to go down the DNA route to get a full appreciation of the families/genus/species that are present and thew way the community gels together. Don't forget to use fungal media as well with antibiotics to knock off the bacteria, and as soon as you get hyphal growth, do single-tip inoculations to get pure cultures and put them into -80 as soon as you can, ,again with multiple backups (-80C, 20% sterile glycerol). Similarly with the microbe slurry: PBS & 20 % glycerol.
Desmond Phillip Felix Auer sir do you have any citation for the above method?
Dear Shubham,
No references per se: this is decades of experience. Look up hydrocarbon-ultilising bacteria for the bulking of those types of organisms if you're interested in those... essentially you inoculate a minimal salts media with about 5 grams of soil and add 1-2 drops of your relevant hydrocarbon. Once cloudy, shift to a solid MSM and seal in a container with 1-2 drops of the same hydrocarbon.... If you get colonies, definitely put them away at -80C in 20% glycerol as soon as possible, since these organisms tend to have these sorts of hydrocarbon-degrading genes in plasmids, not in the chomosome, so you have to keep them under constant hydrocarbon pressure.
Otherwise (remembering that you are ONLY capturing the culturable microbes) serial dilutions and pick out morphologically different colonies and resub to ensure purity. Again, get them stored in -80 asap (with backups) then have fun doing the physiological stuff: phosphatases, IAA's, nitrogen-fixers, and so on and so forth. The big test is when you do seed inoculations and get the plants to seed and then see if you re-isolate your organism... congratulations, you have a seed-borne endophyte!
https://www.frontiersin.org/articles/10.3389/fmicb.2017.00849/full
Article Storage of soil microbiome for application in sustainable ag...
Article Investigating the Impact of Storage Conditions on Microbial ...
hope above mention papers will help you out.
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