I am conducting a trial with a lot of plants inoculated with endomycorrhiza for testing the plant growth promotion effect. The day I have to sample the plants will be too busy to stain the roots right after the sampling for checking the colonization, so I was wondering how to store the roots before the staining, and if this storage could affect the subsequent staining, specially for the arbuscles.
Thanks in advance.
Hello Inaki,
You can store the roots in Formalin–acetic acid–alcohol (FAA). You can keep them in FAA during one year before staining. Be careful, FAA is volatile and potentially noxious. You should work in a ventilated place.
Regards
Hi Inaki,
if you are going to stain roots the day after you collect it, you can also store it in distilled water at +4°C for no more than 24 hours. I had no differences between roots stained soon after collection or after a 24H at +4°C.
If you are going to stain it several days after the collection, so use the protocol suggested by Pablo Adrián García Parisi.
Regards.
Hi Inaki,
if you cool the root samples you want to use for staining (and maybe further for molecular analysis) immediately in liquid N2 or if not possible on dry ice for the transport and afterwards at -80°C, you can store the roots for some days. A staining f.e. with Trypan blue should work.
Good luck
Hi Inaki,
protocols of Pablo Adrián García Parisi, Hani Antoun are well described. You may also store the roots in 50% alcohol until staining. It is not noxious. Wash well before staining.
I think the best way is in alcohol 50% until staining. I fully agree with Partha Pratim Dhar. It is quick and safe. I didn't have problems with this method. Good luck!!
Hi inaki,
You can keep the roots in 50% ethanol at 4°C during 6 Month.
Good luck with your work.
I use 10% KOH; this clears the root sections in 2-5 days (depending on the roots), and you can keep them in the solution for a few days longer, before staining.
The best method is use water to clean the roots and than store at 5C.... .............
But i suggest not store roots segments for longer period of time unless you are very far from your laboratory.. usually the same day you need to processed the samples...this will help in recovering some AM species ..rather than storing the roots for longer time and affecting the viability of rare AM species.... ...Some times suppose you working some rare endemic samples, amount of root samples will less and so u also require some root samples to use for trap culture..so you are sure ... some rare or specific AM species you might recovered trapping them...rather than just processing root for colonization procedure. Half root samples should be use for trap culture and remaining one should use for Colonization process,, In this way you know you can recover some AM species and Processed root samples also......
Thank you very much for all your answers.
Because of the safety and easiness I will go for 70% ethanol. I think that to use KOH could also be useful but I already have set a protocol for this clarifying/staining steps (Trypan blue staining) to get the best results for the subsequent visualization under the microscope.
I was wondering if I should replace the ethanol with water before proceeding with the KOH and other steps, because it may for instance affect in some way to the effectiveness of the stain itself... Any suggestion for this?
You could rinse the roots in distilled H2O, using a fine sieve under the running water in case you lose any of the roots. They can then be placed in an appropriate flask, covered with trypan blue, and left to stain overnight.
I normally tip the ethanol out and leave opened for a while to evaporate the rest of the ethanol. Anything remaining is water.
Helpful :)
I will do the 50% ethanol + 50% distilled water at 4°C storage as suggested, for 5 months.
I will come back to share the outcome.
Thanks
What I am not seeing here is drying the roots (which is done for DNA preservation). I tried this for PRLC and all my samples are coming up blank. I think it made it so that I had to clear them longer with KOH which might have destroyed fungal tissue. Not really sure though.
yes Chris. Longer treatment with KOH Soln may cause such degradation of root.
I need to clear some rice roots, preserved in 70% ethanol, I am using 10% KOH overnight soaking and then heating at 90 degree for 15 to 20 minutes. I am using 10% sheaffer black ink for staining. But in some case the thin roots are degreded but not cleared and the thick roots are not cleared as well. Could anyone suggest me anything which might be helpful?
hi i think you can store the roots at a temperature of 4 degrees or you can use glycerol to preserve them
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