Hello all,
My question relates to pollen that have been collected by a Burkhard type pollensampler. I have access to a historic collection of daily samples dating back to 1969 (we have all pollen counts). We are currently investigating the possibilities to utilize this amazing record and find new trends that cannot be studie with Light Microscopy alone.
One idea is to try and perform metabarcoding/metagenomics on the samples. One possible issue is that the pollen have been stained in safranin (mounted in glycerin, gelatin and phenol). Does anyone know whether it would still be possible to extract DNA from these pollen?
Very curious to hear your thoughts,
Thanks a lot,
Marcel
I would caution against using the physical historical pollen slides for other than record keeping, especially if there is only one set of slides. Imagine if all the records of the actual counts were lost, and the slides have been destroyed in the process of metabarcoding, then you have no way of getting these historical records back.
If you are still eager to do this, then I'm not sure if it is possible regardless. In this recent study from the PollerGEN team (see Article Temperate airborne grass pollen defined by spatio-temporal s...
) we did what you suggested with grasses but using samples from Burkard Multivial Cyclones. The cyclones collects more material than a Burkard Spore trap, hence your material might be lower overall. Sure, metabarcoding can take low levels of DNA into account so this might not be an issue. Have a look at this paper for DNA extraction from samples collected from Burkard Spore Traps, seeArticle Efficient and sensitive identification and quantification of...
The question then becomes about DNA degradation rates. I am not sure about the rates in preserved pollen but I can imagine that if these slides have been stored in room temperature, within slides that might or might not be sealed properly, for a long time, that might degrade the DNA. Older slides are therefore more likely to have more degraded DNA than newer slides, and that might potentially skew your analysis.
I am however not knowledgeable enough to say how the staining chemicals might affect the pollen and DNA, especially after that long time. You might consider contacting either Caitlin Potter , Simon Creer or Georgina Lauren Brennan and ask their opinion on it.
Hi Carl,
Thanks so much for your answer @Carl!
I agree that this destructive method would not be the first choice normally, but in my project I also work on automatic pollen image detection and as such I will be making high-res multifocus digital scans of the whole microscopic slide to be studied. This will ensure we will not loose the 'raw data' completely.
Looking forward to hear other suggestions
Hi Marcel,
Just wondering whether you figured out the effect of safranin in DNA extraction. I'm also trying to extract from pollen samples mounted in microscope slides and stained with safranin, and any advice would be most helpful.
Thanks,
Mireia
Mireia Gómez-Gallego : we have not tested it yet but are currently setting up an experiment with some of our slides to try it. The concentration of the saffranin is very low (0,002%) so we hope that the effects will be manageble.
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