Is there a consensus on the optimal concentration of glutaraldehyde for the preservation of marine phytoplankton? I typically use 1%, but notice that other researchers use from 0.1% to 3%.
I am interested in maintaining as many taxonomic features as possible for LM and SEM with the lowest concentration of glutaraldehyde that is acceptable, since I don't like working with this chemical at sea. I typically work in low chlorophyll high nutrient areas of the Southern Ocean, but may find sea-ice blooms from time to time.
I appreciate you thoughts and recommendations :).
It's to preserve cells on a mooring up to twelve months. We can't use acid Lugol's and alkaline Lugols is generally only good for about 3 months. For sure, 0.25% glut does not do the job. We have made that mistake. The other consideration is that as the volume increases there is less room for sample.
HI.
usually working stock of 10% then between 1% and 2% is where I have usually gone. I have a paper which I used as a good reference for marine samples, ya no lugols, as the colour issue and also the degradation with diatom frustules getting broken down. My lowest would be 1% pretty smelly still, but I make it on board ship in fume hood then you can pre-spike vials keep it in the fridge and then out samples back into the fridge after sample is added, I hope that helps? Ya more is better than less. Also remember that lugols does colour the organelles so harder to identify, much better to use glut.
I hope this helps, let me know if you need the paper?
Di
Thanks Diana and Diane:)
Appreciate you advice, yes Diane, always happy to read more on preservation, if you could send the paper that would be great.
Thinking of the Umitaka voyage here, (no way I'm changing the mooring config Di! )No fume onboard for handling glut as far as Im aware, but I could pre-decant before I go, that's a good idea.
Thanks Ettahiri
I loose some detail with Lugols as Diane pointed out , but its much safer to use at sea.
Many of my coastal samples are preserved in Lugols, but I typically get them processed faster than the Southern Ocean ones. Lots of compromises to be made!
yes look best practice would be make it fresh, but this stuff is super stable so you could make it before you travel (but transporting as a liquid can be tricky,) and pre spike vials and keep in fridge. then use on voyage only taking out just before you use. This is the original old school reference but I think always reliable, pg 72, in pdf, or from the pdf reader menu pg 82. Good luck with the trip.
Di
1% or 0.5% should do the trick. keep them in fridge after. wear proper gloves. Good luck :)
I typically use 1% and refrigerate immediately afterwards. You could always try wearing a mask to diminish the smell... Good luck and have fun! :-)
I generally use 0.25 and 0.5 % for marine and freshwater preservation of protists, Freshwater often needs buffering. No problems for ID by utermohl techniques. For thin sectioning I would use higher conc glutaraldehyde (1-2% for protists) for proper stabilisation. I'm not sure on what organisms Diana based her recommendation for higher concentration glutaraldehyde?
Diatom silica dissolves at 'natural' pH, hence recommendation for acidified lugols. With long term glutaraldehyde preservation I would acidify the samples after (!) initial preservation. Mind you, that will get rid of cocco's and other calcareous scaled organisms.
HTH, René
Hi Rene, Ya I think I mentioned in the beginning, the solution but may have forgotten, the pH of the filtered seawater is buffered and set to 7.5 before the glut is made up, glut sorts out the pH issue, and basically the seawater samples coastal and open ocean, which does have cocco's in there and also damaged diatoms, so therefore the 1% is needed to still preserve, these samples,. Also we run through flow cyt, for picoplankton so are a little more sensitive, hence the higher con. Ans yes I have run both acidified lugols and glutaraldehyde/or paraformaldehyde on the same samples from the same cruise, and compared both sets of samples, and there's a loss of cells due to the acid presence, hence my advice. Great discussion, on this definitely beats, burning through lots of old literature on your own.
Ha, thanks for the clarification Diane! Yes, buffering is not an issue for seawater. But be sure to acidify diatom reference samples for long term storage. I'm interested in your loss of cells with acidified lugols though. Wall-less thingies do funny things in Lugols, but even so often very characteristically (eg raphidophyceae). Some ciliates explode, ending up in blobs (Myrionecta). But generally I haven't got the feeling a lot of things disappear in Lugols. More so in formalin, and especially so in freshwater. Buffering in freshwater is obligatory, and often needs optimization for a certain group in order to appear life-like.
Thanks, best wishes,
René
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