[3dem] Precipitation observed after making freeze substitution cocktail containing 5% water

Wed Jan 15 21:36:59 PST 2020

Hi Miriam,

I do the same thing as Kim, putting the molecular sieve straight in the bottle with the solvent and only use the top to avoid picking up any particles. If it looks particularly dusty, I will wash it a couple of times beforehand using the solvent though.

All the best,
Chris

________________________________
From: Lucas Miriam Susanna <miriam.lucas at scopem.ethz.ch>
Sent: 15 January 2020 13:56
To: Reinhard Rachel <Reinhard.Rachel at biologie.uni-regensburg.de>
Cc: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>; Christopher Peddie <Christopher.Peddie at crick.ac.uk>; aparvate at lji.org <aparvate at lji.org>
Subject: AW: [3dem] Precipitation observed after making freeze substitution cocktail containing 5% water

Dear Reinhard,
dear All,

Thanks a lot for your comments!
Following up on this topic, I'd like to ask, if you are preparing the dry ethanol and/or acetone yourself, and if so - how do you do it?

We are using molecular sieve, which we wash (and store) in alcohol to remove abrasion/dust, then fill it into dialysis tubes (with clips to close both ends) and add this into the bottles in which the alcohol is supplied. We refill a bottle once. However, the handling of these dialysis-tubes is a pain and they tend to burst when introduced into the bottles.
Several suppliers sell extra-dry ethanol or acetone with molecular sieve floating freely in the bottles.

We had a discussion in the lab, weather abrasion dust from molecular sieve is transferred onto the samples, and weather this happens in an extent that it really influences the imaging, e.g. for critically-point dried samples, as well as for resin-embedded specimen.

What's your opinion?

Kind regards,
Miriam

---
Dr. Miriam Lucas
ETH Zürich | ScopeM - Scientific Center for Optical and Electron Microscopy
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-----Ursprüngliche Nachricht-----
Von: 3dem <3dem-bounces at ncmir.ucsd.edu> Im Auftrag von Reinhard Rachel
Gesendet: Freitag, 20. Dezember 2019 11:47
An: Christopher.Peddie at crick.ac.uk; aparvate at lji.org
Cc: 3dem at ncmir.ucsd.edu
Betreff: Re: [3dem] Precipitation observed after making freeze substitution cocktail containing 5% water

>>> 19.12.2019 at 20:41:
> I would agree with the 7-8% stock concentration for UA. Even at that
> concentration, it would take a while to dissolve in methanol.
> Additionally, I would filter it with a 0.22 micron filter, make
> aliquots and then freeze in LN2. Dont know if LN2 was overkill but i
> have never seen cloudy precipitation in my FS mixes and i was using 3%
> OS and 0.5% UA along with some TA and glutaraldehyde.

with some delay, here is a brief comment from my side.
some people do not mention the solvent, in their comments (or in the M&M in the papers).
in theory, you can push the stock solution in pure water to 7-8% , but ...
this depends whether you have CO2 free water (UO2 carbonate might precipitate, first!). The CO2 / carbonate anion is one of our enimies, in these solutions. Just keep this in mind.
Thus, you may boil Water (= get rid of CO2) before solving the UAc. Or use freshly distilled (!!) water. Yes, distilled, not the Millipore stuff: no, never. -- And if you like, you may add minute amounts = a mini drop of glacial acetic acid (which tries to keep the pH such that the CO2 is favored against the bicarbonate anion and the carbonate!)

and solubility depends on the temperature (dissociation constants depend on the temperature! physical chemistry).
Thus, I do not recommend to put such stock solutions into the cold room, below 10 C, or fridge.
Nor does it make sense to freeze them ... why? make small amounts and use them up. Freezing:  you enforce the stuff to precipitate ... and, for the solution at room temp.: If you know of a bug that starts growing in a stock solution of UAc 5% or higher, please let me know; I would be interested (I am also microbiologist).

we and others have tested: UAc dissolves nicely in MeOH, up to 20%. This does not last that long; I would make small amounts, only, which are needed within a week. - but this is easy to do.

Have fun, and to all of you:
merry Christmas - a happy New Year, and all the best!
Reinhard

--
Prof. Dr. Reinhard Rachel
University of Regensburg
Centre for EM / Anatomy
Faculty of Biology & Preclin. Med.
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D-93053 Regensburg - Germany
tel +49 941 943 -2837, -1720
mail reinhard.rachel at biologie.uni-regensburg.de
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