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

Wed Jan 15 12:13:13 PST 2020

Hi Miriam,Regarding your mail to the 3DEM list,I tend to avoid the use of molecular sieve in solvents for freeze substitution. (Fun fact, in the old book 'Crytechniques in biological electron microscopy' Hans Moor warns against the use of molecular sieve with freeze substitution) The main problem (besides the chance of small particles in your sample) is that any trace of the molecular sieve will directly react with any osmium used during the freeze substitution turning it black within a few hours. I've successfully  used glass distilled solvents (EMS), VWR anhydrous solvents and Merck seccosolv solvents (though the latter have been discontinued last year). I keep them dry by flushing the bottle with dry nitrogen gas and storing the bottle in a container with silica pearls under dry nitrogen (And all lids taped off with parafilm).Alternatively if you do not use water in the freeze-substitution mix you can also use acidified 2,2-dimethoxypropane (1-2 drops of 37% hcl in 100ml dimethoxypropane) and add 2 mg of this to 98ml 96% ethanol. Any water present will be converted to acetone  and methanol. In contrast to other methods of making dry ethanol this does'nt  react with osmium.If you're not using omium then the only issue will be the dust particles. You could opt for using the 'porous containers' EMS sells for critical point drying (catalog nr. 70187-20) instead of dialysis tubing.Best wishes!Rob---------Dr. Rob MesmanPost-DocDepartment of Microbiology (postbox. 72)
Institute for Water & Wetland Research
Faculty of Science
Radboud University
Heyendaalseweg 135
6525 AJ Nijmegen
The Netherlands
-------- Oorspronkelijk bericht --------Van: 3dem-request at ncmir.ucsd.edu Datum: 15-01-20  20:28  (GMT+01:00) Aan: 3dem at ncmir.ucsd.edu Onderwerp: 3dem Digest, Vol 149, Issue 14----------------------------------------------------------------------Message: 1Date: Wed, 15 Jan 2020 13:56:47 +0000From: Lucas Miriam Susanna <miriam.lucas at scopem.ethz.ch>To: Reinhard Rachel <Reinhard.Rachel at biologie.uni-regensburg.de>Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>Subject: Re: [3dem] Precipitation observed after making freeze	substitution cocktail containing 5% waterMessage-ID: <2781b74b66cb44f8901ffcdb6a0b7382 at scopem.ethz.ch>Content-Type: text/plain; charset="iso-8859-1"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 LucasETH Z?rich | ScopeM - Scientific Center for Optical and Electron MicroscopyHPM C57.2Otto-Stern-Weg 3CH-8093 ZurichSwitzerlandPhone:?? +41 44 633 4424Fax:???????? +41 44 633 1336miriam.lucas at scopem.ethz.chwww.scopem.ethz.ch-----Urspr?ngliche Nachricht-----Von: 3dem <3dem-bounces at ncmir.ucsd.edu> Im Auftrag von Reinhard RachelGesendet: Freitag, 20. Dezember 2019 11:47An: Christopher.Peddie at crick.ac.uk; aparvate at lji.orgCc: 3dem at ncmir.ucsd.eduBetreff: 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 RachelUniversity of RegensburgCentre for EM / AnatomyFaculty of Biology & Preclin. Med.Universitaetsstrasse 31D-93053 Regensburg - Germanytel +49 941 943 -2837, -1720mail reinhard.rachel at biologie.uni-regensburg.deoffice: VKL 3.1.29member of the IFSM boardNext microscopy conferences:- EMC2020 in Kopenhagen, 23.-28.8. 2020 (European conference)- MC2021 in Vienna (D-A-CH conference)- IMC20 in Busan, South Korea: Sept 25-30, 2022- next Microbiol. conferences:  VAAM 8.-11.03. 2020 Leipzig_______________________________________________3dem mailing list3dem at ncmir.ucsd.eduhttps://mail.ncmir.ucsd.edu/mailman/listinfo/3dem------------------------------Message: 2Date: Wed, 15 Jan 2020 19:27:59 +0000From: "Gibson, Kimberley" <kimberley.gibson at yale.edu>To: Lucas Miriam Susanna <miriam.lucas at scopem.ethz.ch>, Reinhard	Rachel <Reinhard.Rachel at biologie.uni-regensburg.de>Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>Subject: Re: [3dem] Precipitation observed after making freeze	substitution cocktail containing 5% waterMessage-ID:	<BN7PR08MB48848B3B8C3B2E465F316B0DEB370 at BN7PR08MB4884.namprd08.prod.outlook.com>	Content-Type: text/plain; charset="iso-8859-1"Hi Miriam,When using molecular sieves, we would mix them directly in a bottle with Ethanol (i.e. free floating). Once capped, invert/shake the bottle a few times to mix the sieves/Ethanol and let the Ethanol solution sit for at least a day or more before using it. The sieves and any particulate matter should settle to the bottom of the bottle. Afterwards, when pipetting or pouring the pure Ethanol, make sure to not invert or shake the bottle.I've never had any issues with contamination of molecular sieve dust in resin-embedded samples - which would have been very noticeable when sectioning with diamond knives.All the best,Kim________________________________From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Lucas Miriam Susanna <miriam.lucas at scopem.ethz.ch>Sent: Wednesday, January 15, 2020 08:56To: Reinhard Rachel <Reinhard.Rachel at biologie.uni-regensburg.de>Cc: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>Subject: Re: [3dem] Precipitation observed after making freeze substitution cocktail containing 5% waterDear 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 LucasETH Z?rich | ScopeM - Scientific Center for Optical and Electron MicroscopyHPM C57.2Otto-Stern-Weg 3CH-8093 ZurichSwitzerlandPhone:   +41 44 633 4424Fax:         +41 44 633 1336miriam.lucas at scopem.ethz.chhttps://nam05.safelinks.protection.outlook.com/?url=www.scopem.ethz.ch&data=02%7C01%7Ckimberley.gibson%40yale.edu%7Ca11d5d25595946e6f12708d799c2cb1f%7Cdd8cbebb21394df8b4114e3e87abeb5c%7C0%7C0%7C637146934251328073&sdata=CoIE%2BaE6JAoQixsthcSry7fIZEUVQnXp0sP9dvlO7lY%3D&reserved=0-----Urspr?ngliche Nachricht-----Von: 3dem <3dem-bounces at ncmir.ucsd.edu> Im Auftrag von Reinhard RachelGesendet: Freitag, 20. Dezember 2019 11:47An: Christopher.Peddie at crick.ac.uk; aparvate at lji.orgCc: 3dem at ncmir.ucsd.eduBetreff: 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 RachelUniversity of RegensburgCentre for EM / AnatomyFaculty of Biology & Preclin. Med.Universitaetsstrasse 31D-93053 Regensburg - Germanytel +49 941 943 -2837, -1720mail reinhard.rachel at biologie.uni-regensburg.deoffice: VKL 3.1.29member of the IFSM boardNext microscopy conferences:- EMC2020 in Kopenhagen, 23.-28.8. 2020 (European conference)- MC2021 in Vienna (D-A-CH conference)- IMC20 in Busan, South Korea: Sept 25-30, 2022- next Microbiol. conferences:  VAAM 8.-11.03. 2020 Leipzig_______________________________________________3dem mailing list3dem at ncmir.ucsd.eduhttps://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fmail.ncmir.ucsd.edu%2Fmailman%2Flistinfo%2F3dem&data=02%7C01%7Ckimberley.gibson%40yale.edu%7Ca11d5d25595946e6f12708d799c2cb1f%7Cdd8cbebb21394df8b4114e3e87abeb5c%7C0%7C0%7C637146934251328073&sdata=bOylnOfiC9sJCCqs47fQjveeWZJVVfQgS8jOuBn0DH0%3D&reserved=0_______________________________________________3dem mailing list3dem at ncmir.ucsd.eduhttps://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fmail.ncmir.ucsd.edu%2Fmailman%2Flistinfo%2F3dem&data=02%7C01%7Ckimberley.gibson%40yale.edu%7Ca11d5d25595946e6f12708d799c2cb1f%7Cdd8cbebb21394df8b4114e3e87abeb5c%7C0%7C0%7C637146934251338067&sdata=r5Gqt%2Fm4YJEHTyWa7vafNWzROftgmcx2toqGTXr7Bo0%3D&reserved=0-------------- next part --------------An HTML attachment was scrubbed...URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20200115/b1da9e20/attachment.html>------------------------------Subject: Digest Footer_______________________________________________3dem mailing list3dem at ncmir.ucsd.eduhttps://mail.ncmir.ucsd.edu/mailman/listinfo/3dem------------------------------End of 3dem Digest, Vol 149, Issue 14*************************************
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20200115/364f7c97/attachment-0001.html>