My experience is primarily in TEM and I know that concentrated glutaraldehyde tends to polymerize over time and we never let our 3% glut. exceed 4 months in age. I've learned that formaldehyde gets more acidic over time, but when is it too far gone?
Douglas W. Cromey, M.S.,
dcromey@ccit.arizona.edu
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The same discussion has been going on in our lab with regards to 4% paraformaldehyde. We have noticed a definite change in as little as one week after fix preparation, and marked changes after 2 weeks. Our fixatives are generally kept no longer than 4 weeks. The changes we notice are that with transcardiac perfusion, the body gets very rigid within 5 min. when using fix less than 1 week old. It takes longer for body stifeening after 1 week and athe body remains semi-limp after two weeks, but the brain histology still looks fine after overnight immersion. So it might be that there is some diminuition of fixative strength, but not so much that early postmortem changes are not arrested, and it takes longer for thourough fixation to occur.

Glen MacDonald
glenmac@u.washington.edu
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Formaldehyde that is prepared from paraformaldehyde powder is normally made fresh just before use since it very poor shelf life. However, it is a relatively pure form of formaldehyde and is used usually with glutaraldehyde as a fixative for EM studies. Commercially available 37% formaldehyde solution on the other hand has impurities including methanol and therefore is not good for EM studies. But it can be readily used for studies at the light microscopy level. It is quite stable compared to the formaldehyde generated from paraformaldehyde powder. I had the opportunity a few years ago to compare results obtained from cells fixed with a new and a 2 year-old stock of the 37% formaldehyde (for the fluorescent-localization of F-actin with Rhodamine/Phalloidin). I could not detect any difference.

From: "M.V. Parthasarathy"
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Whenever possible we only make enough fixative for that day. That goes for glut., paraformaldehyde or glut/para mixes. If we need to store it before use we keep it frozen. We have not done any longevity test but it seems to be good at least one month. We usually use it up before any longer time. We do the same with 8% glut vials that we have opened and not used completely. We seal the top with parafilm and then place the vial in a screw top jar with a good seal. ..............................................................................................................
Since an aqueous solution of formaldehyde (even made fresh from paraformaldehyde) exists as an equilibrium between methylene glycol and formaldehyde (skewed heavily towards the m.g.), covalent linkage of formaldehyde to tissue molecules is very slow , reaching equilibrium in 16 h @37 C (eg., reviewed in Fox et al. J Histo Cyto 33:845-853, 1985). This argues that BRIEF fixation in formaldehyde (as in immuocytochemistry preps) induces artifacts because of the high concentration of methylene glycol, and that little actual crosslinking occurs. Why use formaldehyde as a fixative for EM?

R. Howard Berg
internet: bergrh@cc.memphis.edu
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If you are really interested in reading about fixatives and how they work there is a chapter devoted to this in the book by G. Griffiths. The ref. is "Fine Structure Immunocytochemistry" 1993 published by Springer Verlag, Heidel : From: Sverker Enestr|m
Subject: Fixative Quality Control
To members interested in fixatives, formaldehyde solution in particular. Here are some additional informations about storage of commercial form- aldehyde.
The principal changes which may take place in formaldehyde on storage are as follows (listed in their order of importance from a practical standpoint):
(1) Polymerisation and precipitation of polymer.
(2) The Cannizzaro reaction, involving oxidation of one molecule of form- aldehyde to formic acid and reduction of another to methanol.
(3) Methylal formation.
(4) Oxidation to formic acid.
(5) Condensation to hydroxyaldehydes and sugars.

The changes are detrimental to product quality but may be avoided or kept at a minimum by maintenance of proper storage conditions. With optimum conditions of storage, commercial formaldehyde will remain unimpaired for long periods of time. In general, proper storage involves avoidance of temperature extremes and the use of storage in glass bottles, inert to corrosion by the mildly acidic solution. Low temperature favor polymer precipitation, high temperatures accelerate the reaction leading to chemical loss of formaldehyde. At improper storage temperatures, a form- aldehyde solution gradually becomes cloudy and eventually solid hydrated polymer separates as a precipitate.
Much more could be said about this highly interesting fixative which possesses many unusual characteristics.
-- * Sverker Enestr*,
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From: desclinj@ulb.ac.be (Desclin Jean)

as an additional measure to all those already mentioned ;-) for keeping formaldehyde solutions as long as possible, I seem to remember that, before those days when we began preparing our formaldehyde immediately before use, we kept it in bottles with a lot of calcium carbonate on the bottom. This took acidification into account, but did not avoid polymerisation, of course ;-) HTH.
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From: DCROMEY@CCIT.ARIZONA.EDU
Subject: More on Formaldehyde QC
Thanks for the many responses to my initial posting Re: Fixative Quality Control. I've done some more reading, called a few suppliers and incorporated the responses from the microscopy listserver to create a memo for the labs I work with. A copy follows for those who are interested:
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We have been doing some research about the shelf life of 37% Formaldehyde. There is no definitive age after which 37% Formaldehyde is no longer useful as a stock solution. Formaldehyde chemistry is moderately complex, but after discussions with other microscopists, manufacturers and reviewing pertinent texts, the following observations are applicable. Formaldehyde should be stored at room temperature, cold temperatures encourage the formation of trioxymethylene with a resulting white precipitate. Formaldehyde should be stored tightly sealed, since exposure to air encourages the oxidation of formaldehyde to formic acid (37% formaldehyde is usually shipped with 10-15% methanol to inhibit this change). Our recommendation is, if the 37% formaldehyde solution is clear, colorless and has no precipitate, and has been stored at room temperature in a tightly sealed bottle that has not been exposed to sunlight, it should be good, however, we still do not recommend using a stock bottle that is older than 1 year, bottles that are already opened should not be used more than 6 months. Consequently, we recommend that labs purchase their formaldehyde more frequently and in smaller quantities than perhaps they have done in the past. *************************************************************
There's more, but this should suffice. The only additional comment is that 37% formaldehyde is not recommended for EM work and that a higher grade "methanol-free" formaldehyde or a solution made from paraformaldehyde should be used instead.
Thanks for your help:-{)
Douglas W. Cromey, M.S.
dcromey@ccit.arizona.edu
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From: "W.L. Steffens"
One fixative that really works much better than it should is McDowall- Trumps, which is a modified Karnovsky-type fix. It is prepared from bulk (reagent grade) glut and commercial formalin in a phosphate buffer. Its popular in clinical laboratories where much of the tissue received is pathological or from necropsy. People around here like it because its cheap (a couple of dollars per gallon) and is stable for months.
I personally like fixatives that incorporate picric acid, for its ability to retain proteins. Picric acid is not a fixative per se, but acts by precipitating proteins, rendering them insoluble. We routinely use 2% GTA- 2% formaldehyde-0.5% picric acid in cacodylate buffer. It produces good contrast in just about everything vertebrate. This "yellow" fix is stable for at least 2 months.