Edited by Thomas E. Phillips University of Missouri

Selected postings from the Microscopy Listserver from March 1, 2017 to April 30, 2017. Obtain complete listings and subscription information at . Postings may have been edited to conserve space or for clarity.

Specimen Preparation: silicon nitride or graphene support fi lms I’d like to make good support fi lms for TEM. I used to be able to make

fi lms without holes in them but my tried-and-true method using 0.25% Formvar in ethylene dichloride hasn’t been working (purchased solution or made in-house). Holey support fi lms have become a way of life and one of the weakest links in my productivity. I would like to learn more about making silicon nitride or graphene fi lms (they are so expensive to buy). Can anyone share a protocol? If necessary, I have access to a nice chemist and a clean room. Beth Richardson Fri Apr 28

I cannot respond to the other fi lms mentioned in your post, but as to Formvar, ethylene dichloride decomposes to release HCl, which impairs Formvar fi lms. Be sure your reagent—or your Formvar supplier’s—is fresh. In addition, it has been my experience that high-humidity environ- ments make fi lmmaking problematic, and Georgia can be very humid this time of year. Bill Tivol Sun Apr 30

Specimen Preparation: formalin

I have a bottle of Formalin that has a white precipitate, probably polymerized formaldehyde from being stored in the refrigerator. Is there anything I can do to return the solution to normal? Jonathan Krupp Wed Apr 26

Technically you can heat the solution to try and depolymerize the presumptive paraformaldehyde oligomers. I do not recommend trying this - it works occasionally, but hot formalin is almost never a good idea, even in a hood. Option 1: Send it to Hazardous Waste and buy another bottle. Option 2: Filter it and use it anyway. I have needed to use option 2 a few times over the years - mostly due to reagent shipping issues and/or “surprise!” samples. In my hands, fi ltered PF works okay for straight histology-level work, so-so (at best) for immuno work, and leads to a range of interesting results for EM samples. I would still recommend option 1—formalin is < $10/liter from Fisher. In a pinch, fi ltering for H&E or other optical microscopy will probably be okay. Aaron Barnes Wed Apr 26

It might depend: Is that bottle of formalin “labelled” (e.g., adhesive label indicating the producing company like Merck, Fisher or whatever company) and has high percentage of CH 2 O or is it only a lower concentrated, “home-made” formalin solution made from concentrated formaldehyde solution? Has it been prepared with buff er or without buff er (= hydrous only) solution? If the former: usually if there has been included a “stabilizer” (besides 10% methanol usually calcium carbonate these days, in former days: “dolomite powder”) the white precipitate originates possibly from the stabilizer. If the latter: it might be part of stabilizer (if the solution had not been fi ltered when it was mixed up) or it might be in fact deteriorating formaldenyde- oligo- polymers (as Aaron Barnes pointed out) or even precipitated phosphate or other ions (if the formaldehyde-solution was prepared


with a buff er). In that case: If it were only one bottle (1 L or eventually also 1 gallon) I would do the following: Either fi lter or take out most of the solution by a pipet without disturbing the precipitate and use only for “normal” Histology as Aaron Barnes pointed out already. T e problem arises here only that you do not know about the real concentration of the fi xative. Or: dispose of (according to your legal and national safety concepts) aſt er “neutralizing” (decomposing) the solution with, e.g., concentrated NaOH (which decomposes formaldehyde by means of the Canizarro reaction to produce formic acid (HCOOH) and methanol (CH 3 OH). Since the blocking of free aldehyde groups (in fi xation of tissues for immunocytochemistry) is done with glycine, sodium-borohydride or even ammonium chloride-solutions (or addition of these substances to formaldehyde solution) you could try to deactivate the formaldehyde solution prior to disposal by converting formaldehyde to alcohol(s). In addition, addition of bisulfi te has been reported to be effi cient. If you would like to look for other possibilities to dispose of old (perhaps deteriorated and of uncertain origin) formaldehyde solutions you might search by Googling for: | formaldehyde OR formalin AND deactiv* AND disposal | (also perhaps read my blog on formaldehyde-safe disposal on:

https://www.researchgate. net/post/How_do_you_neutralize_

formaldehyde ). Another possibility: Many aldehydes are respiratory irritants, and some, such as formaldehyde and acrolein, are quite toxic. T ere is sometimes merit in oxidation of aldehydes to the corresponding carboxylic acids, which are usually less toxic and less volatile. Procedure for permanganate oxidation of 0.1 mol of aldehyde 3RCHO + 2KMnO 4 → 2RCO 2 K + RCO 2 H + 2MnO 2 + H 2 O. A mixture of 100 mL of water and 0.1 mol of aldehyde is stirred in a 1-L round-bottomed fl ask equipped with a thermometer, dropping funnel, stirrer, steam bath, and, if the aldehyde boils below 100°C, a condenser. Approximately 30 mL of a solution of 12.6 g (0.08 mol, 20% excess) of potassium permanganate in 250 mL of water is added over a period of 10 minutes. If the temperature rises above 45°C, the solution should be cooled. If this addition is not accompanied by a rise in temperature and loss of the purple permanganate color, the mixture is heated by the steam bath until a temperature is reached at which the color is discharged. T e rest of the perman- ganate solution is added slowly at within 10°C of this temperature. T e temperature is then raised to 70 to 80°C, and stirring continued for 1 hour or until the purple color has disappeared, whichever occurs fi rst. T e mixture is cooled to room temperature and acidifi ed with 6 N sulfuric acid. (Caution: Do not add concentrated sulfuric acid to permanganate solution because explosive manganese oxide (Mn 2 O 7 ) may precipitate.) Enough solid sodium hydrogen sulfi te (at least 8.3 g, 0.08 mol) is added with stirring at 20 to 40°C to reduce all the manganese, as indicated by loss of purple color and dissolution of the solid manganese dioxide. T e mixture is washed down the drain with a large volume of water. If the aldehyde contains a carbon-carbon double bond, as in the case of the highly toxic acrolein,

doi: 10.1017/S1551929517000566 • 2017 July

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