Dear Wolfgang,

Thank you for excellent point. These crazy regulators are making our life really difficult. Depleted uranium is not really radioactive (just a bit), and does not belong to a group of highly poisonous things. Dumb regulators understand only the word “uranium” and act accordingly. Your poster is great in demonstrating of poor quality of ersatzs (chicory instead of coffee, anybody?)

However, digital TEM cameras are superior to analog film. For example, with digital camera I can sometimes (depending on task) work without any staining at all, OsO4 is sufficient, as in attached photo.

Dear Vlad,

thank you for your valuable comment!

I would like to add here only two points:

This my poster / test-work by no means is/was thought to create an against ANY safety officer in an institution and, by the way, by no means an intention blaming any of the mentioned companies/producers (which I have to thank for leaving their original substances to me as a "trade sample" without any reimbursement of costs for the chemical, handling and/or postage.

The shown images in the enclosed Poster-file are only few (since I had to select), are of monor quality due to transfer into powerpoint and converting to a pdf ( additional magnification via the menue up to 300% should be possible without a blurring or pixeling).

I wondered too about the contrast in the images I got after physical development followed by digitization of the negatives.

You display the micrograph from digital imaging of an (after primary fixation, I guess) only OsO4-treated specimen/ultrathin section with huge contrast (it might depend also on the brand of Digital Camera used, as well as imaging mode and use of a sophisticated NEW TEM).

I shall include in my further study on this matter also imaging of "blank" ultrathin sections from the original series, as to be able to see what "contrast" the e-beam produces through resin only (and naturally also ultrathin sections just fixed and osmicated, nothing added by staining as usual).

Handling Uranylacetate (powder as well as solutions) is easy if one knows several points learned in chemistry, physics and scientific working methods. Preying upon Potemkin villages or manhunt on knowing colleagues IMHO is not necessary.

On the other hand, alternatives [there are some ones too, as I have seen from literature] for staining of sections for TEM should be sought yet, for scientific reasons.

Thank you again for your "plea" in favour of using UO2Acetate and its certainly misunderstood/misinterpreted (hazardous) properties (for example, I haven't heard from any lab that using e. g. KCN for e.g. cell culture experiments cannot be used anymore due to its potential as toxic and hazardous chemical!) .

Very best regards,

Wolfgang

Dear Dr. Wolfgang Muss,

It is finaly a pleasure to read an article that actualy states some hard facts.

As you know my firm (IBILABS.COM) is the only manufacturer of uranyl acetate world wide. Some of our distributors rather than educate the user of the uranyl acetate have decided to advertise a replacement or alternative to uranyl acetate. This has been completly misleading because there is no real replacement. They should be attempting to educate the safety officers on the safe handling of the material but unfortunatley they are generaly not chemist like your self and have by their silence assited in demonising uranyl acteate.

You have posted and excellent article and I hope you get good feedback.

Alex Besenyo PhD

@ Wolfgang,

Of course your poster is not an aggressive pamphlet; It was I who has been aggressive (enough is enough…)

As for contrast on my picture, there is nothing special, just routine picture from 4 years old digital camera attached to much older microscope (CM12).

@ Alex

Many thanks for uranyl acetate.

Dear Vladimir, dear Dr. Alex Besenyo,

glad to have your replies on this.

Since I believe (until anybody "officially" will tell us the contrary) that the issue might be a(n Inter-)National Security Matter (NSM! not “NSA”!), perhaps without further notice to us I found it necessary to place my statement as written above. That there might be some resentments or a kind of apprehension in posting replies officially in this RG-forum to this my question I can deduce perhaps from really positive and consensual replies I have gotten via my private (and work-)mail adress.

I agree completely with your opinion that a low "radioactive" "chemical" used in scientific Labs cannot be used to prepare dangerous or highly risky resources for e.g. bioterrorism in an outrageous manner as the same would apply for hundreds of other lab-chemicals too.

@ Dr. Alex Besenyo: The idea to educate people (whether they are safety officers or working on the lab bench) either by correct working instructions (instead of merely sending an MSDS or technical Datasheet) or by working out precise information how the substance is to be handled I find a good one and perhaps we should try to cooperate intimately to set (new?) standards (if they are / should be needed).

@ Vladimir: I shall try something similar with my specimens. And my photographer will produce a contact copy of my negatives (so I will be able to show the tremendous decrease in contrast over the images taken within the test series for the mentioned substances).

Best wishes and regards,

Wolfgang

Dear professor,

Have you tried with lead citrate ? I know that it was used not in replacement, but as a alternative to uranyl acetate in the staining in the ultrathin sections - I think it may apply also to cutaneous tissue staining.

Dear Professors, couple of years ago, when we tried to order Uranyl Acetate, we have been told that the cost of this product was around 150 euro, plus 800 euros for delivery charges!!!! Luckily, at that time, we discovered that a collegue had dismissed his TEM lab, so we could receive some precious grams of UA from him...we are still using it, in the meantime we are going to try safer alternatives, right now we are starting with UAR-EMS, although your results do not seem very promising...i have couple of questions for you, especially for Prof. Muss.

For aldehyde-fixed (both paraformaldehyde 4% and glutaraldehyde 5% according to Karnovsky), Osmium-post fixed, Acetone-dehydrated, Durcupan-Araldite embedded, 80 nm-thin sections of human tissue staining, do you recommend using concentrate UAR solution? the two dilutions you tested were even less effective than the concentrated UAR, I suppose. EMS data sheet suggests a 4x (or even higher) dilution, therefore I was about to try different dilutions but I found your poster and now I am puzzled...furthermore, I noticed that you used Nichel grids, are copper grids unrecommended with UAR-EMS? 

Thank you very much for reading and, hopefully, for answering to my questions.

Dear Tiziana, I just found your Reply and thank you for telling as well as questioning. I admit that today I am in a kind of timeframe I cannot respond earlier than in the evening (which I shall try). Apologize for that.

For now: best regards and wishes,

Wolfgang

Thank you very much Professor Muss, thanks for replying to me even if you were in a hurry, I will wait for any suggestion from you whenever you will have time, best regards!

Tiziana

Dear Tiziana,

it is a bit difficult for me to reply in depth to your comment and questions. Apologize for being late....I was working hard (despite being retired) in my storage room to prepare for readily take-up of my private things which I had to remove from the lab within the last 4-6 weeks.

OK...I shall try...I remember when it was declared that storage of more than approx. 10 g of radioactive UO2 Ac in the / a lab without special training, course and diploma had been banned. Also the time when we heard about the risk of keeping "radioactive" material said to be used possibly as a basic stuff in preaparing a "dirty, radioactive threat"....etc. We know that nowadays UO2 Acetate is fabricated from depleted Uranium....I have now to dispose of "old UO2Acetate" chemical as well as "Uranyl-nitrate" (which emits about 10 times higher radiation rate than UO2Acetate if my measurements last week were reliable....but  I will prove that today with our special radiation saftey officer).

I will keep you posted on this, if you don't mind.

Regarding the high costs of UO2-Acetate(di-hydrate) nowadays: if you remember: in the 1980ies until approx. 2004/2005 (if I remember correctly) it was possible to purchase 25 g or even 50 g of Uranylacetate...(e. g. from Merck). Starting with the restrictions (= EU regulations) the volume was limited to 10 g so you naturally have additional costs for packing etc.

Other increase in cost (transportation etc.) as a consequence of the severe / rigorous ADR-regulations.....

Concerning use of undiluted or diluted UAR-concentrations:  as you may anticipate there COULD be an effect of concentration of the UAR stuff which I have NOT tested in full with my experimental work as is outlined in my poster (cf. histochemical staining issues using correct dilutions of dyes for positive staining effects) . For replying to this very special aspect please allow some day for thinking over how I can answer your doubts in a right way (my problem is that I am NOT allowed and able to do further preparational and experimental work...."my" EM lab is to be closed, so I can't work further anyway there... One should ask EMS on which base their suggestion for a 4fold dilution of their UAR was made....(I am sorry but until last November 2015 I did NOT see any paper about use of UAR telling about practical use for (T)EM-work... but if I missed such articles I greatly should appreciate receiving some bibliographic hints about such work (which naturally should contain sufficient micrographs demonstrating the effects).

As last point you mentioned my using Nickel grids for testing: this only owing to omit any side effect of Copper / copper grid surface with one of the ingredients of UAR or Blue platinum. Since Nickel should be more inert than copper, I guess(ed). Nothing more about it.

Will come back later on...have to leave now...

best wishes and regards, Wolfgang

Dear Wolfgang,

thank you for finding the time for a reply, in spite of all the work you are doing to fix everything connected to your lab dismissal and retirement. 

I think I will perform couple of tests with UAR concentrated (30-min at RT, in association with 8-min Reynold's lead citrate, that we commonly use), just to have an idea of how different it stains tissues in comparison to UA-Reynolds. We are using TEM for diagnostic (mostly renal biopsies) and research purposes, and we can't accept a loss in definition of ultrastructural details. On the other hand, we face the safety problems of working/correctly disposing UA and we think that a good-performing alternative would be absolutely welcomed. 

Again, thank you very much for your precious suggestions, best regards

Tiziana

Dear Tiziana,

thank you for your understanding and patience.

You are completely right. BECAUSE it has been stated to dilute the UAR I did so in my "experiments" (cf. in my Poster UAR & (=) AND  Blue-Pt-stain, respectively:  I used

Orig.solution = C1 =100%,

Diluted = C2 = 10% = (1pt / vol. C1 + 9 pts / vol. fresh A. bidest.) and

Diluted = C3 =    1% = (1pt / vol. C2 + 9 pts / vol. fresh A.bidest.). 

Results with UAR as well as Blue Pt (it could be it was an old formula) were not satisfying (at least regarding "optical" visibility on the screen of my analog TEM as mentioned.) You intend to do staining series which is hopefully a promising  beginning.

Just to tell: I used in my stainings after 0.05% TA and 1%methanolic UO2-acetate (15 min) only 3 min Pb-citrate (Venable Coggeshall 1965, at least ONE DAY OLD) because from my experience UO2Acetate (1%methanolic, pH around 3.4/3.6) staining followed by leadcitrate with high pH of approx.11-11.5 LONGER Pb-citrate staining times than 3 minutes always resulted in "low and lower" contrast (sort of "bleaching" *) UO2-acetate staining.

*)"bleaching" for sure is not the right term... IMHO it is 'neutralization and/or destaining' effect on Uranyl-stain due to the action of the fresh - highly alkaline- Venable-Coggeshall-Pb-citrate.

The treatment and priming of Uranyl-acetate waste solution used in staining sections for electron microscopy for disposal  is quite EASY and cheap (yes!)....  but unfortunately disposal of the end product (which is some grams on filter paper which is yellow, radiant, and somewhat like Uranylhydroxide, respectively) will cost nevertheless a lot (due to restrictions in transport and costs to hand it to national authorities...at least in Austria).

NB: I have gs of Uranylacetate-Dihydrate (unopened in the original plastic container.... how much in total I can not communicate in this forum) as well as Uranyl-nitrate in the Lab's chemical safety cupboard  and might have to dispose of the whole stuff at really high costs withi the next days..... 

I greatly should appreciate your kind post in this RG-thread about the results of your staining experiments/tests! Really important task for which I wish you all the best and good luck, 

cordiali saluti/cordially yours,

Wolfgang,

Salisburgo-Austria (privato: [email protected])

I have used the UAR (uranyl acetate "substitute") in concentrated form without success. I will not be using it again. Cannot waste time or sections, so I went back to saturated uranyl acetae in 50% ethanol for Spurr's embedded tissues. Reynold's lead citrate is my counter-stain.

The restrictions on uranyl acetate are ridiculous and the dissemination of information on the handling and disposal of the substance is practically non-existent. I do know that URAC is not a glass or even a plastic penetrator. That is not to say that handling and disposal is not important - it is! But I had to get all information about this from our Environmental Safety people, as not much else was available except for the MDMS.

I know I haven't really provided any solutions to your problem - wish I could! As Dr. Kurzeluk mentioned above, staining with lead citrate only will give you more contrast in the membranes, but will not bring out the  protein and nucleic acid inclusions in the cytoplasmic ground substance or in the nucleus.

Best of luck to you, My Dear Tiziana.

Dear colleagues,

I have used UA for several years with excellent results (triple staining method: Reynold's lead citrate - saturated UA 50% methanol/water - lead citrate) . I work in a microscopy lab, so I have to stain almost every week working with very different samples, from bacteria to plant and animals. I stopped using UA because its disposal is very expensive and its use in the lab is subjected to a really strict regulation. And, not less important, my Geiger counter went to full scale when I pointed to the mounth of the open bottle! I tryed several positive staining protocols, starting from that written in the sheet from the package, trying to obtain an acceptable result. After months of daily trying, I ended up to a triple staining method, which gave me the best results with UAR. I used to observe my samples with the analog screen and takig micrographs by a digital camera. Sometimes I obtained good results, but not always. I always use to adjust contarst and brightness after acquiring, as I did before with the UA. My protocol is the following:

1) Reynold's lead citrate for 2-3 minutes; 2) drying grids for some minutes; 3) UAR diluted 50 % with methanol for 30 minutes (shorter times gave poor contrast); 4) Reynold's lead citrate for some minutes. I would like to share this triple method and I hope I wil get a feedback from you all. Good luck to everyone.

Sergio, good to see that there is a solution - at least this one....(knowing there to be some other successful staining recipes used with UAR or other "replacement" chemicals....Hopefully we will end up with some more experiences from the practice at benchwork....(:-)) Thank you for your comment I acknowledge....and will annex into my electronic /digital "EM-LM-METH's&TECHn's&Dye Works collection....(:-)) Best wishes and regards WM.

Dear Prof., thank you very much for your fast replying. I think we all have to continue working and trying to achieve better results. We have to share our partial success in order to improve the methodology. I know UAR is not so effectve as UA, but I hope to improve. The problem is that uranyum has a very big nucleus, which is very effectve at scattering the electron beam, most of all for its great size. Another problem is that we used saturated UA solution. But UAR is supplied already dissolved. I also add methanol to improve the penetration inside Spurr resin, so I further dilute the stain solution. That is a further problem. Don't you agree? The best wishes.

I have attached some details of a micrographs, showing thykoids in a chloroplast, after a correct TEM preparation, staining with UAR (see my protocol in a previous message), acquiring with a TEM Philips EM208S (it's a simple TEM microscope), and adjusting of brightness and contrast with a Photoshop program. I hope the image will be well visible. I think it's enough.

I believe the UAR is some sort of molybdenum compound. It may take some time to work out a good protocol, but I really like what you are doing so far. The image above shows crisp, well-defined lamellar membranes and little or no background, which would be completely acceptable to me. Nice work!

I have finally resolved my staining issues by en bloc staining with 0.1% tannic acid after primary fixation, followed by saturated aqueous URAC stain after osmication.

Let's all keep pushing forward in the pursuit of excellence!

ok... had my 15 -30 min. 'elaborate' deleted by chance within a second.... by a wrong click! .... so I have to try again...

@Debra: UAR (Uranyl Acetate Replacement, also 'UAR-EMS') might be a trade mark-name of EMS (Electron Microscope Sciences = EMSDIASUM, HATFIELD PA, USA, Stacey KIRSCH; also cf. their catalogue: e.g. @ http://scienceservices.de/en/uranyl-acetate-replacement-stain-uar-ems-25ml.html).

As of their web-information ('last updated: 05/22/2014) to be found @ laboratoryresource.com.au/?navaction=getitem&id=79 their UAR "stuff" consists of a mixture of the two (non-radioactive) lanthanide salts 'samarium triacetate and gadolinium triacetate', which according to their instructions are delivered at a higher concentration and have to diluted approprietly for application on samples for negative or positive staining (i.e. ultrathin sections). Their Instructions are quite less informative for practical working (and without having experience leave a lot to be desired) so one has to start with test series...(as I did in 2014 and 2015, cf. my poster on UO2Ac - UAR - Platinum blue [= also Blue Platinum]

@Conference Paper Uranylacetate as superb staining reagent used in diagnostic ...

For convenience of all further readers I add the URL for a more recent article on the matter, which RAJ et al published 2016 in an OA-Journal [https://www.clinmedjournals.org/, Scottsdale, AZ, USA] (pdf cf: https://www.clinmedjournals.org/articles/ijmnr/international-journal-of-medical-nano-research-ijmnr-3-013.pdf). The authors report on good results (comparing UO2Ac - 'UAR'- and MUAR ("Modified UAR"). Interesting article and results - not knowing whether further efforts in the method(s) resulted in a better staining/contrast formation by the e-beam.

Another thread on RG can be found @ https://www.researchgate.net/post/Has_anybody_tried_the_UAR_uranyl_acetate_replacement_stain_to_carry_out_staining_of_a_virus_for_TEM;

all Questions containing at least "Uranyl acetate" in their text cf.: @https://www.researchgate.net/topic/Uranyl-Acetate-Stain

@ 'Prof.' Sergio....(I am no 'Professor' at all! No need for such an entitling!)....Thank you for posting/uploading your valuable comments and results as per your "practical" and 'own-hands-on' experience. Great. I - for sure - would love to comment further on your method(s) (which should be published at least as a "short communication" or "Technical comment"!") if you could provide more necessary data (i.e. "whole processing protocol" from primary fixative via dehydration to embedding (resin type) to staining [purchased from which company? EMS or other??), perhaps also the TEM you use and naturally e-beam conditions (kV, diameter of aperture(s), type of digital camera, etc.,etc. )....

Last but not least: From the legend of your nice image: ""thykoids" or "thylakoids"?....and: you haven't used tannic acid treatment of your sections? (or used it as an en bloc mordanting stuff- as Debra did)...Sorry no possibility here for highlighting some words or passages by bold font due to the lenghtiness of my reply (can't see the diverse font-icons ...APOLOGIES....if there is something lacking, I shall get back and add.... nevertheless: have a good carnival-weekends' reading...

(:-)) Regards WM.

Aah... yeah....regarding saturated UO2Acetate....this IMHO is not really necessary! Far too much powder ...10-100fold oversupply to the binding sites of the embedded tissular structure....(just like/very similar to the oversupply with 1%-4% Osmiumtetroxide solution...)!

I always used 1% UO2-Acetate in 100% methanol to which 1-2 drops of concentrated acetic acid were added (/ 50 ml) for better 'nuclear' stain and perhaps also "softening" of the epoxide sections' surface for a better reaction (and penetrating a bit into the section surface) and to maintain the quality of the stain on a more acid pH (than usually should be achieved depending on the UA you might use).

Usually, centrifuging the working solution (as often recommended) before use /application did not prevent staining artifacts (precipitation of UO2-microneedles) - so the solution needed was pipetted from the upper portion of the solution as contained in a 50ml amber glass bottle always stored in the dark and under ambient conditions.

My Staining Procedure at least the last 15 years of my professional bench-work was a triple staining technique (reference for this see my publications/posters):

i) 0.05% (hydr.) TANNIC ACID [low molecular weight(!)]

(stock solution 0.5% - ultrasonic dissolution necessary, filtered, and diluted 1:10 for use: inverse incubation= section down on a drop of stain posittioned on parafilm(R) , @ RT for 8-10 min), followed by

ii) UA-Staining (1% methanolic): generally 15' staining at RT, darkness provided, adequate and sufficient jet washing with A.bidest., blotting grids with the edge of piece of a lintless filterpaper] - total drying the grids in between UA and Lead should be omitted -

iii) Lead citrate (Venable & Coggeshall,1965): 1- max 3.5 min (depending on the "freshness" of the solution made:

used same day: 1-1.5 min;

used on 2nd day: 2 -2.5 min

used on 3rd day: 3-3.5 min. - (Tapping the full potential of CO2-prevention - NaOH-pastilles - recommended!)

now: I am finished....Best 'carnivalistic' regards (;-))

Only for convenience:

Regarding disposal: cf: https://www.researchgate.net/post/What_precautions_and_safety_guidelines_to_be_followed_for_staining_samples_with_Uranyl_acetate_for_TEM

Thanks for the correction to the molybdenum statement.

I looked up your reference to the lead citrate, which has always been a bane to me. It's "quick lead" with actual instructions on how to make and alter the solution (if necessary) ! You are a gold mine of information and I genuinely appreciate it.

Sometime soon I will try your staining protocol, as en bloc staining is always risky.

Dear Debra, thank you for your kind words. You know for sure that I did not want to 'teach' you anyhow! (But now you know that I had - unfortunately too short in left time before my retirement - some "practical experiences" with the UAR (and platinum blue) stuff....unfortunately I was not able therefore to complete the test-work. Regarding the Venable&Coggeshall recipe: it is really easy to perform....BUT

you should keep in mind the following:

i) to me the amount of lead citrate (p.A.) always was a bit critical to measure. you'll find out that no weighing is practical but just to add some powder grains from a knifeblades top are sufficient (for 10ml solution).

ii) For making (and further storing) the solution I marked the upper meniscus of the filled in volume of 10ml HOT WATER on a small 15 / 20 ml (clear) glass vial by means of a grinding head clamped on a minidrill. Don't use a diamond for scratching (since the glass vial would break when filled with hot A.bidest!). In case you don't have such a device you could use the edge of a grindstone (usually used also to scratch the neck of glass ampoules prior to cracking or to sharp some instrument used for preparation) These small glasses with a plastic 'snap cap', those which are sold in packages of 50 or 100 glass vials (TAAB, AGAR AIDS, etc.) aren't expensive....and -fitting a specimen rotator - can be used also for specimen preparation (fixation etc.).

iii) before the first use of such a marked glass for making the Pb-citrate working solution you should have the following on the bench ready:

- 10N NaOH (stored in and 'micro'-pipetted from a plastic flask/plastic stopper. [100µl pipette and pipette tips)

- 250ml Beaker glass filled with~100 ml A.bidest, gas / bunsen burner or thermic heating plate to heat and and let boil the water to preclean the 15 / 20 ml glas vial + the plastic cap (some 2-3 min). grasp glass vial (caution: HOTTTT!) and snap cap (plastic lid) with a previously appropriately cleaned pair of tweezers

- a second (50-100ml) beaker glass ready to heavily boil 25-30 ml A.bidest for 2-3 min to facilitate a complete removal of CO2 ).

- Fill in the hot boiled A.bidest to the 10 ml -mark into the hot glass vial, and place the lid (but dont lock/close fully the cap). Then add 'fewer than a pinch' (from the tip of a knife or a spatula) - just very few PB-citrate powder grains - into the hot A.bidest, close the plastic cap and shake vigorously for some strokes...Then

iv) be prepared to add 100 [-150-200µl are possible] 10N NaOH solution with the 100µl tip-pipette without taking away the whole plastic cap (just only half of the lid to prevent CO2 intrusion/aeration. Then

v) close the lid (be prepared to feel the heat of the solution on your "naked" thumb or forefinger! so use a so called heat/frost-protective glove) and SHAKE VIGOROUSLY for at least 1 minute.

vi) check thoroughly if the added leadcitrate grains have solved totally, otherwise another minute or two. (if after 3 minutes Pbcitrate hasn't solved the solution should be discarded and another trial (with clean glass vial etc.) be started.

vii) If the solution IS clear on inspection against a light background then cool the solution (with closely locked plastic lid) carefully (and smoothly shaking the vial) under cold water stream from tap.

viii) The solution will be ready then for use (up to 5 days and more if kept in the dark and caveats against CO2-uptake have been set and followed). The first hours the alkalinity of the solution is really high - so the incubation time should be in the range of 1-1.5 min for staining....storage in a plastic container/plastic stoppered plastic flask) might be an alternative but has not been test by myself (and might be impractical....) (If these practical hints are too much -apologies again...) Article VENABLE & COGGESHALL 1965, and protocols/comments from my collections attached...Feel free to ask further questions.... REGARDS AND A BEAUTIFUL WEEKEND1