Is there any paper or protocol to fix envelop viruses for EM? Can we fix envelop virus with formaldehyde or formalin?
We use 4%-Paraformaldehyde solution for a lot of viruses. It is possible to use it for enveloped viruses (poxviridae, etc.).
Good luck!
Hi Denis,
Can you suggest your publication in which used this. just as a reference.
Thank you
Hi Asraf Hussain,
There are a lot of articles about the fixation of viruses by paraformaldehyde. For example:
http://www.who.int/csr/disease/smallpox/ryabch.pdf
http://edoc.rki.de/oa/articles/reHSnBZiKGC7A/PDF/242x32SNu0eyc.pdf
Good luck!
After conventional processing of your Virus sample for a typical negatively stained preparation, you can improve the overall appearance of the viruses (and obtain a certain three-dimensionality) by additional metal shadowing (after the sample is completely dry; Tungsten) of your grids. You can easily find out the appropriate angle of shadowing and the amount of metall deposit. Start at low shadowing angle and continue with samples shadowed at higher angles.
Hello, dear Asraf Hussain,
regarding your request and the replies as given until now (FA=formaldehyde- - formalin- fixation?, 4%-Paraformaldehyde solution, negative staining + following decoration by metal "shadowing") I would like to comment as follows:
i) I guess you won't fix vital viruses (it would be interesting to know ...) with only.... at least BUFFERED freshly prepared 2-4% PFA (paraformaldehyde)-fixative should be used.
[ is/has been a trivial trade name of MERCK Company for a hydrous solution of 'formaldehyde gas' (which solves +/- solidly only at max. 38(-40) %(v/v) concentration. To be sold as commercially available solution this is stabilized by approx. 10% w/v Methanol and to buffer forming components {= formic acid due to polymerization during longer storage in the long run would induce } (in earlier times fine "dolomite powder" ) calciumcarbonate powder (was) is added to the solution.]
ii) question of processing hazardous material at your working place .... any (personal or official) precautions?
(!! cf: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4353909/ !!)
iii) why not use a combination of properly buffered formaldehyde-glutar(di)aldehyde fixative? (needs check of pH and tonicity]
iii) you unfortunately did not say if you want to observe virus particles in plastic resin sections or as negative stained particles....
NB: Find some publications on searching e. g. for < GELDERBLOM and virus > and < Gelderblom AND virus AND EM-fixation >
Best wishes and good luck, Wolfgang
Dear Denis,
thank you for agreeing with my thoughts by your upvote.
Unfortunately yesterday I did not have had the time to look immediately into the sources you provided in your Re#03, where you provided access to the article of Lars Möller et al, 2015 already.
Only one wish open: would you mind (for convenience of furher readers only) to mention / cite the bibliographic data of the abstract of Ryabchikova et al (publication source, year) you also provided? Thank you.
With my compliments and best regards, Wolfgang
Dear Dr Muss,
Thank you very much for your upvote.
The abstract of Ryabchikova et al is from here:
WHO Advisory Committee
on Variola Virus Research
Report of the Seventh Meeting
Geneva, Switzerland
10 –11 November 2005
http://www.who.int/csr/disease/smallpox/abstracts2005/en/
Small world :)
Professor Ryabchikova has taught me to work with the TEM (many years ago).
We receive the letters and specimens of the viruses from Dr. Möller every year - the program EQA-EMV. Usually we have the result of diagnostic 5/6. I like this work! :)
Best regards, Denis
Dear Dr Wolfgang, Dr Frank and Dr Denis thanks for your valuable suggestions. I was just exploring some articles and found Glutaraldehyde and/or Osmium tetroxide to fix enveloped viruses.
How about using Glutaraldehyde and/or Osmium tetroxide and the Conc.?
Dear Asraf, you're welcome.
If you are going to process viral specimens (not knowing whether you are dealing with viral suspensions from experiments / viral cultures or virus containing, diseased human or animal tissues) for TEM:
as Denis also tried to tell, any chemical fixative usually used in transmission electron microsopy can be choosen and should fit.
Important only:
i) primary fixation by aldehyde fixation : can be formaldehyde, made freshly from para-formaldehyde powder, glutar-(di-)aldehyde (EM-grade, better "certified quality" / almost "monomeric"!)), or mixture of GA&FA (= e.g. KARNOVSKY-Fixative full or 1/2-strength) but can also use acrolein....and others (one could think of). Secondary fixation with (naturally buffered) 1% OsO4-solution
ii) right concentration of fixative(also to be personally on the "safe side") (at least 2% if Glutaraldehyde 4 % if FA only, at least 2 hrs at room temperature,
iii) NATURALLY: all the fixatives have to be buffered accordingly (not knowing the species of viruses you would like to process).
iiii) Negative staining as kind of "fixation" too can be done with some specifically prepared heavy metal solutions, the most appropriate (also pH is one parameter to consider and / or to obey) will be:
UO2-Acetate,
(Sodium-)tungstate (or phosphotungstic acid = PTA )
but there exist other special heavy metal solutions for such task which are /can be / should be used if results for UO2Acetate and PTA are unsatisfactory or ineffective.
Besides chemical fixation you might have seen all those really gorgious papers on physical proicessing = leading to 3D reconstructions of viral capsids and HR-virus ultrastructure .
Best regards, Wolfgang
- Badges
- Science topic
- Similar topics
- Mathematical Sciences
- Graphs