There are a lot of good books about microscopy of viruses (most of them are pretty old, because the classical TEM with negative staining was developed about 50 (!) years ago, but this simple method is still actual). It is just impossible to describe all things in this format (small remark), RG answers cannot replace literature. But I can highlight a few things which are important in this area:

1. Concentration. Usually - from 10^6 to 10^9 virus particles per 1 ml.

2. Purification. It is a very important part of preparation. it is necessary to remove small (smaller than virions) "garbage" too, because it can dramatically decrease quality of the pictures.

3. Thickness of the film (usually - formvar + carbon). It should be as thin as possible.

Retroviruses are one of the most difficult viruses in imaging - it is very tricky to prepare the samples with proper concentration and purification. I would recommend you to start (just for practice) from some easier objects like bacteriophages.

Usually EM facility operates only with fixed (4% paraformaldehyde, etc.) samples. Discuss this issue with your local biosafety officer. There are some strict regulations and it is not a good idea to joke with these things.

I spent a few years in microscopy of viruses, don't do it now, but sometimes I use negative staining. E.g. recently I just repeated one old protocol for ultrathin formvar grids preparation for good purified T4 bacteriophage.

Good luck!

You have to generate your samples in a BSI facility. For infectious retrovirus, it is the law in any country that I can think of; whether the retrovirus is directed against human or animal (or usually both).

I had a collaboration with people generating infectious HIV as primary cultures from human subjects in a BSI facility. I supplied them the fixative (EM grade formaldehyde in PBS). They would fix the virus samples in the BSI facility and then do a tissue culture infection test with the fixed samples to ensure the samples were non-infectious. They would then put the samples in 1.5 ml eppendorf tubes and subject the tube's exterior to ultra-violet light before exiting the BSI facility. By law they had to do these tests to ensure nothing leaves the place that is infectious. I would not have touched this material if these controls were not in place.

I have tested general virus procedures subjected to TEM using bacteriophages. If you prepare the samples for cryo-TEM and image the samples for a couple of hours in the TEM, you can remove the grid, thaw it, and when placed into tissue culture, see that there are still live/infectious bacteriophage. If you negative stain the bacteriophages, image them in the TEM, and then test for infectivity, it is non-infectious. NOTE: this does mean I am advocating not using a BSI facility to generate your sample !

@ Saloni Sharma : dear Saloni, from your profile I take the 'message': you're an "Electron microscopist"....welcome in our "family" of the mysteriously working scientists (nobody knows what we are/were doing the whole day...) and 'kudos....'

Brent has pointed in one direction... and at the end of his post, he (if I understood his last sentence right) points to the /an alternative. Which way to go will depend on the legislatiion of your country, the head of your facility or (we love/loved them!) the safety officers or safety regulations in your institution / lab surroundings.

It also will depend on whether you are working in a research ('only') or a [e.g., hospital] diagnostic (T)EM-facility (your employer and the institution which I was not able to find out from your personal RG-profile).

Naturally we "Eletron Microscopists" must have some deeper insight and knowledge of working with hazardous chemicals (since we deal with them every day: e. g.: OsO4, UO2Ac, acids, bases, resins, etc.) AND for sure we know about the infectiosity of and the increased risk we are exposed and perhaps subject someone in our working area) of some specimen / material we (have to) examine.... [be it that we were told about the infectiosity - as this should always be informed / recorded / communicated instantly and correctly - or be it because we were mind- and thoughtful (thinking always the GAU= worst case scenario when working with bacterial or viral specimens, be it from patients' blisters or from cell culture)].

Deactivation ("Disinfection") of vivid viral specimens can be achieved by fixing samples at least with (un-/buffered) 0.2%-1% formaldehyde solution (made freshly from PFA=paraformaldehde -powder).

You might have a look for publications/articles published by Hans GELDERBLOM and coauthors (e.g. Stefan BIEL, MADELEY C.R., and others) and find valuable information on how to deal with treating such difficult biological matter, also with regard to handling unknown specimens from suspected bioterroristic attacks ( there are some articles, unfortunately only in German, but with illustrations which an 'EM-ist' easily will understand what the deal / handling issues will be and is / are.

There is at least ONE article by Massimo GENTILE, and Hans R. GELDERBLOM (2014) which I will enclose here for convenience of your rapid information only, naturally begging you for respecting the 'Copyright' of the publisher and authors.

You could expect more specific information and hints if you would be able to provide more details of your Lab situation (Institutions, hierarchy, existing/non-existing safety regulations and benevolence (= restrained rules and regulations) or relentlessly acting safety officers. [e.g. to my e-mail-address: [email protected]). Cheers and best wishes / good luck, Wolfgang

EDITorial note: The name of the file I wanted to enclose:

but in the first, 2nd & 3rd attempt to upload / enclose the mentioned article: there was an automatic display for:

383.65 KB: "UPLOAD ERROR" - REMOVE (without removal I can't save the reply....) so I'll try with a very short file name [GENTILE_and_GELDERBLOM_2014) again... (4th attempt).

IF THAT will not work too:

Dr. Saloni Sharma: would you mind to send me an e-mail to: [email protected] (rendering your, and make it possible to send the file(s) to your personal or institutional mail-address...)?

(do have some other articles which might be of interest and value to you....)

OK now the attachment procedure for the file was possible....

Happy reading!

There are a lot of good books about microscopy of viruses (most of them are pretty old, because the classical TEM with negative staining was developed about 50 (!) years ago, but this simple method is still actual). It is just impossible to describe all things in this format (small remark), RG answers cannot replace literature. But I can highlight a few things which are important in this area:

1. Concentration. Usually - from 10^6 to 10^9 virus particles per 1 ml.

2. Purification. It is a very important part of preparation. it is necessary to remove small (smaller than virions) "garbage" too, because it can dramatically decrease quality of the pictures.

3. Thickness of the film (usually - formvar + carbon). It should be as thin as possible.

Retroviruses are one of the most difficult viruses in imaging - it is very tricky to prepare the samples with proper concentration and purification. I would recommend you to start (just for practice) from some easier objects like bacteriophages.

Usually EM facility operates only with fixed (4% paraformaldehyde, etc.) samples. Discuss this issue with your local biosafety officer. There are some strict regulations and it is not a good idea to joke with these things.

I spent a few years in microscopy of viruses, don't do it now, but sometimes I use negative staining. E.g. recently I just repeated one old protocol for ultrathin formvar grids preparation for good purified T4 bacteriophage.

Good luck!

Well said, dear Denis! Unbelievable fascinating "structure" you've documented above! Best, W.

Dear Wolfgang, thank you so much. I am very glad :)

Cheers, Denis

Thanks for the suggesions Denis :)

Dear Brent as I understand you are suggesting me to do sample preaparation without BSl lab.Right? Thanks for all the other information

Dear Wolfgang thank you so much for putting so much effort and sharing journal. i got that.Rest information I am sharing on your email.Please check..

best

Saloni

Sorry for the delayed response. NO, you should NOT do the sample prep without BSI lab unless you know for sure your particular virus does not infect humans (mammals).

Thanks Brent . We are not accepting such samples.