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Fixation of virus for TEM


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#1 Penguin

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Posted 11 May 2011 - 02:27 AM

Hi!

Does anyone have any experience of fixing virus samples before negative staining for transmission electron microscopy? I work with a pathogenic virus that has to be fully killed before I can take it out of the lab. Usually I would does this by heat inactivation but this causes alterations in the virus particle so clearly this is not possible for EM studies. I think formaldehyde is also too harsh so I think I need to use glutaraldehyde. Any suggestions on concentration, time and temp of incubation? I need to fully inactivate the virus but maintain structure!

Thanks,
P

#2 David C H

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Posted 12 May 2011 - 06:32 AM

I haven't done it, but I was just reading about this. Both methods hit RNA more than the protein; if done right, virus is inactivated without losing structure.

UV light:

A. Kleczkowski and A. D. McLaren, Inactivation of Infectivity of RNA of Tobacco Mosaic Virus during Ultraviolet-Irradiation of the Whole Virus at Two Wavelengths. J. gen. ViroL (1967), 1, 441-448

Nitrous Acid:

O. P. Sehgal. Inactivation of Southern Bean Mosaic Virus and its Ribonucleic Acid by Nitrous Acid and Ultraviolet Light. J Gen Virol 18 (1973),

Jean, J.-h. and Sehgal, O. P. (1976), Efficacy of Various Procedures in Isolating Ribonucleic Acid from Plant Virions Exposed to Selected Chemical or Physical Agents). Journal of Phytopathology, 87: 6473. doi: 10.1111/j.1439-0434.1976.tb01721.x

If you have an X-ray source on hand, that can also be used.

#3 David C H

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Posted 12 May 2011 - 06:44 AM

Oh, sorry -- I saw you wanted to fix the samples -- Fixation in formaldehyde or glutaraldehyde can make at least minor structural changes. In either case, shorter fixation is better. If that is acceptable, it may be easier to inactivate the virus without fixing, as noted above.

#4 Penguin

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Posted 12 May 2011 - 07:50 AM

Oh, sorry -- I saw you wanted to fix the samples -- Fixation in formaldehyde or glutaraldehyde can make at least minor structural changes. In either case, shorter fixation is better. If that is acceptable, it may be easier to inactivate the virus without fixing, as noted above.


Hi David!
Thanks, I think I will try 2.5% glut for a range of times, 10-60 minutes probably, then check my virus is dead, then take the samples to the EM to see if structure is affected,
P




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