Fixation of virus for TEM
Posted 11 May 2011 - 02:27 AM
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!
Posted 12 May 2011 - 06:32 AM
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
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: 64–73. doi: 10.1111/j.1439-0434.1976.tb01721.x
If you have an X-ray source on hand, that can also be used.
Posted 12 May 2011 - 06:44 AM
Posted 12 May 2011 - 07:50 AM
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,