Hi, I am working on virus concentration and purification.
I have read papers about sucrose gradient purification and the use of formalin to inactivate a virus.
And here is my question.
What would happen if I change the experimental step?
Every paper did sucrose gradient purification first, and then they inactivated virus at the end of the experimental process.
Is there any reason the inactivation step should be done after concentration and purification step?
I think it would be safer and more convenient to perform the experiment after first inactivating it.
Please let me know why people do 'formalin inactivation step' at last.
Hi Jong,
I cannot give you a definitive answer to your question, however I have several concerns about this:
1) Are you sure fixation does not alter the density or fragilty of the virion? If it does, you may have trouble recovering the virus (or you may completely destroy the virion)
2) Are you sure formalin is compatible for sucrose-gradient centrifugation?
3) Is there a possibility that centrifugation would reverse or partially-reverse the inactivation (which is possible when the fixative is removed)
Someone may be able to confirm that none of these are an issue, but otherwise I would not consider altering the order.
Ultracentifugation of high titre material in a perfectly safe technique when the appropraite training, facilities, approved procedures and equipment are in place and is an invaluable procedure for concentrating viral stocks. The only technique I have performed which requires inactivation of ultra-purified virus is preparing samples for electron microscopy - everything else utilises the concentrated virus for infectious techniques. Even in those instances, I would purify enough material to provide both fresh infectious stocks AND samples for inactivation for electron microscopy, so I don't think I would ever fix then purify even if it was possible.
I think the most important think to say about the idea is that you need to be 100% sure that fixing first then purifying is as safe as the current method. I would need to see published data before I would consider changing the order as there are too many risks - yes, if it works then things may be slightly improved, but if it doesn't then the consequences are significant
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