Hello, everyone. I am currently working on oncolytic herpesvirus 1. "To guarantee the genomic homogeneity and the purity of a virus stock, the initial inoculum has to derive from a single infectious particle isolated by a limiting dilution procedure." This is what a protocol (Herpes simplex viruses_methods and protocols, 2nd edition, Springer Protocols) told me. But its procedures made me confused (please check the attached picture for it). Furthermore, when I searched "limiting dilution", I only got protocols for isolating cells or testing titers of virus stocks.
In step 6, it said, "Identify and mark the wells containing single plaques." However, since the cells are not overlayed by methylcellulose or agarose, the released viruses can move freely in the medium. If there is a well containing a single virus at the beginning, progeny viruses produced will infect other cells and ultimately make multiple plaques. So I wonder how can wells containing single plaques exist.
I cannot find more information by myself. What's your experience with virus isolation and could you provide me with any information you have? Thank you for your time.
The protocol calls for cells to be infected in suspension and then grown in a 96-well plate. Microscopically, you identify a well with only a single plaque, that is, a well that originally contained only a single infected cell, and hence, a single virus particle. All virions present in that well should be descendents of that single particle (i.e., clones).
Of course, there is always a finite chance that two infected cells settle so close together that they form a single plaque. Or that a cell got infected with two virions (although I don't know wether in that case both would propagate). If you want to be sure, simply repeat the cloning process.
all is in the first part of the protocol= to infect cells (in suspension) with few, well individualized (so sonication to remove aggregates) pfu =low MOI (20 pfu for 2 million cells= 1 infected cells for 100 000) then the cells are diluted and distributed in p96 wells (100ul of 2 milion cells in 10ml= 20 000 cells/wells so only 1 well out of 5 will get one infected cells (the probability to have more than one infected cell in one well is very low...)
another way to do it would be to infect cells in p96 with serial dilution of the virus
and then take one infected well of the highest dilution; at that virus dilution you should have less than 1 infected well out of 5 ....
Thank you for your advice, everyone. I have received a reply from one of the authors of this protocol. She told me that it is only possible to identify single plaques at the very beginning of the infection. As Didier Poncet has mentioned, the probability to have more than one infected cell in one well is very low. But if you want to make sure there is only one plaque in an infected well, you should try to identify it at the beginning of the infection.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus