titration - (Apr/11/2006 )
Can anybody say me how to titrate a noncytopathic viral suspension whose titre is not known..I want to know about the protocol to follow to know the titre of virus.
What is the virus and what does it infect?
Do you have antibodies to the virus?
You can infect the organism, blot the colonies onto a membrane, treat with antibody, and count the spots. This has been used for mammalian viruses etc.
It is also worth understanding the difference between biological (infectious particles) and physical titre (total particles):
Not every viral particle you purify will be viable. You can measure the OD 280nm and get an idea of physical titre. You need to do a standard curve and titrate in the purified virus to calculate an extinction coefficient etc.
Presumably the following is not an option?
If bacteriophage and E. coli:
1. Make serial dilutions of the phage.
2. Mix with bacteria.
3. Add this mix to low melting point (LMP) agar (just enough to cover a plate).
4. Pour the LMP agar/Bacteri/phage mix onto a normal L-Agar plate and allow to cool.
5. Incubate at relevant temperature for relevant time (e.g. overnight @ 37 C for E. coli)
6. Count the number of zones of clearing (plaques). These are areas in the lawn of bacteria where the bug has been infected by the virus and has been lysed. In theory, each zone of clearing is derived from a single infection event, so you can calculate the biological titre of the phage. You will find that your most concentrated phage sample will completely lyse all the bacteria and the weakest will only result in a few plaques.
If a mammalian virus that infects tissue cultur cells you can do a similar experiment, but you do not need any agar. Just add you diluted virus to the DMEM (liquid media) in the dish when the cells have covered the plate. Then count plaques etc.
Let me know some more information and maybe i can help out!
Dear Tuckern,
Thank you very much for the reply.It seems you can be of help to me.The sample I am having is a tissue sample from the dead pig and it was suspected to be suffering from classical swine fever virus so I have to diagnose from the tissue whether pig really suffered from classical swine fever virus(CSFV member of pestivirus family) and then calculate the titre of the virus from tissue with the help of tissue culture.By now I have got swine kidney cell line and porcine kidney cell line.If you any idea about how should I go for the diagnosis and further titration of virus?By now I have monoclonal antibody against E2 region of CSFV and antimouse HRPO.Can you suggest me how to go further since CSFV is a noncytopathic virus.
Looking forward for a valuable reply or suggestion from you
Do you have antibodies to the virus?
You can infect the organism, blot the colonies onto a membrane, treat with antibody, and count the spots. This has been used for mammalian viruses etc.
It is also worth understanding the difference between biological (infectious particles) and physical titre (total particles):
Not every viral particle you purify will be viable. You can measure the OD 280nm and get an idea of physical titre. You need to do a standard curve and titrate in the purified virus to calculate an extinction coefficient etc.
Presumably the following is not an option?
If bacteriophage and E. coli:
1. Make serial dilutions of the phage.
2. Mix with bacteria.
3. Add this mix to low melting point (LMP) agar (just enough to cover a plate).
4. Pour the LMP agar/Bacteri/phage mix onto a normal L-Agar plate and allow to cool.
5. Incubate at relevant temperature for relevant time (e.g. overnight @ 37 C for E. coli)
6. Count the number of zones of clearing (plaques). These are areas in the lawn of bacteria where the bug has been infected by the virus and has been lysed. In theory, each zone of clearing is derived from a single infection event, so you can calculate the biological titre of the phage. You will find that your most concentrated phage sample will completely lyse all the bacteria and the weakest will only result in a few plaques.
If a mammalian virus that infects tissue cultur cells you can do a similar experiment, but you do not need any agar. Just add you diluted virus to the DMEM (liquid media) in the dish when the cells have covered the plate. Then count plaques etc.
Let me know some more information and maybe i can help out!