Is there anybody who has done mean death time (MDT) in embryonated eggs. I am in urgent need of help because I did it using allantoic fluid 106 - 109 dilution but all the eggs died. So, do I have to dilute more or not at all? Also how I can calculate it?
What virus are you working with? Are you sure that embryos have died due to virus and not due to contamination? If embryos have died too quickly (e.g 24 hours), then it could be bacterial contamination. Confirm viral presence by Haemeagglutination Assay or AGPT.
For MDT, make serial 10-fold serial dilutions and inoculate each dilution into embryonated eggs. Mean embryo death time (MDT) is calculated as the mean time in hours required for the minimum lethal dose (MLD) to kill all embryos for a given dilution. Briefly, for the MDT, a series of 10- fold dilutions of infected allantoic fluid (0.1 ml) are inoculated into the allantoic cavities of 9-day-old eggs and incubated. The eggs should be examined once every 8 hour for 7 days, and the time of embryo death should be recorded. The MDT will be determined as mean time (h) for the minimum lethal dose of virus to kill all the inoculated embryos.
Dear Zaheer Nizamani, I have not non specific death (within 24h) nor bacterial contamination also I confirmed the virus presence. Can I get the reference paper for this method
This paper may be of help to you:
"Replication, Neurotropism, and Pathogenicity of Avian Paramyxovirus Serotypes 1–9 in Chickens and Ducks" by Kim et al - 2012
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0034927
Hi, I am facing the same problem as well where all my eggs inoculate with the virus are dead. My dilution even starts from 10^-5 and 10^-9. Bacterial contamination can be excluded since the allantoic fluid is clear and the eggs take more than 24 hours to die.
Looking forward for the solution!
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