Chicken Egg Virus Harvest (Yolk Problem) - (Aug/30/2012 )
Chicken Egg Virus Harvest (Yolk problem)
I am a masters student working on the Sendai virus pathogenesis. I was wondering if anyone had any experience propagating virus in chicken eggs. As this was my first time inoculating and collecting allantoic fluid; Not surprisingly, I ran into a problem.
While collecting the virus from the allantoic fluid the yolk sac ruptured and some of my collections have yolk in suspension (I collected roughly 15-20mls of allantoic fluid) making the collected allantoic fluid yellowish in color.
I centrifuged the 50ml tubes at 4000 rpm for 5min 2X. I only got a small pellet (consisting of blood cells and some other stuff), the yolk was still thoroughly mixed in the allantoic fluid.
I am planning to filter the allantoic fluid next. But I am wondering if anyone has a better idea on how to remove the yolk from the allantoic fluid. Or let me know if the yolk will have any detrimental effect on the virus (I read that chick antibody production starts in the yolk).
Also I have read a few protocol with inconsistencies concerning whether the inoculated eggs should be rotated or not before harvesting virus. So if anyone has experience propagating virus (it would be super helpful if the virus was Sendai) in chicken eggs, I would like to know if inoculated eggs should be rotated or not before virus harvest.
Any help would be amazing, Thanks!
How old are the chicken eggs? we use 9-day old eggs, why do you have yolk? Is it greenish? it could be contamination. Were all your eggs like that? what was your virus titre that you injected? Sometimes when our titre is high the embryos die and the alantoic fluid gets cloudy. If it is very cloudy we don't collect it because it could be due to contamination. Alantoic fluid is usually clear. Make sure you collect several tubes so that if one is contaminated or has blood can't contaminate the rest of the collected alantoic fluid. Do not collect alantoic fluid that has blood either.
We infect the chicken eggs when they are 10 days old. Yolk is in the samples because, I must have ruptured the yolk membrane, allowing the yolk to spill out into the allantoic/amniotic fluids. The yolk is not greenish and my PI did not think there was a contamination. Only a few of our samples have yolk in them. I am not sure of the virus titer; however, the virus stocks that were used for infection, were chosen by our lab’s PI (Therefore they should have been appropriately selected).
If you have a digital protocol of your egg infection/virus harvest, I could I bother you to email it to me. I’d like to review what your lab does differently than ours.