This may be a result of

1. The overgrowth of the cells because 5 days are too long time to get plaques for influenza. The optimum time is different according to the strain and its replication efficiency (normally from 48-72 hrs)

2. The overlay medium was not solid enough because you did not completely remove the virus seed after adsorption step or the concentration of agarose is low ( i am using it 1-0.8% in the final medium). to escape this problem you can try semi-solid overlay like micro-crystalline cellulose instead of agarose

Thanks Ahmed. I think you might be right with regards to the overlay not being solid enough. I can see the cells coming off when I am removing the plug so perhaps I am not fully aspirating off the wash prior to the agarose overlay. After you overlay your agarose, how long do you let it stand before incubation?

10-20 min at RT

You can switch to the semi-solid micro-crystalline cellulose. Works much better and no issues with temperature and cells detaching. Good paper by Matrosovich et al. (Virol J. 3:63, 2006). We are currently using in 96-well plate format.

Thanks Thomas and Ahmed. I did read that paper and ordered some Avicel last night. thanks again, very helpful.

Hi, I agree with the two points Ahmed raised in his answer. I do use 1% agarose and incubate 48-72h depending on the strain and works great. Also, one key thing is to prepare your plate 60-72h in advance to avoid cell detachment. Even if they may seem confluent at 48 after plating, leave them another 12-24h before infection to strenghten the monolayer by cell-cell interactions. And also check that your agarose is not too hot.,

And when I overlay my agarose, I let stand for 30-45 mins at RT before putting them back in the incubator.

Thanks Andres. At what cell density do you plate in the 6 well to leave for 60-72 hours? I currently plate at 10^4 for 12-15 hours.

I usually plate between 1-1,5E6 cells in a whole 6 or 12-well plate (I prefer 12-well). What I do is resuspend a 80-100% confluent F75 flask of MDCK, MDBK or ST6GalI-MDCK cells in 12 ml of medium. For each plate, I take 2 ml of resuspended cells and add 11 of medium (final volume 13ml) and plate either 1ml per well for a 12-well plate or 2ml for a 6-well plate. Let stand for 60-72h and perform your titration.

Hope it works.

Thank you Andres. I will try the above and let you know how it turns out.