What virus are you working with? Is it a DNA or RNA virus? You have left cell culture flask at what temperature? Anyways, titer will surely drop but re-isolation should be possible.
I am working on RNA virus. I kept flask on 37 C with 5% of carbon dioxide.
As long as cells survive, virus should survive inside cells. Even if all cells undergo lysis due to cytopathic effect of virus, some viruses may survive in the supernatant.
It is worth trying!
Yeah.. that i know. During sub culture, with in fourth day, media color get changed and morphological changes start, cells started facing some stress condition, and on next day u will found dead cells floating on the medium. So if cells are dead then how virus will survive?
Viruses vary in survival times at 37 degrees in medium with cell debris. RNA viruses are less stable. Immediately freeze your flask at -80 degrees and prepare a new flask with relevant cells. Once cell layer is semi-confluent, remove medium and add 3-4 ml of your thawed old culture with virus (in a T75 flask, for instance), and incubate for one hour. Next, add around 10-15ml of fresh medium. Watch for CPE for next 3-4 days and hope for the best!
Better to follow the protocol that you used initially for infecting the cell layer.
Good luck!
Thankyou.
I am following the same protocol. I have collected the super natant from that 9th day old flask, i will try to infect cell with the collected supernatant. Lets see. I know virus titre will be low, but whatever result came, will show the efficiency of virus to infect cell at its low titre also.
Also, you should always include a negative control flask to compare CPE and cell morphology.
It depends on the virus studied and the cell line used. It's often possible to recover a virus from a cell culture 9 or more days after inoculation if the cells remained viable;
It depends on what virus is studied, what condition of the cell monolayer (destruction or no), pH-medium. Need to study it.
Would mycoplasma affect virus propagation anyway if my cells show such signs? would i need to grow new set of cells or i would still be able to isolate? in my case i am dealing with influenza
I actually not sure about this but the logic may be,, the thing is that..mycoplasma needs nutrition to grow, so some how it will consume the nutrition, that will effect the cell.. and if cells are getting affected like they are not getting nutrition then of course viral propagation will suffer and you may get the low titre of virus.
What is the ambient temperature for your virus? Check the temperatures and the duration needed to heat inactivate this virus . For example: If the usual time of appearance of CPE is by 4 days and if you have not harvested till 9th day you still can get CPE when you re-inoculate your harvested virus because even the smallest quantity of virus can replicate in the new cells. So, if the virus in question can be heat killed at 37 degrees by 2-3 days, most of your virus will be inactive.
je pense que le virus de l'encéphalite japonaise se multiplie aussi sur souriceau nouveau né et qu'il est possible de le produire in vivo, de récolter la matière cérébrale après apparition des paralysies, il faudrait broyer la matière cérébrale puis faire une centrifugation à 4000 rpm à (4°C) récolter le surnageant il doit être riche en virus,le titrage se fera certainement sur souriceaux. après extraction de la matièrecerébrale travailler sous glace, ceci protège votre virus. Lors de votre travail ajouter du PBS et des antibiotiques antimycotiques. Bonne chance
lors de votre travail ajouter du tampon PBS et des antibiotiques antimycotiques
treat your cells with Plasmocyin (Invitrogen) and passage your cells for 3-4 weeks. This will not affect viral growth or titers (no effect on naked adenovirus nor enveloped HSV). As for the 9-day old isolation of infectious virus...typically not effective. As Oliver said, dead cells release proteases which are not advantageous to virus survival. In addition, dead/dying cells lower the pH of the media/environment which is just as detrimental to the virus hence why red media containing phenol-red turns yellow due to conversion of phenol to phenolic acid. I would start over with fresh mycoplasma-free cells and a fresh viral inocula....better to be safe and waste a couple of days to decrease your experimental variables than weeks of experiments with non-optimal virus.
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