The method of virus isolation

Generally isolated viruses can be isolated by passing cells,
general cells
or directly with animal inoculation or chicken embryo inoculation, only in the absence of suitable for the growth of the virus of primary cells or pass-through cells, the use of animal inoculation or chicken embryo inoculation methods to isolate the virus.
process should be like this:
first to obtain virus isolation samples, this requirement is relatively high, if not immediately after collection must be low temperature preservation, and as soon as possible sent to the laboratory operation. In addition can also be frozen, in general, the virus is not easy to freeze to death. Of course, with the exception of certain special viruses, this requires access to relevant information.
second virus isolation operation, will obtain the sample grinding, and freeze and thaw at least once, of course, the grinding process must be low temperature. At the same time, the grinding should be sufficient, in the grinding can be added physiological saline or PBS or directly add cell
culture
liquid, grinding completely after freezing and thawing one to three times, the purpose of freezing is to allow the cell to completely rupture the virus released into the cell into the solution. Then low-speed centrifugation, take the membrane clear with 0.22 microns
filter
, to filter the liquid inoculated cells.
It is important to note that not all viral vaccinations require cells to be completely full of cell bottles, generally viruses that do not produce cell disease are best inoculated when cells grow to about 40%, and those that can produce cell lesions need to be fully filled with cell bottles before being vaccinated against the virus. Inoculation process to avoid contamination can be added to the double resistance, or
other
.
Third post-vaccination observation, some viruses that can produce cell lesions are easy to observe, directly under the
microscope
to see if the separation is successful, if the virus does not produce cell lesions, it needs to be detected by other methods, such as fluorescent
antibody
staining,
PCR
or other methods.
most of the time, the first generation is not easy to see or the effect is not good, then you can pass on a few more generations to try. If it doesn't happen four or five generations later, congratulations, you've failed, or there's something wrong with your method, or there's no virus you're going to isolate at all, or your virus is dead during the separation process.
The separation of viruses with cells is probably this process, of course, some viruses may not have the corresponding generation or primary cells, if you want to isolate can only use susceptible animals, operations are similar, nothing but sampling, sample processing, inoculation, post-vaccination detection and observation, collection of viruses only.
the methods mentioned above are all separated from animal tissue, of course, if it is not tissue, such as you want to separate from feces or urine or swabs taken, it can be directly diluted with physiological saline, PBS or
cell culture
liquid (to shake as well as possible if it is a swab should be more squeeze and shake) and then centrifugally remove the filtered inoculated cells.