HSV-2 virus culture question and answer

: Now I'm doing HSV-2 training
training
. The inoculated cells are Hep-2 cells and are sensitive to HSV-2. Once copied, 3 days after the emergence of a typical CPE, the effect is very good. But this time do not know what is going on, 4 days or no CPE,
the
are almost yellow. Has pH had a great effect on the adsorption and replication of viruses? What am I supposed to do? What's with the blind pass?
: Isn't the two batches of the same amount of poison? If the amount of poison is small, it may appear late, but it may also appear, and the cell culture is related. Blind transmission is in the case of do not know whether there is a virus, or in the amount of poison is very small, and you can not be sure, still receive the poison, and then continue to receive the poison, consecutive batches, if the poison will appear CPE, if not will not appear.
: Thank you! Inoculation of the virus is the same, in addition, I used 100 ml of
culture bottle
, in order to get a high level of virus suspension, I only added 2 ml of maintenance fluid. Today the culture fluid is about to turn yellow. Does the pH affect the replication of the virus? I harvest viruses to make molds. So the higher the titration, the better.
: 1, maintenance liquid plus too little, so yellowing, generally our 10ML culture bottle is plus 1ML of maintenance fluid, maintain the pH of the liquid has a lot to do with, but this must be based on their own cells and viruses to touch the conditions;
it's normal to turn yellow, and when you take the culture bottle to room temperature, if it's red, it's certainly not a problem. If you want to get a high-titration virus, you must first give it enough nutrition, otherwise it will not grow long, or the growth rate must be slow. With regard to pH values, I am sure it will affect virus replication, but within a range the virus can still replicate.
HSV is the herpes virus department, may be in the germination way to complete the assembly process, as to whether in the cell can be found, I consider it possible to find complete virus particles, perhaps can find not assembled nuclear shell. I'm not sure, because I haven't done it, but I think you can try it, with viral fluid and cells two separate electro-mirror observation, you can know.
to remind you that the second transmission of poison will have the possibility of frozen insoluble ineration? Or just partial indulation, resulting in insufficient inoculation of the virus. How long has it been before you copied the successful "once"? Viruses stored in a 70-degree refrigerator are also best preserved once every 2 years. If the change in PH after vaccination is not particularly noticeable (one or half a day turns yellow), it should be a small problem. Cell growth metabolism itself produces acid, not to mention you only use 2 ml of maintenance fluid. If the PH change is particularly noticeable, the CO2 concentration may not be allowed. You can correct it once and try it. The media can use HEPERS to tune PH, so that the buffering ability is stronger, not easy to become acid.
2, take the cell precipitation directly to see the electric mirror, I think the difficulty may be a little. It does not have the same effect as
tissue
-slice
, nor does it require the concentration and purity of
purified virus particles with overspeed
centrifugal enrichment." Before looking at the electric mirror, first find out the proliferation characteristics of the virus, and then consider what method to use it. When looking at the electric mirror, it is best to find an electric mirror master to help see, if the concentration of the virus is not enough, it is likely that half a day can not find the virus particles.
3, hsv-2 in vero (green monkey kidney cells) or with bhk cells (ground mouse kidney cells), cpe than hep-2 obvious, 12h or so can be observed obvious cpe. Our laboratory used vero culture hsv generally 12h to observe the obvious cpe, while the hep-2 cpe is not vero's cpe pattern is good to observe. It is recommended that you switch to vero.