I would like to know more about staining cell culture wells infected with virus in TCID50 test. I noticed that some of them use methylene blue or trypan blue. Most of them use these stains for plaque count tests. However, I also noticed that some of them use even for virus that is not forming plaques but have to check cpe only. In this case, how these stain can be used? e.g. mixing with any other reagents proportionally for methylene blue and for trypan blue? For virus that is not forming plaques, we will not add agar. My query here is will these stains remain on the monolayer of the viral infected cells in TCID50 without a layer of agar on cells? When we wash the TCID50 culture plates with pipe water after staining, will this monolayer of the cells in the wells slough off from the wells? Does fixing with formalin help remain the cell layers? or Is it just to disinfect? Thanks.
Typically (and in theory) TCID50 are +/- assays. That is, a well will either be infected (show CPE) or will absolutely not be infected (no CPE). Staining cells in a well with the dyes you mention will provide a quick visual aid to determine whether the cells are there or not. If they are there, then one must assume there was no CPE. Similarly, no dye uptake = no cells = CPE positive. In practice, some viruses may not exhibit complete CPE, and other methods should be used to verify. In my opinion, nothing beats visual inspection of the wells under a microscope to determine CPE, and this can (and probably should) be followed by another way to check (e.g., with a dye you mention, or MTS for a colorimetric readout, or even mixing the supernatant with red blood cells for haemagglutination.) The specific stains you mention have the added benefit of providing a preserved physical record of the assay, whereas otherwise you may be relying on one person's interpretation and hand written record.
You will have to provide more information on your assay in order to determine the best staining or other methods for determining TCID50:
1)What cell line are you using?
2) What virus are you trying to determine TCID50? Many viruses have alternative methods for determining TCID50, which could include staining for specific viral proteins.
If you do not plan to use the cells after plaque formation, you may use fixing/staining solution for few minutes (I generally leave for 1 hr) at room temp. drain the staining solution and wash gently with water, air dry and you can store the plates as long as you want. 0.01% coomassie blue and 10% Methanol and rest water. By this way you can visualize plaques very easily in 6 well plate format and also visualize whether CPE or no CPE (if cytopathic virus) in 96 well plate format for TCID50 assay. Obviously you have to first remove agar overlay before using the fixing solution. Hope this works for you need.
TCID50 tests show whether your cells are infected or not. They may not necessarily form plaques. For a plaque assay, the results are shown as PFU, plaque forming units. The TCID50 is the 50% Tissue Culture Infectious Dose, where the endpoint is determined as the dilution resulting in 50% of your cells being infected. For TCID50, infection can be determined by: CPE, %positive by staining for a particular viral protein, or MTT or other dye which looks at cell death or ATP incorporation.
Staining plaque (in plaque assay) and staining CPE (in TCID50) basically are the same. I usually use 2% crystal violet in 20% methanol (or ethanol). Methanol or ethanol will help to fix the cells. Plaque assay is used for virus that forms plaque (lytic to cell), while TCID50 is used for virus that doesn't form plaque (only cell death).
This is what I usually do for influenza and NDV:
- Remove the media from TCID50 plate and add formalin. For agar media in plaque assay, add the formalin without removing agar. Formalin is used to inactivate and fix the cells.
- Incubate at RT for 30 minutes or at 4C overnight.
- Remove formalin and wash plate under tap water with the help of your fingers to distribute the flow of water. Most adherent cells won't slough off during the washing. If you use agar medium, the agar will be removed this way without having the cells scratched.
- Remove extra water by tapping plate on paper towel.
- Add crystal violet and incubate at RT for 5 minutes.
- Remove crystal violet and wash plate under tap water. Don't worry the cells won't slough off+
I was doing FCoV with CRFK cells. However, it was hard to distinguish whether there were syncytial cell formations (CPE) at 24 hours and 48 hrs PI. Noticed that some researcher waited till 72 hrs. So how are we going to know how long to wait to get CPE as kind of syncytial cells formations. Some even wait for 5 to 6 days. If that long, anything happened to cells might be other causes instead of the virus we inoculated. Anybody know how many hours max to wait to see CPE.
How do you guys know the cells are syncytial cells? When I checked them, it was really hard to say. I did check web images. video clip and pictures of published papers. However, how to confirm things I saw in my infected flask were syncytial cells.
I received virus stock passage 7 (2014) kept in LNT from my friend. I used 100 ul, 200 ul and 400 ul, no changes were found at 24 hr PI and 48 hour PI in all the flasks. What I noticed was that there were floating cells more and more only. What should I do next? Just harvest after 48 hrs no matter what and go for next viral passage with another confluent flask for about 5 to 7 passages? or Must I run qPCR to check the virus stock has real virus or not? Another query is that do we need to add any reagent to prevent from RNA degrading when we stock up these RNA virus for long term?
well, regarding the syncytium, its multinuleated cell which is very obvious. It seems that your virus letting cells detached and then floating. what you need as I believe is checking the TCID50 for each passage rather than qPCR since the latter can inform about the whole virus load you have and not the titer or infectious dose
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