I am trying to determine the virus titre by agarose overlay assay and staining with 0.1 volume MTT (5mg/mL in PBS). The result I got was not small white dots on the blue background. The background was more like blackish, spots were very little and I would not call them white. The interesting part was pictures I got from my microscope. Can anyone tell me what are those snowflake-like crystals in the top two pictures? Is it some contamination from bacteria or are those viruses?
Which virus are you looking for ? The CPE depends of targeted virus.
The crystal-like structures could result from 1) your MTT substrate; 2) fungal contamination. I prefer other staining like purple crystal.
It does not seem to result from cell lysis by virus infection
Hi Katrina,
please tell me more details about your protocol... Virus incubation time, host cell line... It really doesn't look like a typical MTT-Virus-titer result.
ps: the snow-flakes look like christal from a dried saline buffert
Thank you very much! It is my first experience with the Virus Plaque Assay and I have tried it for months already with no good results. Firstly, I tried 5 different agaroses but cells already were dead the very next day (I guess they didn't like the particular agaroses that much...). Then I finally got UltraPure agarose and it seems cells at least can survive the necessary incubation time.
In this case I am working with Adenovirus type 12 and A549 cells, I used this protocol: http://www.virapur.com/protocols/Virus%20Plaque%20Assay%20Protocol.pdf
As I see for Adenovirus, plaque formation time is 7-10 days, so I incubated for 9 days and then stained with MTT, kept for 3 hours as written in the protocol, and then visualized it.
I am afraid this particular virus doesn't show CPE, that could be the problem. When I infected cells for virus cultivation purposes no special effect on the cells could be noticed comparing with the control (at least in my opinion). I also work with Adenovirus type 2 and it is possible to see more swimming cells and different outlook of the cells (they seem bigger). So, now I am also doing a parallel Virus Plaque Assay with Adenovirus type 2 to see if that was the problem.
If Adenovirus type 12 really doesn't show CPE how can I measure virus concentration other way?
Dear Katrina,
if Ad12 doesn't show CPE on A549 cells, you can try on 293A cells.
Otherwise, you can measure virus production by real time PCR
Alternatively, you can use a antibody to detect virus infection. The protocol we use is here:
http://adz.cf.ac.uk/content/protocols
I would recommend using PCR or ELISA to measure virus production (whichever technique you are most familiar with and can acquire the reagents i.e primers, antibodies etc.). It is much easier to create standards for either of these techniquies and I am sure you will have more consistent results. Plaque assay would be my last choice to measure virus production.