Virus air spot experiment question and answer

asked:
I am ready to do the virus air spot experiment, do not know how to configure methyl cellulose cover
culture
liquid, please teach
A:
1, I do this:
24 holes Board VERO, HSV1
1) 24-well plate pre-board
2)24h, discard the culture solution, wash with hanks liquid 1 time
3) inoculated virus liquid 0.1 ml/hole
4) Put in 37 degrees 5% CO2 culture box 1h, during every 15min slow shake, so that the virus fully adsorbed
5) add 1% methyl cellulose (1 ml/) hole covering
culture base
, put 37 degrees 5% CO2 culture box continues to culture
6) 3 days later, absorption culture base
7) add 5% formaldehyde 1 ml / hole fixed 4min, discard formaldehyde, add crystalline purple 0.8ml / hole dyeing 20min
8) Tap water slow buffer washing dye, calculate the number of spots
2, my teacher short spot test is also with methyl cellulose, but the concentration is 2%, configuration method is: methyl cellulose: 3 grams dissolved in 150 ml distilled water, the final concentration is 2%; The final concentration of 5% is very good. However, methyl cellulose to be configured long in advance, about 2 weeks, after high pressure into the refrigerator, before use sterile operation to add culture fluid, mixed into the refrigerator, after a day to take out, shake hard, so that cellulose to achieve a homogeneous state, sit until the blister completely disappeared after use. Staining can be incubated with a neutral red 0.1% (w/v) temperature box for 2-3 hours, if the 6-well plate per hole plus 1 ml; After dumping, count.
Ask:
"1% methyl cellulose (1 ml/) hole covering medium, put in 37 degrees 5% CO2 culture tank to continue culture" refers to 1% methyl cellulose directly into the medium-placed hole, or methyl cellulose and medium mixed into the cell? What is the formula ratio? Methyl cellulose is configured a long time in advance, about 2 weeks why? When can I count? By what standard?
:
1, 1% methyl cellulose (1 ml/) hole coverage culture should refer to the amount of 1 ml/hole plus methyl cellulose coverage culture. Methyl cellulose-covered medium should refer to Viscous medium, which is a specialized medium. Simply put,
cell culture
added methyl cellulose to the liquid to provide viscosity, methyl cellulose should be non-nutrients. Because methyl cellulose is insoluble, it is enough to disage and sterilise the methyl cellulose solution first. Then in sterile conditions to add other media required ingredients, note that MEM is accompanied by 10×, so to add MEM solution liquid accumulation is much less. I press protol to match Viscous medium with a final total volume of about 400 ml, so 10× MEM only takes 40 ml. The addition of other ingredients is based on a 400 ml culture base conversion. So methyl cellulose is not separate from the culture, methyl cellulose is just one component of this culture.
As to when you can count to see what happens after infection CPE, this depends on the specific test of the virus, at first every day to insist on observation and counting is, until no new spots appear, probably within a week. You can fumble it out if you do it again.
2, I also used Viscous medium to do virus air spot experiments, methyl cellulose cover culture specific formula for reference: In a glass bottle, combine 8.8 g methyl cellulose (4000 centipoise, Sigma) with 320 ml distilled water. Stir with a large magnetic stirrer. Autoclave 30 min at 121 ℃, leaving the magnetic stirrer inside the bottle. Remove from autoclave while still hot and stir again until methyl cellulose has completely dissolved (usually a few hours). Add 40 ml 10× MEM or 10× DMEM, 40 ml FBS, 40,000 U penicillin, 40 mg streptomycin, and 4 ml of 1 M HEPES. Add L-glutamine and sodium bicarbonate according to the medium supplier's recommendations. Store up to 6 months at -20 degrees C. Configuring this Viscous medium is really cumbersome, with more than

reagents used, methyl cellulose is insoluble, but I've dissolved it in about two or three days under the above method. As long as the methyl cellulose solution is ready, the other steps are simple.
asked:
also want to ask for advice, no matter what culture base, how to sterilise? With high-pressure words
agargae
out soon after solidification ah if the high temperature added to the cell will be burned to death, that agar slightly
heating
dissolved are quickly solidified, please tell.
:
can use low melting point agarose ah, after pressing on the 42 degrees insulation culture base 37 degrees insulation, with half an hour of mixing will not condense. I did the air spot experiment is to use a low melting point agar sugar, the effect is very good. Of course, it is also recommended in the literature because I am doing rod virus-insect expression system, the upper covering is only to prevent the virus from spreading with the liquid.