When I make 10-fold dilutions of my influenza virus for a plaque assay there is often only about 5-fold more plaques in the well meant to contain 10-fold more viral particles.
This does not seem to be due to overlapping plaques. If I make 2-fold dilutions of my stock and then dilute those 10-fold (to get a manageable number of plaques per well) the plaque counts conform with the 2-fold dilution but not the 10-fold dilution when I compare wells with a similar number of plaques. The only difference in pipetting/mixing is the volume of the diluent.
Contact my graduate student he has been working on the plaque assays for influenza: email him, [email protected]
This is very odd and seems to indicate a procedural issue, but It is hard to answer without more information. Can you provide the method you are following.
Hi Leonard, I have attached some data & provided a method outline below.
The attached data shows the average number of plaques from three wells of the same dilution plotted against the average from three wells of the previous dilution. I have included data for two viruses and an idealized data set to indicated the expected values. I have included trendlines that intersect at 0,0. The data range (2-112) is the similar for both data sets. I see that the slope for the 1.5:1 dilution is a little steeper than the ideal slope and that the slope for the 1:10 dilution is much steeper.
confluent layer of MDCK cells in 12 well plate
minimum transfer volume 100μL, 100-900μL diluent (PBS)
transfer 100μL, 2x mix 200μL volume using calibrated electronic pipette
repeat until desired dilutions are made
remove culture medium and add 200μL diluted virus
incubate at 35°C for 1 hour, gentle rocking every 15 minutes
remove inoculum
overlay with EMEM / 0.8% agarose & 1μg/μL TPCK trypsin
incubate 33 or 35°C for 2-3 days
fix with ethanol or methanol & stain with crystal violet
"transfer 100μL, 2x mix 200μL volume using calibrated electronic pipette"
Ewan, it reads to me like you're not mixing the dilutions enough. Does the quote above mean pipetting 200 µl up and down twice? In a volume of 1ml I'm not certain that's enough, and that could be introducing a systematic error. If you use smaller total volumes for the lower dilutions, the effect will go away, as you seem to see. Just be a bit more brutal when you mix - two pulse vortexes, or pipette a bigger volume up and down.
Paul
Thank you Paul,
I lot of people agree that the mixing is too little and leads to the transfer of more virus. To have a similar error between different 10-fold dilutions for different viruses indicates that I am consistently collecting the sample from a similar section of the deep well plate if the virus assuming the virus settles/floats/sticks to a specific area after mixing. I see the same effect looking at 10(-2) to 10 (-4) dilutions for a DI preparation and 10(-6) to 10(-8) dilutions for a regular preparation.
Ah - you're doing the dilutions in the multi well plate? My lot always do them in separate tubes. Easier to mix with a vortexer. The virus won't settle as such but if the media it's in is denser than PBS it may effectively pool. It might well bind to the plastic though. Another advantage of doing dilutions in polypropylene tubes, not styrene plates perhaps.
paul
After some very productive discussions with Jianping Li I am going to look into the effect of the meniscus.
I do the infection with 200μL of virus in each well of a 12-well plate. A lot of that volume is not in close contact with the monolayer so this would affect the attachment kinetics. Larger area plates and proportionally a smaller inoculum should address this.
Also if you have a large concentration of virus you may have carry over and may consider changing tips between dilutions.
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