Why do my plaque assays look so weird?

09 December 2020 2 7K Report

I'm growing hCoV-OC43 in Vero E6 cells but whenever I do a plaque assay to assess viral titer the plaques appear as dark spots on a purple background rather than lighter spots. I attached some pictures of assays from four separate batches of virus grown over the last few months. The "plaques" consistently look like this and decrease 10fold with a 10x dilution and aren't present in the negative control, but every picture of a plaque assay I've ever seen looks like blank white circles on a dark purple background.

We've been using a 0.9% methylcellulose overlay, fixing with 5% formalin, and then using 0.1% Crystal Violet to stain. My colleague is growing SARS-CoV2 and staining with the same protocol and getting the same results so I'm assuming it must be something we're doing. Does anybody have any insight?

This is how I've been staining the cells, if that helps:

Fixation and Staining:

Discard the methylcellulose. To do this, attach a 1000 ul wide bore tip (or a serological pipet with the cotton removed) directly to the aspirator. Hold the plate at a 45 degree angle. Remove as much as possible without disturbing the monolayer. Aspirate into freshly diluted 1:14 bleach (anything containing virus must be inactivated for at least 10 minutes before disposal)

Overlay the remaining cell layer with 1 ml ice cold 5% neutral buffered formalin by placing the tip of the serological pipette against the side of the well and dispensing slowly.

Place the plate in the 4°C refrigerator for at least an hour to fix. (You supposedly can leave it overnight at this step, but I haven't tried that yet.)

Wash out the aspirator before removing the formalin.

While being extremely careful not to disturb the monolayer, aspirate the formalin layer, then wash with 1-2 ml cold PBS. Aspirate again (carefully). Make sure you dispose of formalin waste according to proper hazardous waste disposal protocols.

Set up a blue pad in the chemical fume hood for the crystal violet staining. You will need a bottle of water to rinse with and a waste container for the crystal violet-containing rinsate, as well as several disposable pipettes. (Note: This is more because crystal violet is messy and it will get everywhere and ruin everything it touches than any actual need to prevent breathing fumes)

Using a disposable pipette, add ~0.5 ml of 0.1% Crystal Violet Staining Solution to each well (you can eyeball this -- just use enough to cover the bottom completely).

After sitting in stain for at least 30 seconds, aspirate the crystal violet solution.

Gently rinse with water, then aspirate the rinsate water with another disposable pipette into a hazardous waste container. Repeat the rinse 4 times for each plate (until removed water is no longer tinted purple).

Invert on paper towels to drain, propping up the plate on top of its lid at an angle so they don't get separated and mixed up. Air dry completely before counting.

Xiaoyu Niu

I don't think the plaques are always necessarily to be light spot in plaque assay. For the OC43 project, did you observe your plate under microscope before fixation? you can have a try and confirm whether the dark spots are plaque you are looking for.

Shih-Heng Chen

Hi' Zadia,

I tried Vero E6 before and not working. Now I use MRC-5 cells, seeding 1E+06 cells/well in 6-well plate. Culture at 34 degree C for 6 days, and the plaques are clear and easy to count. Please see the attached protocol.

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