Contamination free HTP assays utilizing 384 well plates

Hi all,

I run a bacterial cell culture assay in 384 format flat bottom plates with 50 ul total volumes and use a Vantage to load the wells using the 96 head. Using non-treated 384 plates, we have an acceptable rate of contamination of <5% over hundreds of plates. After accidentally performing a multi-plate run with tissue-culture treated 384 well plates, we found a significant increase in contamination of 20+% across all plates and in a predictable pattern on each plate.

It was an easy fix to make sure not to use TC treated plates. I would love to know the reason for the significant increase in contamination so I can be more informed regarding appropriate use of these plates in future workflows. I have read that TC treated plates make the surfaces hydrophilic rather than hydrophobic in order to encourage cells to attach.

I’m curious if anyone else has experienced the same issue, and if anyone has ideas regarding what exactly is happening. Does the hydrophilic surface encourage wicking up the side of the square wells perhaps, or otherwise encourage migration into adjacent wells?

Bonus question:
I would also like to know if anyone can recommend a 384 format plate sealing strategy for bacterial glycerol stocks that they use confidently for storage at -80 C with a low rate of intraplate contamination. Every -80 C rated foil seal I have tried eventually peels up at that temperature.

Thanks in advance!

Can’t comment on the contamination, but Brandel have a plate sealer which uses adhesive seal as apposed to heat sealing: Brandel: Plate Sealer

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Curious what the rest of your process is. What plates, the pattern observed, rough protocol and the points where you see contamination occurring, etc. Are the tips traversing the deck without droplets? When you see contamination in the non-TC treated plates, is that something that’s predictable?

Can’t speak to the increased hydrophilicity of the coatings causing causing wicking. I wouldn’t imagine this would be the case given the working volume doesn’t tend to change due to the treatment. 50uL is within the typical 384w plate working volumes of 10-100uL unless there’s a low-profile version of the plate you’re using.

Are the tips fully submerged during the dispense causing the liquid to breach the top of the wells?

Just some thoughts to help troubleshoot.

I have experienced this sort of contamination with small molecules using TC treated 384-well plates. The problem was solved by either not using treated plates or using plates with cylindrical wells. The wicking occurs almost exclusively in the corners of the well. It’s enough that we could see contamination well-to-well with small molecules, so not surprised at all that bacterial cell culture would have a lot of contamination.

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My imagination stands corrected. Also found this:

I wonder what the upper limit on size for transport is.

I’m not sure why we were using treated plates for small molecules, it may have been an assay plate we were triaging. But most were surprised about the amount of wicking that was happening. It’s way more than anybody thought we would see. Like the article you linked, you can actually see it with just simple tartrazine dye in some cases.

There are also some plate manufacturers that make square well, 384-well plates with fillet (rounded) corners for this exact reason. It’s that hard 90 degree angle in the corners that allow the wicking to occur. Even a small fillet will reduce the wicking to almost nothing.

Interestingly, completely removing all automation from the picture and carefully loading the TC treated plates (flat bottom, square well, 110 ul max volume) with 50 ul dilute culture in a checkerboard pattern with a multichannel pipettor before leaving the lidded plate on a bench overnight undisturbed resulted in the same pattern of contamination, spanning the upper horizontal third and the middle vertical third of the plate. I am assuming the pattern is related to how the hydrophilic treatment is applied to the plates, perhaps those areas receive a more thorough treatment, but just a guess!

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