Hi Everyone,
I am establishing liquid culture techniques to grow worms in a 96-well plate at 20C. Our 20C environmental chamber has significant air flow from the fan which is leading to substantial evaporation of our liquid media. We’ve used the following approaches to try to reduce evaporation but none have proven to be effective:
-plastic lid on plate
-plastic lid on plate and put in plastic worm culture box
-plastic lid on plate and seal bottom plate and lid with parafilm
-seal plate with parafilm “lid” (no plastic lid)
We have a few other permutations we’ll try to prevent evaporation but I’m curious what other folks use to reduce evaporation in environmental chambers that have strong fans/air flow? Our funds are limited so we are looking to use common lab supplies to seal the plates while allowing for adequate gas exchange. If you suggest microplate sealing tapes, can you please provide a brand and catalog number that has worked well for you?
Thanks!
Two no-cost solutions: 1) disconnect the fan; or 2) increase the humidity in the chamber by adding beakers of water.
Another method I’ve seen to increase humidity is to soak a stack of paper towels in water. The stack of wet paper towels can then be placed in a plastic box next to the stack of 96-well plates. I think the plastic box can then be parafilmed, though I wasn’t closely involved and my memory is imperfect.
Helllo
As described in Quantitative and automated high-throughput genome-wide RNAi screens in C. elegans (http://www.ncbi.nlm.nih.gov/pubmed/22395785)
we use AeraSeal cellular culture film from Dutscher (http://www.excelscientific.com/aeraseal_content.html)
Thanks for all of the suggestions! We’ll try them.
We ran into a similar issue while working with C. elegans cultures in 96-well plates at 20°C. The airflow from our incubator’s internal fan accelerated evaporation, especially along the plate edges. A few things that helped us:
We used Breathe-Easy sealing membranes (Diversified Biotech, cat. #BEM-1), which strike a good balance between gas exchange and evaporation control. They’re more cost-effective in the long run compared to constantly replacing parafilm, and they’ve worked better in our hands.
We also placed the plates inside a secondary container with dampened Kimwipes or paper towels to raise the local humidity, similar to what others here suggested. We sealed the container loosely with parafilm to limit evaporation while avoiding anaerobic conditions.
One more tip: when designing our experiments, we started using a 96 well plate template to better map out control vs. edge wells. This helped us identify edge effects caused by uneven evaporation, so we now use the outer wells as buffer zones with water or media. That adjustment alone improved our reproducibility a lot. You can find some helpful layout tools and guides online like this one: 96 well plate template.
Hope this helps! Curious to hear if you find a perfect solution, these airflow-heavy incubators can be tricky.
One thing that sometimes gets overlooked is reducing the vapor pressure gradient around the plate instead of only trying to make the seal tighter. A humidified secondary container can help more than parafilm alone. Even a clean plastic box with a small open reservoir of sterile water inside can create a much more stable microenvironment while still allowing gas exchange.
Another practical trick is to avoid using the outer wells for experimental samples and fill the perimeter wells with sterile water or buffer. That won’t stop evaporation completely, but it often reduces edge effects enough to improve consistency across the inner wells.
When comparing different sealing approaches, keeping a 96 Well Plate Template at:96wellplatetemplate.com to map plate positions and record evaporation patterns by row and column can also make it much easier to identify whether the fan is creating directional bias or if only the edge wells are being affected. That kind of tracking can help determine whether changing the chamber setup or switching to a breathable membrane is worth the added cost.
If you end up testing sealing films, it would be interesting to hear which breathable membrane performs best at 20°C over multiple days, since long-term liquid culture can be much more sensitive to gradual evaporation than short assays.