Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

When we're studying aging and sea elegans we're typically following a population of animals over time. This gives us a glimpse of what that process you're studying is doing in the population at each time point but it doesn't tell you how the process is changing in individual animals over time. These single animal culture environments allow us to follow dynamic changes in molecular processes and physiological processes in individual animals over time for hundreds of animals in parallel.

We find that there are two major things that can really impact the quality of these multi-well culture systems. One is either over or under drying the wells and the second is contamination. And we try to note throughout the protocol specific steps where you can try to mitigate these problems.

Demonstrating the protocol today will be Emily Gardea. She's a technician in my lab and has done a lot of work to optimize this protocol. After preparing all the reagents, mix 60 grams of PDMS base and six grams of curing agent per mold in a large disposable way boat using a disposable spatula.

Place the PDMS mixture in a vacuum chamber for 30 minutes at minus 0.08 Mega Pascal to remove bubbles. Pour and fill the PDMS mixture into each 3D printed mold. Put the filled mold and any extra PDMS mixture in the vacuum chamber for 25 minutes at minus 0.08 Mega Pascal.

Remove the filled mold from the vacuum chamber and pop any remaining bubbles with a disposable spatula. Starting with one edge, slowly lower a flat acrylic sheet on top of the PDMS filled mold. If bubble formation occurs proceed as described in the manuscript.

Place a 2.5 pound weight on the acrylic and place the weighted molds in the oven. Let them cure at 55 degrees Celsius overnight. At the end of the incubation, remove the weights and the molds from the oven.

Next, work a razor blade between the acrylic and the cured PDMS To break the seal Using a razor or a metal spatula, carefully loosen the sides of the cured PDMS and remove them from the mold. Wrap the PDMS device in aluminum foil. Seal it with autoclave tape and sterilize by autoclaving on a dry cycle for at least 15 minutes at 121 degrees Celsius and 15 pounds per square inch gauge pressure.

To prepare the multi-well device, preheat a dry bead bath incubator to 90 degrees Celsius. Unwrap an autoclaved multi-well device and cut a small notch in the top right corner of the device. Place up to three unwrapped devices into the plasma cleaner with the wells facing up and run the plasma cleaner.

Sterilize one single well polystyrene tray per device by spraying the inside of the tray with 70%ethanol and wiping it dry with a task wipe. Remove each device from the plasma cleaner with a gloved hand and place it in a cleaned tray. Then place a 25 milliliter disposable pipette basin in the bead bath incubator preheated at 90 degrees Celsius.

Take one tube of solidified LMNGM pre-mixture per device to be filled and place it in a 200 milliliter glass beaker. Remove the cap and Parafilm and microwave until the media melts sufficiently to pour. Pour the molten LMNGM pre-mixture into another sterile 200 milliliter beaker.

Multiple test tubes can be combined in this beaker if more than one device is filled at a time. Microwave the LMNGM pre-mixture for an additional 20 seconds. If the pre-mixture begins to boil over stop the microwave and allow it to settle.

Remove the molten LMNGM pre-mixture from the microwave and cool it to 60 degrees Celsius. Add the remaining LMNGM ingredients to the pre-mix mixture and pour the molten LMNGM into the 25 milliliter basin in the bead bath. Fill the device wells using a 200 microliter multi-channel repeater pipette.

Set the repeater to dispense aliquots of 14 microliters and mount five pipette tips. Load the tips with molten LMNGM and dispense the first 14 microliters back into the LMNGM containing basin. Moving quickly but carefully, dispense 14 microliters into the inner wells of the device and dispense the remaining LMNGM back into the basin as the final aliquot is typically less than 14 microliters.

Next, set the repeater to dispense aliquots of 15 microliters and fill the outermost ring of wells. Using a 200 microliter pipette, dispense 200 microliters of copper sulfate solution into the device moat in each corner. Avoid overfilling the moat and ensure that the copper sulfate does not touch the top surface of the wells.

If the copper sulfate does not flow easily through the entire moat, use a short platinum wire pick to help break the tension and drag the copper sulfate through the moat. After the copper sulfate has flowed through the entire moat, remove as much copper sulfate as possible using a 200 microliter pipette or aspiration with a vacuum. Using a squeeze bottle, add the water crystals in the spaces between the device and the tray walls.

Close the tray lid and wrap all four sides with a piece of Parafilm. Add two additional Parafilm pieces to seal the plate completely. Using a repeater pipette, spot each well with five microliters of concentrated bacteria.

Avoid direct contact with the LMNGM surface. Dry the devices using a custom-built drying box which can be constructed at minimal cost using computer case fans and HEPA filters. Under a dissection scope, inspect wells to ensure all wells have dried successfully.

Using a platinum pick, transfer the animals from the prepared worm plates and add one worm per well. Add a drop of prepared detergent solution to the inside of the tray lid and rub with a task wipe until the detergent solution has dried. Wrap the tray with three pieces of Parafilm as described in the manuscript.

The lifespan extension from daf-2(RNAi)is blocked by the daf-16 null mutation. The health span also decreased in the presence of daf-2(RNAi)The three-day rolling mean of activity across the lifespan is reduced by daf-16 mutant and daf-2(RNAi)The variation in lifespan and health span across individual animals compared in either absolute terms or as a fraction of the total lifespan is shown. The cumulative activity across the lifespan for individual animals correlates better with lifespan than the activity for individual animals on any specific day across the lifespan.

It's important to fill the wells and to add the worms as quickly as possible. Otherwise the wells can dry out and the worms won't survive. This system is compatible with automated imaging which can be used to gather high throughput data such as lifespan, activity and body size or shape.