Agarose pads dried at room temperature or in an oven tend to wrinkle, and microscopy is more difficult when the worms are not on a flat surface. However, heating fresh 3% agarose pad slides on a hotplate above the melting temperature of agarose gives a glassy-smooth surface, as well as drying them thoroughly to ensure rapid absorption of liquid medium.
You will need: 0.6 g low-melting agarose, ~95 ml DI H2O, a 25-ml Erlenmeyer flask, a 100-ml beaker, a 5” Pasteur pipet, a pipet bulb, about 6 dozen slides (half a box), a single-edge razor blade, a hotplate, two empty boxes from slides, a desiccator or tightly-fitting food container (such as Rubbermaid Lock-its, 5 cup size), and some Drierite.
1. Add 0.6 g agarose to 20 ml DI H2O in a 25-ml Erlenmeyer flask with a stirbar.
2. Heat on a stirring hotplate until all agarose dissolves (or melt very carefully in a microwave without boiling over). Also heat ~75 ml of DI H2O in a 100-ml beaker.
3. Place slides side by side in two groups of three on a clean countertop in front of the hotplate. Place two slides crosswise at opposite ends, leaving a 1” gap in the middle.
4. Use a Pasteur pipet to place 5 drops of agarose on each slide, quickly covering puddle with a second slide before agarose solidifies. The pad will shrink when drying, so it should be 1.2-1.6 cm across.
5. Flush the pipet with the hot DI water and place it in the beaker on the hotplate.
6. After the agarose pads congeal and become cloudy, separate the slides.
7. Before placing the slides on the hotplate, do a quick quality check. If the pads are not circular, that’s OK. Rotate pad so its longer axis is parallel to the longer axis of the slide. If there are a few small bubbles, that’s OK too. (If you get a circular pad with no bubbles, frame it for posterity!) If the pad is nearly as wide as the slide, cut off one edge to make room for the Vaseline line. If the pad is too messy, slide it off and reuse the slide.
8. Place the slides in a row along the edge of the hotplate with about 1 cm sticking off the edge for handling. The pads will dry in a few minutes, depending on how close they are to the central heating element. Check the first batch while drying to make sure the hotplate is not too hot (bubbles form under pads) or not hot enough (pads do not melt and dry with wrinkles) and adjust accordingly.
9. While the slides are drying, repeat steps 3-5. Do not remove the top slide until there are open spaces on the hotplate, as pads on a cold countertop attract dirt.
10. As the pads dry, place them in the empty box they came in, two per layer, alternated Lincoln-Log style with an empty 1” square in the center. (About 32-36 fit in box.)
11. Place more pads on the hotplate as spaces open up.
12. Repeat the process until all agarose is used up. Try not to stop in the middle; the melted agarose has a limited working time. If it is heated too long and the pads look matte or wrinkly, they will not work properly, so it’s time to quit and throw away any leftovers. The bad pads can be hydrated and scraped off to salvage the slides.
13. Make sure the desiccator or food container has fresh Drierite, and store each box of slides as it is filled. It is OK to stack the boxes in the container.
