Agarose immobilization of C. elegans for time lapse imaging

Model Organism: Worms Methods & Reagents
1

I was trying to follow the protocol - http://www.wormbook.org/wbg/articles/volume-18-number-1/agarose-immobilization-of-c-elegans/comment-page-1/#comment-30983
by Fang-Yen et.al
I tried making a 10% agarose solution but it is very hard to dissolve and could only make 5%.
Also, when I used the polystyrene beads (Polysciences 00876-15, 2.5% w/v suspension) the beads are all over the slide and in some cases too many to do proper imaging.
Is there something I can do differently?
Thanks.

2

for 10% agarose pads I was taught to lay out all of your spacer slides/bottom slides first, then microwave your agar slurry.
The second your hear the first crackle (15-25 seconds) take it out and quickly make each slide. I can usually get about 20 pads made before it goes solid,
I’ve never had success trying to reheat it so I just make it fresh everytime.

3

I recently had a go at making 10% agarose pads, and it actually wasn’t too tricky. This is what I did:

  • Make up 50ml 10% agarose in small conical flask, vortex to really get an even suspension, and then microwave in little bursts.
    It’ll bubble up really quick, and when it does stop it, let it calm down, then heat it again.

  • Get a hot plate to around 100C. I then put on an aluminium block (about 12x8cm) to heat up.

  • Find something smooth and flat, roughly the same size as the aluminium block. I used a gel casting tray.
    Put a couple of layers of electrical tape down the two long edges of the casting tray.

  • Pour some of the hot 10% agarose on to the hot aluminium block, and squish it out into a big pad using the gel casting tray.

  • Carefully take the aluminium block off the heat block to cool down for a while. I put it on some wet paper towels to speed this up.

  • Slide off the gel casting tray. You should be left with a giant agarose pad still stuck to the aluminium block.

  • Take the lid of a sharpie marker pen and use it like a cookie cutter to cut out little circular pads, roughly 1cm in diameter.

  • Once you’ve got as many pads as you can out of the slab, use tweezers to lift off each pad. Store them in M9.

I made up around 20 or 30 of these the other day, and have been happily using them since then.
I just carefully take one out with tweezers, dry it on a tissue, add 1ul M9 + beads, put on a worm,
place coverslip on top and then seal the edges with wax.

I hope this is clear enough!

Jake

4

Here is our protocol:

  • Prepare 100mL 10% agarose in water in a 400mL bottle ; heat in a microwave at minimum power for at least 20-30’ (no or little bubbling - using 400mL bottles and minimum power prevents any problem)

  • Aliquot in 5mL tubes (we use bacteria culture tubes). These tubes can be kept for a few months.

  • To make slides just heat one tube in the microwave and keep it in a heating block at 95°C - this tube can be used for a few hours. Do not try to re-heat the same tube the next day.

  • Make slides as for 2% agarose. We use pure beads with very small drops (<5microL).