Agarose immobilization of C. elegans

C. elegans can be immobilized for imaging by applying anesthetics, gluing to a substrate, or clamping in microfluidic channels, each method having its own advantages and disadvantages. Here we describe a protocol for immobilizing worms using agarose and polystyrene beads. Our method has the advantages of being inexpensive and technically simple, does not expose animals to toxic substances, and allows recovery of the animals.

1. Prepare pads composed of 10% agarose in M9 via standard methods (see Methods in Cell Biology). The solution will be too viscous to pipette. To transfer agarose, scoop with a small spatula.
2. Add 0.25-0.5 µl of 0.1 µm diameter polystyrene microspheres (e.g. Polysciences 00876-15, 2.5% w/v suspension) onto the pad.
3. Transfer worm(s) of any developmental stage to the pad using a platinum wire or eyelash pick.
4. Gently cover with a coverslip. The worms are ready for imaging (Fig. 1).
5. To recover worms, lift the coverslip without sliding. Add a small amount of M9 to a worm and remove by pick or pipette.

How does it work? The maximum force resisting the worm’s movements is the product of (i) the normal force compressing the worm between the pad and coverslip and (ii) the friction co-efficients between the worm, agarose pad, and coverslip. The stiffer agarose pad increases the normal force (relative to ~2% agarose pads that are typically used for imaging), while the polysty-rene beads may increase the friction coefficients.

We have used agarose immobilization for fluorescence imaging, laser killing of neurons, laser cutting of axons, and calcium imaging of neurons (Fig. 2). Worms can be immobilized for many hours with no apparent harm if coverslips are sealed with wax to prevent dehydration.

Figures

Fang-Yen_Fig1
Figure 1: Agarose-immobilized worm.  The worm is slightly compressed in the direction of imaging. 
Figure 2: 90 min time lapse movie of region in dashed box in (a), showing fertilization of oocytes. Click to view full size; requires Quicktime -- eds.
Fang-Yen_Fig2
Figure 3: (a) AFD thermosensory neuron expressing calcium indicator YC3.60 in an agarose-immobilized worm.  (b) Calcium signals in AFD.  Blue: YFP/CFP ratio. Green: temperature. Worm grown at 20°C.

Comments

sarfarazhussain farooqui January 12, 2010 at 7:08 am

I would be glad if you can put some longer movies which show us cell divisons or migrations of the cells between L3 and adult stage of worms, because its pretty hard to follow the cells at this stages of development.

ahmet apaydin March 11, 2010 at 3:39 am

10% agarose seems very hard to prepare. Do you have any technical tips for that?

Claudiu May 14, 2010 at 2:32 am

Hi Ahmet,
After trying several ways to make 10% agarose here’s what I came up with:
50 mL M9 in Erlenmeyer flask
5 g agarose
Add first 2.5 g of agarose, parafilm, vortex until no clumps.
Add additional 2.5 g agarose, parafilm, vortex again until no clumps.
Microwave just like when you make agarose for DNA gels. I microwaved in several (2-4) rounds (end a round when the bubbling ends up at neck of flask) with vortexing in between. The solution will ultimately become translucid. Use it fast for casting the pad. If agar cools microwave again.
Cast pad with plastic pipette cut so that mouth is about 5 mm diameter.
Hope this helps!

Christopher Fang-Yen May 24, 2010 at 5:50 pm

To prepare 10% agarose I mix the slurry in a 50 ml Erlenmeyer flask and microwave until bubbly.

Skye April 18, 2011 at 2:37 pm

Have you tried this with agar?

R July 21, 2011 at 4:20 pm

I’ve tried to use agar instead, but it does not work. Melting the 10% agarose is a pain and re-heating only seems to work once or twice. I’ve found that I can make a bunch of small pads and store them in m9. I put 10ish pads in a conical tube to avoid contamination. This seems to work really well. I use the cap of a Sharpie pen to cut out the pads so that they are all the same size.

Sal January 22, 2014 at 6:47 pm

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?

Christopher Fang-Yen April 2, 2014 at 5:05 pm

You could try diluting the beads suspension.

FYI, in our hands 0.05 µm beads (Polysciences 08691-10) work better than 0.1 µm.

Joshua Judkins June 9, 2014 at 9:22 am

I can use 5% agarose with the polystyrene beads to get immobilization, but I’m having problems with recovery – even with very brief time spent under the cover slip, the worms don’t survive.

Do you have recommendations, or know where I may be going wrong? I’m inclined to try diluting the bead solution

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