Nematode Slide Preparation: A Method to Mount Animals on an Agar Pad

Overview

During live imaging of C. elegans, an agarose pad can be used to hold the nematode still without damaging or drying it.  This video describes how to prepare a simple mounting pad for microscopy.

Protocol

This protocol is excerpted from Teoh et al, Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans, J. Vis. Exp. (2019).

Preparation of slides for imaging

1. Prepare 3-4% w/v agarose stock in a microwave-safe bottle (3-4 g in 100 mL of ultrapure water).
   NOTE: Agar can be used in place of agarose at the same concentration.
2. Melt the agarose carefully in a microwave oven, taking care not to over boil, until all agarose dissolves (clear solution). Keep the solution at 70 °C to prevent solidification.
   NOTE: Agarose stock can be used several times. Reheat before use if the agarose has solidified.
3. Using a Pasteur pipette, place a drop of melted agarose onto a microscope slide.
   NOTE: Avoid having air bubbles in the drop of agar as these disrupt the integrity of the pad.
4. Immediately press down the agarose droplet with another microscope slide, gently flattening the agarose into a pad between the two slides.
   NOTE: Any leftover bubbles should be removed at this stage. If not, new slides should be made as animals can easily get stuck in the bubbles. Avoid overflow of agar to the side when pressing on the slide.
5. Leave the agarose to dry for 1 min.
6. Carefully separate the two slides to avoid damage to the agarose, while leaving the solidified pad on one of the slides.
   NOTE: Slides with agarose pads should always be prepared fresh just before the experiment as they can dry out. Make sure the agarose pad has a consistent thickness. There should be no air bubbles or cracks in the pad as this may interfere with imaging.
7. Add a small drop (5-10 µL) of 0.05% tetramisole hydrochloride (anesthetic) to the center of the agarose pad.
8. Using heat-sterilized pick, quickly transfer about 10 – 15 animals from the stock plate to the anesthetic droplet before the solution dries out. Avoid transferring too much OP50 bacteria as this makes the solution cloudy, which can interfere with imaging.
   NOTE: If the anesthetic solution dries out while picking, simply pipette a second drop of 0.05% tetramisole hydrochloride onto the animals. The animals tend to lay on their lateral side in the anesthetic solution.
9. Apply a coverslip by positioning it just above the agarose pad and gently dropping it down. This will prevent the formation of bubbles. Do not apply pressure to the coverslip as this may crush the animals.
10. Label the slides with the strain name.

Materials

Name	Company	Catalog Number	Comments
Agar-agar	Merck	1.01614.1000
Agarose	Invitrogen	16500-500
Glass coverslips #1	Thermo scientifique	MENCS22221GP
Glass coverslips #1.5	Zeiss	474030-9000-000	Made by SCHOTT
Glass slides	Thermo scientifique	MENS41104A/40
Pasteur pipette	Corning	CLS7095D5X-200EA
Platinum wire	Sigma	267201-2G
Tetramisole hydrochloride	Sigma	L9756-5G