Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 376

viii. Brush embryos out of M9 into the center of slide using eyelash. Position
embryos in a single layer side-by-side. We find that especially in the case of
embryos in which only a percentage show a phenotype of interest (e.g., homo-
zygous mutant progeny from heterozygous mutant mothers, weak RNAi, etc.),
that a large contiguous grouping of embryos is useful ( Fig. 1H ).
ix. Set the edge of a coverslip at the side of the agar pad opposite theM9 and slowly
drop so that the coverslip lands on the embryos prior to the M9. Use a Kimwipe
to wick excess buffer from edges of coverslip and wick air bubbles from under
coverslip.
x. Trim excess agar from edges of coverslip using a razor blade. Seal edges of the
coverslip with melted VALAP using a paintbrush.

Troubleshooting

i. Problem: Embryos fail to develop.
Solution: One-cell embryos are especially vulnerable to mechanical stress and
are challenging to mount without killing. If studying a later stage of develop-
ment, the likelihood of embryos surviving is markedly increased if two-cell or
later-stage embryos are used to make the mount. Groupings larger than 15-20
embryos often display increased lethality due to oxygen starvation. By keeping
groupings of embryos to less than 20 embryos, oxygen starvation should not be a
problem.
ii. Problem: The agar pad dries on the slide before it can be used.
Solution: Make the pad immediately before use. Stereomicroscopes with light
sources mounted under the stage have the potential to heat the stage after long
use, which can quickly dry agar pads. Using a stereomicroscope with an external
bulb or a cool temperature bulb will reduce this problem.
iii. Problem: When coverslip is placed on slide, all the embryos wash to the edge of
the coverslip.
Solution: Too much M9 buffer is used and the M9 buffer is hitting the embryos
before the coverslip can land on them and hold them in the agar.
iv. Problem: The slide has air bubbles under the coverslip.
Solution: Use more M9 buffer. This will allow M9 buffer to completely wash
under coverslip. However, too much M9 buffer will cause embryos to wash away
(see previous Problem).

Discussion

Mounting C. elegans embryos on agar mounts provides a stable, long-term envi-
ronment for microscopic analysis of development. The slight compression from the
coverslip will result in embryos reproducibly positioned with either the left or right
side facing toward the objective lens. During later stages of embryogenesis embryos
turn such that left-hand views become dorsal views and right-side views become
ventral views. Embryos on agar mounts will survive and hatch from the eggshell on