Cell Identification and Cell Lineage Analysis - Caenorhabditis Elegans: Molecular Genetics and Development - page 334

1. Troubleshooting
The joy of observing a well-behaving worm is balanced by the frustration of a
badly behaved worm that persistently heads for the edge of the agar pad, only to end
up in the immersion oil or vaseline. To avoid such frustration it is important to check
your worm frequently (every 10 min) and to learn how to recover the worm intact
from the slide mount. Practice sliding off the coverslip and getting the worm into a
buffer-drop from which you can suck it back into your capillary.
If the worm stops moving and cells lose contrast, the animal may be dying, or it
may be entering lethargus, the 1-2 h period of inactivity that precedes each molt. If
the developmental stage makes the latter explanation unlikely, there may be too
much bacteria under the slide, leading to hypoxia. The worm can be revived by
removing it from the slide (slide off the coverslip and use mouth pipette + drawn-out
capillary to retrieve the worm). Place the worm on an NGM agar plate to recover for a
few minutes, then remount.
The microscope DIC optics should be optimized (Kohler illumination). A heat
filter must be used to prevent specimen heating under the prolonged observation.
Immersion oil should be used between the objective and coverslip and between slide
and condenser top lens. Ensure there are no bubbles or debris in the agar pad; once a
worm crawls into a bubble, it will not come out again.
VII. Discussion
Cell-lineage analysis allows rigorous definition of cell ancestries and positions,
mutant phenotypes, and gene expression patterns to single-cell resolution. The labor-
intensive nature of lineage tracing from live samples has tended to limit its popu-
larity. The recent development of automated lineage analysis promises to reduce the
effort needed for early embryonic lineage studies, but lineage tracing in later
embryos and in larvae remains a labor of love. Further computational advances
may help to return lineage studies to the center of C. elegans developmental biology.
Acknowledgments
The authors thank members of their lab for comments and the C. elegans community for their
development and sharing of the techniques mentioned here. Our work on embryonic development has
been supported by an NIH NRSA Award to CAG and NIH R01 GM54657 to ADC.
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