- Start with a drop of buffer solution on a microscope slide and transfer intact worms to the droplet. Once the worms are in place, use a lint-free tissue to soak up and remove the buffer. Fix the sample by bathing the specimens with 90% ethanol several times, allowing it to evaporate after each application.
Once the ethanol has evaporated after the final rinse, add a solution containing the nucleic acid dye, DAPI, to the sample. DAPI binds preferentially to adenine and thymine bases in the minor groove of DNA. When bound, the DAPI-DNA complex absorbs UV light and emits visible blue light.
Next add an anti-fade preservative to extend the shelf life of the sample. Apply a cover slip and seal it to the slide. Then use a fluorescent microscope with a blue filter to visualize DAPI bodies, which, depending on the cell and its cycle status, can represent single chromosomes, sister chromatid pairs, or whole nuclei. In this experiment, we will count DAPI stained sister chromatid pairs in oocytes, to identify tetraploid strains of Caenorhabditis elegans.
- Tetraploid strains have 12 chromosome pairs, which can be validated by counting the DAPI stained bodies in unfertilized oocytes. To do so, drop five to ten microliters of M9 buffer onto a slide and transfer six to 10 putative tetraploids into the drop. Under a dissecting microscope, carefully soak up most of the M9 into a lint free cleaning tissue.
Then drop 10 microliters of 90% ethanol onto the worms and watch the ethanol evaporate completely. As soon as the evaporation finishes, repeat the process of adding ethanol and watching it evaporate. In total, add 10 microliters in four applications. After the last drop evaporates, add six microliters of DAPI at two nanograms per microliter, or a similar stain.
For long term storage of the slides, mount the worms in a commercially available or homemade anti-fade. Then add a coverslip and seal the edges with nail polish. After the nail polish has dried, the slides can be scored. Use a fluorescent microscope and 100 x magnification.
First, find the most mature unfertilized oocyte, which is immediately adjacent to the spermatheca, and has not yet entered either the spermatheca or the uterus. The DAPI bodies here are presumably single chromosome pairs. Next, focus on the nucleus of the oocyte and use the microscope's fine focus to slowly scan it, from top to bottom, while counting the DAPI bodies. Then recount the DAPI bodies in the same nucleus by moving the focus from bottom to top.
Wild type oocytes have six DAPI bodies on average. The presence of 12 DAPI bodies indicates that the animals in this strain are either partial or complete tetraploids. Analyze at least 10 animals per strain. Often chromosome pairs will be very close or touching so the number of DAPI bodies is often smaller than the actual number of chromosome pairs.