Why does null hypothesis testing matter for DAPI-based ploidy validation?

Null hypothesis testing ensures that observed differences in DAPI body counts between strains are statistically significant, supporting confident validation of engineered ploidy states in genetic models.

How does independent variable isolation fit the ethanol fixation workflow?

Isolating the variable of ploidy by controlling fixation and staining conditions allows direct attribution of DAPI body count differences to genetic manipulations, strengthening mechanistic interpretation.

What do quantitative DAPI body measurements enable in genetic model assessment?

Quantitative DAPI body counts provide objective, reproducible metrics for distinguishing diploid from tetraploid strains, enabling robust model selection and downstream experimental design.

Why are replication requirements critical for cross-functional scoring of oocyte DAPI bodies?

Replicating DAPI body counts across multiple animals and scorers ensures data reliability, supports cross-team confidence, and reduces subjective bias in genetic model validation workflows.

What statistical analysis capabilities are required before implementing DAPI-based ploidy scoring?

Teams must be able to perform statistical comparisons of DAPI body counts across strains, assess variance, and determine significance to validate ploidy manipulations before broader implementation.

Ethanolfixatie en DAPI-kleuring: een methode om DNA in C. elegans te visualiseren

JoVE Encyclopedia of Experiments

Biology

0 views • 3:32 min • April 30th, 2023

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- 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.

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Last updated: 4 July 2026

Ethanolfixatie en DAPI-kleuring: een methode om DNA in C. elegans te visualiseren

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