Lee&#160;and Kang, <a href="https://www.jove.com/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>,&#160;J. Vis. Exp. (2017).
1. C. elegans&#160;Egg Laying Assay
<ol>
	<li>Incubate the age-synchronized eggs on the NGM plate containing DMSO or etoposide for 64 h (before first birth) until the young adult stage at 20 &#176;C. 
	NOTE: It is optional to use the worms from the growth measurement experiments. It is convenient to do the growth measurement and egg laying assay together.</li>
	<li>Transfer 5 adult worms to the new NGM plates without chemicals (condition 1, chemical treatment within a certain period of time, from eggs to young adult stage) or new NGM plate containing the same chemical pretreatment (condition 2, continuous exposure of chemicals through the overall experimental period), and then incubate them for 24 h. 
	NOTE: It is recommended to make replicates using 3-5 NGM plates for each treatment; these replicates can be used for statistical analysis.</li>
	<li>Transfer to the new NGM plate as described above and count the number of eggs every day. Count the worms that crawled off, died, and were internally hatched.</li>
	<li>Repeat these steps until the worms lay no more eggs, usually in 5 days.</li>
	<li>Calculate the number of eggs laid per worm from each plate, and sum these values every day for 5 days to determine the total number of eggs laid. 
	NOTE:&#160;Exclude the number of worms that crawled off and internally hatched from the calculation.</li>
</ol>

<table><tbody><tr><td>Caenorhabditis elegans&amp;nbsp;N2</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td>Wild type</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma, USA</td><td>D2650</td><td></td></tr><tr><td>Escherichia coli&amp;nbsp;OP50</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td></tr><tr><td>Etoposide</td><td>Sigma, USA</td><td>E1383</td><td></td></tr><tr><td>35 &amp;times; 10 mm Petri dish</td><td>SPL Life Sciences, South Korea</td><td>10035</td><td></td></tr><tr><td>Stereo microscope</td><td>Nikon, Japan</td><td>SMZ800N</td><td></td></tr></tbody></table>

egg laying assay: a method to quantify the egg-laying behavior of c. elegans

Source: Lee, S. Y. and Kang, K. <a href="https://www.jove.com/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2017).
C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. 
...

- Experiments to quantify egg laying have revealed the neurological components and environmental cues that impact this behavior. Changes in egg laying behavior may indicate toxic effects on the neuromuscular or reproductive systems. To test for toxicity, raise developmentally-synchronized eggs on nematode growth media plates with and without the compound of interest. These experiments use heat-killed bacterial worm food to prevent the bacteria from metabolizing the compound. Allow this generation to grow until they become young adults. Then move worms onto new plates under the appropriate control or experimental conditions, and leave them to lay eggs for 24 hours. Each day, transfer the adults to a new plate, and count the eggs on the previous plate. Continue until the adults stop laying, about five days for wild-type worms. Calculate the number of eggs laid per adult worm each day, and add up the total at the end of the experiment. In the example protocol, we will use the C. elegans egg laying assay to test etoposide-- a chemotherapy drug-- for reproductive toxicity. - Incubate the age-synchronized eggs on the NGM plate containing DMSO or etoposide at 20 degrees Celsius for 64 hours, until the young adult stage. Transfer five adult worms to new NGM plates without chemicals or new NGM plates containing the same chemical pre-treatment, and incubate them for 24 hours. Transfer the adults to new NGM plates, as just demonstrated, and count the number of eggs every day. In addition, count the worms that crawled off, died, or were internally hatched. Carry out calculations according to the text protocol.

egg-laying-assay-method-to-quantify-egg-laying-behavior-c

Research

JoVE Encyclopedia of Experiments

Biology

Caenorhabditis elegans (worm)

Basic Methods

C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Overview

Protocol

<table><tbody><tr><td>Caenorhabditis elegans&nbsp;N2</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td>Wild type</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma, USA</td><td>D2650</td><td></td></tr><tr><td>Escherichia coli&nbsp;OP50</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td></tr><tr><td>Etoposide</td><td>Sigma, USA</td><td>E1383</td><td></td></tr><tr><td>35 &times; 10 mm Petri dish</td><td>SPL Life Sciences, South Korea</td><td>10035</td><td></td></tr><tr><td>Stereo microscope</td><td>Nikon, Japan</td><td>SMZ800N</td><td></td></tr></tbody></table>

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

In This Article

Overview

Protocol

Materials

Name	Company	Catalog Number	Comments
Caenorhabditis elegans N2	Caenorhabditis Genetics Center (CGC)		Wild type
Dimethyl sulfoxide	Sigma, USA	D2650
Escherichia coli OP50	Caenorhabditis Genetics Center (CGC)
Etoposide	Sigma, USA	E1383
35 × 10 mm Petri dish	SPL Life Sciences, South Korea	10035
Stereo microscope	Nikon, Japan	SMZ800N