The authors thank CGC for kindly sending the C. elegans. This work was supported by National Key Research and Development Program of China (#2018YFC1603102, #2018YFC1602705); National Natural Science Foundation of China Grant (#31401025, #81273108, #81641184), The Capital Health Research and Development of Special Project in Beijing (#2011-1013-03), the Opening Fund of the Beijing Key Laboratory of Environmental Toxicology (#2015HJDL03), and the Natural Science Foundation of Shandong Province, China (ZR2017BF041).

The protocol follows the animal care guidelines of the Animal Ethics Committee of the Beijing Center for Disease Prevention and Control in China.
1. Chemical preparation
<ol>
	<li>Obtain chemicals (Table 1 and Table of Materials).</li>
	<li>Determine the highest and lowest dosage of the individual chemicals for a minimum concentration of 100% lethality (LC100, 24 h) and a maximum concentration of 100% nonlethality (LC0, 24 h) to worms. Use at least six dilut...

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+rank+order+water+soluble+compounds+according+to+their+toxicity+using+Caenorhabditis+elegans,+a+Complex+Object+Parametric+Analyzer+and+Sorter,+and+axenic+liquid+media.">A method to rank order water soluble compounds according to their toxicity using Caenorhabditis elegans, a Complex Object Parametric Analyzer and Sorter, and axenic liquid media.</a> Food and Chemical Toxicology. 47 (4), 722-728 (2009).</li><li>Wang, D., Xing, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+locomotion+behavioral+defects+induced+by+acute+toxicity+from+heavy+metal+exposure+in+nematode+Caenorhabditis+elegans.">Assessment of locomotion behavioral defects induced by acute toxicity from heavy metal exposure in nematode Caenorhabditis elegans.</a> Journal of Environmental Sciences (China). 20 (9), 1132-1137 (2008).</li><li>Leung, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans:+an+emerging+model+in+biomedical+and+environmental+toxicology.">Caenorhabditis elegans: an emerging model in biomedical and environmental toxicology.</a> Toxicological Sciences. 106 (1), 5-28 (2008).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+chemical+acute+toxicity+between+the+nematode+and+the+rodent.">Correlation of chemical acute toxicity between the nematode and the rodent.</a> Toxicology Research. 2 (6), 403-412 (2013).</li><li>Boyd, W. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+Effect+of+the+ToxCast+Phase+I+and+Phase+II+Chemicals+in+Caenorhabditis+elegans+and+Corresponding+Responses+in+Zebrafish,+Rats,+and+Rabbits.">Developmental Effect of the ToxCast Phase I and Phase II Chemicals in Caenorhabditis elegans and Corresponding Responses in Zebrafish, Rats, and Rabbits.</a> Environmental Health Perspectives. 124 (5), 586-593 (2016).</li></ol>

The authors have nothing to disclose.

The advantages of C. elegans have led to its increasing usage in toxicology9, both for mechanistic studies and high-throughput screening approaches. An increased role for C. elegans in complementing other model systems in toxicological research has been remarkable in recent years, especially for the rapid toxicity assessment of new chemicals. This article provides a new assay of high-throughput, quantitative screening of worm phenotypes in a 384-well plate for the automatic ident...

Along with the rapid development of chemical compounds applied to industrial production and people&#39;s daily life, it is important to study the toxicity testing models for the chemicals. In many cases, the rodent animal model is employed to evaluate the potential toxicity of different chemicals on health. In general, the determination of lethal concentrations (i.e., the assayed 50% lethal dose&#160;[LD50] of different chemicals) is used as the traditional parameter in a rodent (rat/mouse) model in vivo, which is time-consuming and very expensive. In addition, due to the reduce, refine, or replace (3R) principle that is central to animal welfare and ethics, new methods that allow for the replacement of higher animals are valuable to scientific research1,2,3. C. elegans is a free-living nematode that has been isolated from soil. It has been widely used as a research organism in the laboratory because of its beneficial characteristics, such as a short lifespan, easy cultivation, and efficient reproduction. In addition, many fundamental biological pathways, including basic physiological processes and stress responses in C. elegans, are conserved in higher mammals4,5,6,7,8. In a couple of comparisons we and others have made, there is a good concordance between C. elegans toxicity and toxicity observed in rodents9. All of this makes C. elegans a good model to test the effects of chemical toxicities in vivo.
Recently, some studies quantified the phenotypic features of C. elegans. The features can be used to analyze the toxicities of chemicals2,3,10 and the aging of worms11. We also developed a method that combines a liquid worm culturing system and an image analysis system, in which the worms are cultured in a 384-well plate under different chemical treatments12. This quantitative technique has been developed to automatically analyze the 33 parameters of C. elegans after 12-24h of chemical treatment in a 384-well plate with liquid medium. An automated microscope stage is used for experimental video acquisition. The videos are processed by a custom-designed program, and 33 features related to the worms&#39; moving behavior are quantified. The method is used to quantify the worm phenotypes under the treatment of 10 compounds. The results show that different toxicities can alter the phenotypes of C. elegans. These quantified phenotypes can be used to identify and predict the acute toxicity of different chemical compounds. The overall goal of this method is to facilitate the observation and phenotypic quantification of experiments with C. elegans in a liquid culture. This method is useful for the application of C. elegans in chemical toxicity evaluations and phenotype quantifications, which help predict the acute toxicity of different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.&#160;In addition, this method can be applied to the toxicity screening and testing of new chemicals or the compound as the food additive agent pollution, pharmacautical compounds, environmental exogenous compound, and so on.

<table border="0" cellpadding="0" cellspacing="0" style="width:959px;" width="957">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">2-Propanol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">59300</td>
			<td style="width:407px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384-well plates</td>
			<td style="width:171px;">Throme</td>
			<td style="width:175px;">142761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Bacto</td>
			<td style="width:175px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Atropine sulfate</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">PHL80892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Bleach buffer</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td>0.5 mL of 10 M NaOH, 0.5 mL of5% NaClO, 9 mL ofultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cadmium chloride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">202908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Calcium chloride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">21074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">CCD camera</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:175px;">AxioCam HRm</td>
			<td style="width:407px;">Zeiss microscopy GmbH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cholesterol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">C8667</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Copper(II) sulfate</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">451657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Ethanol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">24105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Ethylene glycol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">324558</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Glycerol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">K-Medium</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">3.04 g of NaCl and 2.39 g of KCl in 1 L ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">LB Broth&#160;</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">10&#160;g/L&#160;Tryptone,&#160;5&#160;g/L&#160;Yeast&#160;Extract,&#160;5&#160;g/L&#160;NaCl&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Magnesium sulfate heptahydrate</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">63140</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:207px;">NGM Plate</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">3 g ofNaCl, 17 g ofagar, 2.5 g ofpeptone in 1 L of ultrapure water, after autoclave add 1 mL of cholesterol (5 mg/mL in ethanol), 1 mL of MgSO4 (1 M), 1 mL of CaCl2 (1 M), 25 mL of PPB buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Bacto</td>
			<td style="width:175px;">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Potassium chloride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">60130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Potassium phosphate dibasic</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">795496</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Potassium phosphate monobasic</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">795488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">PPB buffer</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">35.6 g of K2HPO4, 108.3 g of KH2PO4 in 1 L ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">shaker</td>
			<td style="width:171px;">ZHICHENG</td>
			<td style="width:175px;">ZWY-200D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Sodium chloride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">71382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Sodium fluoride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">s7920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Sodium hydroxide</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">71690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Sodium hypochlorite solution</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">239305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">The link of program</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">https://github.com/weiyangc/ImageProcessForWellPlate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">T7293</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">Y1625</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Zeiss automatic microscope&#160;</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:175px;">AXIO Observer.Z1</td>
			<td style="width:407px;">Zeiss automatic microsco with peproprietary software Zen2012 and charge coupled device(CCD) camera</td>
		</tr>
	</tbody>
</table>

a high-throughput assay for the prediction of chemical toxicity by automated phenotypic profiling of caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

We have tested the phenotypes of worms exposed to different concentrations of more than 10 chemicals12. In the test, 33 distinct features were quantified for each chemical compound at three time points (0 h, 12 h, and 24 h). Previously, a comparison between a manual and an automatic analysis of a lifespan assay was done11,12. In this assay, we found that chemicals and concentrations can influence the worm p...

Watch this Scientific Journal Video about A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans at JoVE.com

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

C.elegans toxicological assay is valuable. This protocol describes how to treat C.elegans with chemicals in a 384-well plate, capture videos, and quantify toxicological-related phenotypes. This method could help to identify and predict a chemical's potential acute toxicity.This quantitative technique has been developed to automatically analyze the 33 parameters of C.elegans after 12 to 24 hour of chemical treatment in a 384-well plate with liquid medium. C.elegans is valuable as a good model of rapid toxicity assessment for new chemicals. As mice, it can be applied to the toxicity screening and testing of new chemicals, or the compound as the food additive agent pollution, pharmaceutical compound, environment exogenous compound, and so on.Read the protocol carefully and always follow the with step by step, then you will be the good one. Visual demonstration is easier for user to see the operation of each step, and the result of the operation. Demonstrating the procedure will be Wenjing Zhang and Gaochao Han.They are graduate students from my laboratory. To begin, conduct a preliminary worm lethality test for a new chemical, to determine the highest dosage, and the lowest dosage, which are the minimum concentration of 100%lethality, and the maximum concentration of 100%non-lethality. For this experiment, prepare seven gradient concentrations of cadmium chloride.To prepare two times the highest concentrated aqueous solution, dissolve 92.8 milligrams of cadmium chloride solid powder in a centrifuge tube filled with eight milliliters of K medium. Vortex to mix. After the powder is fully dissolved, use a pipette to fill up to 10 milliliters.Prepare the other concentration levels by dilution with K medium. In a super clean bench, use a sterile tip to pick a single colony of E.coli OP50 from the streak plate, and place it into the flask with 100 milliliters of LB broth to inoculate the colony. Grow it overnight at 37 degrees Celsius in the incubator shaker.Pour NGM into a 90 milliliter plastic Petri plate. Seed each plate with 300 microliters of E.coli OP50 solution. Incubate N2 worms on the NGM plates with OP50 at 20 degrees Celsius for about 2 to 3 days, until most of the worms have reached the adult stage.With sterile water, wash down gravid worms into a 15 milliliter sterile conical centrifuge tube to harvest. Let the worms settle down for at least 2 minutes. Aspirate the water and add five milliliters of bleach buffer.Vortex the tube for five minutes, and then put the tube in a centrifuge to spin at a relative centrifugal force of 1300 for 30 seconds to pellet the eggs. Use a pipette to aspirate the supernatant. Add five milliliters of sterilized water into the tube to wash the eggs, and vortex the tube for five seconds.Centrifuge the tube for 30 seconds, at a relative centrifugal force of 1300. Remove the supernatant and add five milliliters of sterilized water to wash again. Pipette the eggs onto a new NGM plate with OP50.Incubate them at 20 degrees Celsius overnight. The next morning, the hatched worms reach L1 stage. After approximately 40 hours, the worms reach the L4 stage.With K medium, wash the L4 stage worms off the Petri plates into a 50 milliliter sterile conical tube. Pipette 50 microliters of the worm liquid from the tube to a glass slide. Under a stereo microscope, use a pipette to adjust the concentration of worms to approximately 40 animals per 100 microliters of K medium.Add 50 microliters of the prepared medium into each well of the 384-well plate. These synchronized L4 stage worms are ready for following treatment by chemicals. Before adding the chemicals, place the 384-well plate, with the synchronized worms on the automatic stage, and set up video camera with the programmed acquisition procedure.In the 384-well plate, worms are treated with six to seven dosages of each individual chemical, with eight parallel wells containing 50 microliters of the two times chemical solution for every dosage. Prepare at least three groups of eight parallel wells of K medium as a control. Add 50 microliters of the two times chemical solution for each well.Set the time as the zero-hour point. Then, put the 384-well plate in the incubator shaker set at 20 degrees Celsius, and 80 revolutions per minute. After a desired time, remove the plate from the incubator, and transfer it to the automatic stage.Take videos of each well of the plate, at 12 hours, and at 24 hours to check the phenotypes of the worms. To start processing the experimental video, transfer the video files to the computer. Via the graphical user interface, click on the Select button, to choose the source images directory.Add the middle result directory. The middle results include the segmented images, which are useful for the visual observation of the processed images. Add the final result directory in the interface.Set the average worm size parameter at 2, 000, in the Worm Size text box in the interface. Set the threshold of moved ratio at 0.93 in the interface. Click on the Analyze button to start the image processing.The program developed can recognize worms and quantify phenotypes automatically. In this experiment, 33 distinct features were quantified for cadmium chloride treatment at three time points, zero-hour, 12 hours, and 24 hours. Experimental images show that the worms died more quickly as the chemical concentration increased.In the beginning, there was no significant difference between the control and chemical treatments. After 12 hours of treatment, the worms treated with a high concentration became straighter and less curved than at lower concentrations or in control groups. The major axis length of worm body covered region increased as time increased.There is also a gradient trend from lower to higher chemical concentrations in both the major axis and in the minor axis length. The worms'motility, based on the area and the ratio, showed similar patterns. No significant difference was observed at the beginning.As time passed, the worms in the control group showed a stable decrease in motility. After 12 and 24 hours, the worms that were treated with cadmium chloride showed significant differences in motility, compared with control groups. In addition, the worms under higher concentration treatments showed a weaker motility, compared to the worms under lower concentration treatments.In summary, this technique paves a way of rapid toxicity assessment in multiple areas. Researchers that could apply the method to the emergency analysis of toxicity in food borne toxicosis, the safety evaluation of pharmaceutical compounds, as well as the acute toxicity screening, and the detection of new chemicals, and the environmental exogenous compounds. The cadmium chloride is a low toxic substance.Please wear gloves and a mask during your handling, and follow the operating instructions.

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Research

JoVE Journal

Environment

7.1K visualizações.  Beijing Center for Disease Prevention and Control/Beijing Center of Preventive Medicine Research, China. O ensaio toxicológico de C.elegans é valioso. Este protocolo descreve como tratar C.elegans com produtos químicos em uma placa de 384 poços, capturar vídeos e quantificar fenótipos tóxicos relacionados. Este método poderia ajudar a identificar e prever a potencial toxicidade aguda de um produto químico.Esta técnica quantitativa foi desenvolvida para analisar automaticamente os 33 parâmetros de C.elegans após 12 a 24 horas de tratamento químico em uma placa de 384 poços co...