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.
