The overall goal of this procedure is to measure the effect of chemicals, such as the topoisomerase inhibitor etoposide, on the growth and reproduction of the model animal Caenorhabditis elegans. This method can help answer key questions in the toxicology field, such as toxicological evaluation for the development of drugs and risk assessment of environmental toxicants. The main advantage of this technique is that the toxicity of various chemicals can be checked in non-mammalian systems, so this technique is convenient and cost-effective.
Demonstrating the procedure will be So Young Lee, a graduate student from my laboratory. To begin, prepare two clean, 200-milliliter glass bottles by adding 0.6 grams of sodium chloride, 0.5 grams of peptone, 3.4 grams of agar, 195 milliliters of distilled water, and a magnetic stirrer to one bottle. Then add a magnetic stirrer to the other empty bottle.
Autoclave the bottles for 15 minutes at 121 degrees Celsius, and then cool down the bottles in a water bath for 30 minutes at 55 degrees Celsius. Next, to the filled bottle, add 0.2 milliliters of one molar calcium chloride, 0.2 milliliters of five milligrams per milliliter cholesterol, 0.2 milliliters of one molar magnesium sulfate, and five milliliters of one molar potassium phosphate. Then mix the solution by magnetic stirring on a hot plate at 55 degrees Celsius.
Pour half of the mixed NGM medium into the empty 200-milliliter bottle. Then add one milliliter of DMSO to one bottle, and use magnetic stirring to mix it again. Store the other bottle in a water bath at 55 degrees Celsius.
Then aliquot three milliliters of the DMSO-containing medium to 35-by-10-millimeter Petri dishes. Remove the other bottle from the water bath, and use magnetic stirring to mix it. Then add one milliliter of 75-millimolar etoposide.
And after mixing again, repeat the aliquoting procedure. Cool down the chemical-containing NGM plates by leaving them at room temperature for approximately three hours, and store them at four degrees Celsius until use. For the chemical test, spread 100 microliters of heat-inactivated E.coli OP50 on each NGM plate.
Allow the plates to dry overnight in a C.elegans incubator at 20 degrees Celsius. Transfer the age-synchronized C.elegans eggs to an NGM plate supplemented with 1%DMSO or 750-micromolar etoposide. Place the C.elegans in an incubator at 20 degrees Celsius for four days.
Observe and take microscopic images of C.elegans under stereo microscope every day. Also, take a microscopic image of the microscope stage micrometer for worm size calibration. To measure the body length of C.elegans treated with DMSO or etoposide at each time point, in ImageJ, open the microscope stage micrometer image.
Then using the Straight Line tool, drag a line of 1, 000 micrometers on the image. Click Analyze and then Set Scale, and input 1, 000 and micrometers into the Known Distance box and Unit of Length box, respectively. Then check Global and click OK.Open the worm images using File and then Open.
Then, using the Freehand Line tool, draw a line along the feature of each worm. Obtain the body length data by clicking Analyze and then Measure. Repeat these steps for 50 worms.
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 pretreatment, 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. As shown here, the treatment of etoposide at 24 to 96 hours significantly retarded the growth of C.elegans.
After 96 hours of incubation, etoposide-treated worms grew to 0.86 millimeters in body length while the vehicle-treated worms grew to 1.04 millimeters. Growth retardation was also observed under stereo microscope observation, as seen in these images of etoposide-treated worms, as compared to control-treated worms. After 72 hours of incubation, eggs from the vehicle-treated worms were observed.
In contrast, eggs were observed after 96 hours of etoposide treatment, raising the possibility that etoposide treatment delayed the first birth time of C.Elegans. As depicted in these graphs, a decrease in total egg number by etoposide treatment was observed when the young adult worms were cultured, either in the presence or absence of continuous etoposide treatment. Once mastered, this technique can be done in one week if it is performed properly.
After watching this video, you should have a good understanding of how to evaluate chemical toxicity in C.elegans, a model nematode.
