Method for counting embryos developing in a uterus of caenorhabditis elegans

A method for counting embryos developing in a uterus of Caenorhabditis elegans, comprising the following steps:

• step 1:
• preparation of an E. coli solution:
• selecting freshly prepared E. coli OP50 as feed for Caenorhabditis elegans, which is divided into two parts according to its intended use, wherein one part of the E. coli solution is used to coat an NGM medium and the other part is used to feed Caenorhabditis elegans daily when exposed to triphenyl phosphate, which is name as TPHP;
• wherein preparing process the NGM medium in step 1 is as follows:
• adding 3 g of NaCl, 17 g of agar, 2.5 g of peptone, 0.111 g of CaCl2 and 0.24 g of MgSO4 to 1 L of deionized water, stirring a resulted mixture for dissolution, sterilizing the mixture at 121° C. for 25 minutes, and after sterilization, cooling the medium to 55° C. to 60° C., adding 1 mL of cholesterol solution and 25 mL of potassium phosphate buffer solution, stirring the mixture for even mixing, and loading 15 mL of medium onto a 7 cm petri dish with a peristaltic pump, to obtain the NGM medium after cooling and solidification at room temperature;
• wherein the 5 mg/mL cholesterol solution is prepared with absolute ethanol and the process for preparing the potassium phosphate buffer solution is as follows:
• adding 10.83 g of KH2PO4 and 4.7 g of K2HPO4 to each 100 mL of ultrapure water, stirring a resulted mixture for dissolution, and sterilizing the mixture at 121° C. for 25 minutes;
• step 2:
• coating of E. coli:
• drawing an appropriate amount of E. coli for coating onto a surface of the NGM medium with a pipette, then coating E. coli at the center of the petri dish evenly with a sterilized coating rod or bottom of a test tube, and after E. coli is completely attached to the NGM medium and forms a thin film, placing the petri dish upside down in a 20° C. dark biochemical incubator for later use;
• step 3:
• culture of Caenorhabditis elegans:
• inoculating an appropriate number of Caenorhabditis elegans on the surface of the NGM medium with E. coli, observing a growth status of Caenorhabditis elegans daily, and if E. coli is consumed completely, replacing the medium in a timely manner until most Caenorhabditis elegans are pregnant;
• step 4:
• lysis of Caenorhabditis elegans:
• rinsing pregnant Caenorhabditis elegans under culture from a petri dish into a 1.5 mL centrifuge tube with K solution 3 times, discarding a supernatant after centrifugation, adding 1 mL of lysis solution into the centrifuge tube, subjecting a resulted mixture to vortex oscillation vigorously for 1.5 minutes, then discarding a supernatant after centrifugation again, adding the lysis solution, subjecting the resulting mixture to vortex oscillation vigorously until the pregnant nematodes are completely lysed and the eggs are completely exposed, collecting the eggs and washing the eggs 3 to 5 times to remove the residual lysis solution;
• step 5:
• synchronization treatment of Caenorhabditis elegans:
• adding nematode eggs dropwise to the NGM medium containing no E. coli, culturing the nematode eggs at 20° C. for 16 to 20 hours under dark conditions, at this time, Caenorhabditis elegans are in the L1 stage, and rinsing Caenorhabditis elegans at the stage into a 1.5 mL centrifuge tube with the K solution 3 times;
• step 6:
• preparation of exposure solutions:
• weighing 0.1000 g of TPHP solid accurately, dissolving the TPHP solid in dimethyl sulfoxide until a concentration reaches 10 g/L, then diluting TPHP solutions with dimethyl sulfoxide again until concentrations reach 10 mg/L, 100 mg/L, 1000 mg/L and 5000 mg/L to be used as mother liquors later, using dimethyl sulfoxide as the mother liquor of the blank control group, further diluting the mother liquors by 100 folds with the K solution and then diluting the diluted mother liquors by 100 folds with a K+ solution to finally obtain exposure solutions of Caenorhabditis elegans containing 0 μg/L, 1 μg/L, 10 μg/L, 100 μg/L and 500 μg/L TPHP.
• step 7:
• exposure of Caenorhabditis elegans to a TPHP solution:
• adding a prepared TPHP exposure solution to a sterile 6-well plate with 5 mL of prepared TPHP exposure solution per well, then evenly adding Caenorhabditis elegans at the L1 stage to the 6-well plate after the synchronization treatment, feeding Caenorhabditis elegans with 100 μg/L E. coli daily, and culturing Caenorhabditis elegans for 72 hours;
• step 8:
• selection of Caenorhabditis elegans at an exposure stage:
• after exposure to TPHP, transferring Caenorhabditis elegans to the NGM medium containing E. coli OP50, culturing Caenorhabditis elegans until pregnancy, then transferring pregnant Caenorhabditis elegans to a 1.5 mL centrifuge tube and washing the pregnant Caenorhabditis elegans with the K solution 3 times;
• step 9:
• preparation of NaClO solution:
• taking NaClO with effective chlorine content of 5.5% to 6.5% and diluting NaClO at a volume ratio of 1:4 (NaClO:K solution) to obtain an experimental NaClO solution for later use;
• step 10:
• transparent treatment of Caenorhabditis elegans:
• adding the prepared NaClO solution to a sterile 24-well plate with 1 mL of the prepared NaClO solution per well, then adding 10 to 20 Caenorhabditis elegans to each well, and after 5 minutes of standing at room temperature, observing states of Caenorhabditis elegans every 2 minutes until nematode bodies are transparent;
• step 11:
• counting of embryos in the uterus of Caenorhabditis elegans:
• putting the 24-well plate containing transparent Caenorhabditis elegans under an optical stereomicroscope, and counting exposed embryos of each Caenorhabditis elegans;
• and
• step 12:
• data analysis:
• analyzing data by using SPSS software, setting a confidence interval of samples to 95%, and calculating an intra-group mean and mean error of the numbers of embryos developing in nematodes in different treatment groups.