This protocol for determining brood size and embryonic viability is used by many C.elegans labs. Decreases in brute size and embryonic viability can indicate defects in important developmental processes such as myosis, fertilization, and embryogenesis. This technique provides clear instructions for novice C.elegans researchers.
It is relatively simple to set up and execute, and is a great starting point when working with new mutant strains. The biggest challenge for this technique is embryo versus unfertilized embryo identification. New researchers should practice the identification of embryos, oocytes, and the different larval stages before beginning these experiments.
To begin, label the back of the plate, transfer an individual L4 stage hermaphrodite down to the plate. Ensure no embryos or other worms are transferred onto the plate. Allow the worms to develop into adults and lay self progeny for 24 hours at the standard culturing temperature of 20 degrees celsius.
Score the plate on day three. On the next day, label a set of new plates and transfer the day one individual worm onto the new plate. Allow the worms to lay embryos for 24 hours at 20 degrees celsius.
Score the plate on day four. On day three, label a set of new plates and transfer day two individual worms onto the new plate. Allow the worms to lay progeny for 24 hours at 20 degrees.
Score the plate on day five. Draw a grid pattern on a 35 millimeter lid using a fine marker. Place the gridded lid under the test plate for counting to keep track of worms previously counted.
Using a differential cell counter, count the live larvae and unhatched embryos within the individual square. For worms on the square borders count based on the location of the worm head. Count worms with heads touching the top and left edges of the square.
Record the number of live larvae and unhatched embryos in a laboratory notebook. On day four, score the day two plate by counting the live larvae and unhatched embryos and record them in a laboratory notebook. On day five, score the day three plate by counting the live larvae and unhatched embryos, and record them in a laboratory notebook.
After labeling the back of the plate, transfer an individual L4 hermaphrodite worm onto the plate. Ensure no embryos or other worms are transferred onto the plate. Transfer a single L4 male worm onto the plate containing an L four hermaphrodite.
Allow the worms to mate and lay progeny for 24 hours at 20 degrees celsius. Score the plate for live larvae versus unhatched embryos on day three. The next day, label a set of new plates and transfer the hermaphrodite and male onto the new plate.
Ensure that the hermaphrodite has reached adulthood. Allow the hermaphrodites to lay progeny for 24 hours at 20 degrees celsius. Score the plate for live larvae versus unhatched embryos on day four.
On day three, label a set of new plates and transfer the hermaphrodite and male onto the new plate. Allow the worms to lay progeny for 24 hours at 20 degrees. Score the plate for live versus unhatched embryos on day five.
Using a differential cell counter, count the live larvae and unhatched embryos from the day one plate and record them in a laboratory notebook. On day four, count the live progeny and unhatched embryos from the day two plate and record them in a laboratory notebook. Check the day one plate for males.
If mating has occurred, the expected genetic ratio of hermaphrodites to males is 50 to 50. If the day one plate does not contain any males, mating between the male and hermaphrodite did not occur. Discard this mating pair and record the observation in the laboratory notebook.
On day five, count the live larvae and unhatched embryos from the day three plate and record them in a laboratory notebook. The embryonic viability assay for N2 yielded a viability percentage of 98.9%while both him-5(e1490)and spo-11(ok79)showed a reduction in embryonic viability with a percentage of 74.9%and 0.8%respectively. The average brood size of N2, him-5(e1490)and spo-11(ok79)were determined to be 217, 105 and 219 respectively.
Recognition of the various stages of C.elegans development is important for accurate and reproducible data. We recommend becoming familiar with worm development using pictures and a dissecting microscope. This procedure is a great first step to determine if a gene is involved in a developmental process and can be followed by cytological analyses to determine which process is disrupted.
